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19 Kempton, J.H. (1920) Heritable characters of maize V. Adherence, J. Hered.
11, 317–322
20 Becraft, P.W., Stinard, P.S. and McCarty, D.R. (1996) CRINKLY4: a TNFRlike receptor kinase involved in maize epidermal differentiation, Science 273,
1406–1409
21 Jenks, M.A. et al. (1996) Mutants in Arabidopsis thaliana altered in
epicuticular wax and leaf morphology, Plant Physiol. 110, 377–385
22 Lolle, S.J., Hsu, W. and Pruitt, R.E. (1998) Genetic analysis of organ fusion in
Arabidopsis thaliana, Genetics 149, 607–619
23 Sinha, N. (1998) Organ and cell fusions in the adherent1 mutant in maize, Int.
J. Plant Sci. 159, 702–715
24 Sinha, N. and Lynch, M. (1998) Fused organs in the adherent1 mutation in
maize show altered epidermal walls with no perturbations in tissue identities,
Planta 206, 184–195
25 Lolle, S.J., Cheung, A.Y. and Sussex, I.M. (1992) Fiddlehead: An Arabidopsis
mutant constitutively expressing an organ fusion program that involves
interactions between epidermal cells, Dev. Biol. 152, 383–392
26 Lolle, S.J. and Cheung, A.Y. (1993) Promiscuous germination and growth of
wild-type pollen from Arabidopsis and related species on the shoot of the
Arabidopsis mutant, fiddlehead, Dev. Biol. 155, 250–258
27 Lolle, S.J. et al. (1997) Developmental regulation of cell interactions in the
Arabidopsis fiddlehead-1 mutant: a role for the epidermal cell wall and cuticle,
Dev. Biol. 189, 311–321
28 Aida, M. et al. (1997) Genes involved in organ separation in Arabidopsis: an
analysis of the cup-shaped cotyledon mutant, Plant Cell 9, 841–857
29 Koornneef, M., Hanhart, C.J. and Thiel, F. (1989) A genetic and phenotypic
description of eceriferum (cer) mutants in Arabidopsis thaliana, J. Hered. 80,
118–122
30 Levin, J.Z. et al. (1998) A genetic screen for modifiers of UFO meristem
activity identifies three novel FUSED FLORAL ORGANS genes required for
early flower development in Arabidopsis, Genetics 149, 579–595
31 Neuffer, M.G., Coe, E.H. and Wessler, S.R. (1997) Mutants of Maize, Cold
Spring Harbor Laboratory Press
32 Becraft, P.W. (1998) Receptor kinases in plant development, Trends Plant Sci.
3, 384–388
33 Hülskamp, M. et al. (1995) Identification of genes required for pollen–stigma
recognition in Arabidopsis thaliana, Plant J. 8, 703–714
34 Heslop-Harrison, J., Knox, R.B. and Heslop-Harrison, Y. (1974) Pollen-wall
proteins: exine–held fractions associated with the incompatibility response in
Cruciferae, Theor. Appl. Genet. 44, 133–137
35 Verbeke, J.A. (1989) Stereological analysis of ultrastructural changes during
induced epidermal cell redifferentiation in developing flowers of
Catharanthus roseus (Apocynaceae), Am. J. Bot. 76, 952–957
36 Preuss, D. et al. (1993) A conditional sterile mutation eliminates surface
components from Arabidopsis pollen and disrupts cell signaling during
fertilization, Genes Dev. 7, 974–985
37 Taylor, L.P. and Hepler, P.K. (1997) Pollen germination and tube growth,
Annu. Rev. Plant Physiol. Plant Mol. Biol. 48, 461–491
38 Wolters-Arts, M., Lush, W.M. and Mariani, C. (1998) Lipids are required for
directional pollen-tube growth, Nature 392, 818–821
39 Bell, P.R. (1995) Incompatibility in flowering plants: adaptation of an ancient
response, Plant Cell 7, 5–6
40 Conner, J.A. et al. (1998) Comparative mapping of the Brassica S locus
region and its homeolog in Arabidopsis: implications for the evolution of
mating systems in the Brassicaceae, Plant Cell 10, 801–812
Susan J. Lolle* and Robert E. Pruitt are at the Dept of Molecular
and Cellular Biology, Harvard University, 16 Divinity Avenue,
Cambridge, MA 02138, USA.
*Author for correspondence (tel 11 617 495 0568;
fax 11 617 496 6702; e-mail lolle@billie.harvard.edu).
Molecular genetics of DNA repair in
higher plants Anne B. Britt
Damage to DNA occurs in all living things, and the toxicity and/or mutagenicity of the damage
products are reduced through the activities of one or more DNA repair pathways. The mechanisms of DNA repair are best understood in microorganisms and mammals, but the field has
recently expanded to include both plants and lower animals. These recent advances in our
understanding of the molecular and classical genetics of DNA repair in higher plants include
such aspects as the repair of UV-induced pyrimidine dimers, the correction of mismatched
bases, and the rejoining of double strand breaks.
T
he genomes of all living things are constantly subject to damage and decay. Some of the damage occurs as an inevitable
consequence of the chemical nature of DNA and its aqueous
environment, or as a result of errors of metabolism (i.e. the donation
of a methyl group to DNA rather than its intended target, or the
presence of stray radicals mistakenly formed during respiration or
photosynthesis). This endogenously generated damage is often
termed ‘spontaneous’ DNA damage to distinguish it from damage
due to exogenous sources, such as UV radiation and chemical DNA
damaging agents.
The wide variety of DNA damaging agents results in the formation
of an equally wide variety of damage products. Although DNA
damage is often associated with mutagenesis, the actual biological
consequences of these damaged products depends on the chemical
nature of the lesion. For example, the widely used artificial mutagen,
20
January 1999, Vol. 4, No. 1
ethyl methane sulfonate (EMS) can donate its alkyl group with varying efficiencies to almost all of the nitrogen and oxygen atoms
present on all of the bases1. Its most frequently induced product,
N7-alkylguanine, is also its most innocuous; this lesion apparently
has no biological consequences, and behaves as a proper guanine
both in terms of its ability to form base pairs and its accuracy in
being read as a ‘G’. In contrast, the second most frequently induced damage product, N3-alkyladenine, is highly toxic; because it
is incapable of being recognized at all by DNA polymerase it acts as
a block to DNA replication. This is in contrast to yet another alkylated base, O6-alkylguanine, which base pairs very efficiently during
replication, but with poor accuracy; DNA polymerase is as likely to
insert a T as a C opposite this lesion. The consequence of this tendency of O6-alkylguanine to mispair is readily visible in the dozen
or so EMS-induced alleles that have been sequenced from plants:
1360 - 1385/99/$ – see front matter © 1999 Elsevier Science. All rights reserved. PII: S1360-1385(98)01355-7
trends in plant science
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O
O
O
HN
CH3
CH3
HN
CH3
NH2
HN
O
O
N
O
N
N
H
H 3C
N
O
N
Fig. 1. Two common UV-induced photoproducts: a cyclobutane
pyrimidine (in this example T-T) dimer and a pyrimidine [6-4]
pyrimidinone (in this example T-C) dimer. The dimers are formed
between adjacent (59 and 39) bases on the same DNA strand.
all of these mutations are point mutations involving the transition of
G to A. Thus, a single DNA damaging agent can produce lesions
that are inconsequential, mutagenic (due to mispairing), or cytotoxic (due to the blockage of transcription or DNA replication).
Of course, the toxicity or mutagenicity of any particular lesion also
depends on the efficiency with which it is eliminated by the cell.
All organisms employ a wide variety of strategies to either reverse,
excise, or tolerate the persistence of DNA damage products. Even the
extremely compact genomes of bacteriophage, where space is at a
premium, often encode a repair enzyme. Some of these damage
resistance mechanisms are ancient and almost ubiquitous, whereas
others seem to be of more recent origin. Although repair and damage
tolerance mechanisms have been thoroughly described in E. coli,
Saccharomyces cerevisiae, humans and rodents, remarkably little is
known about these processes in plants. However, in recent years, increasing interest in the effects of enhanced UV-B radiation has provided a lot of information on the repair of UV-induced pyrimidine
dimers. Similarly, interest in the development of genetic engineering
techniques with which to manipulate the plant genome, has resulted
in promising advances in research into the repair of double strand
breaks and homologous recombination. The potential ‘applications’
of these DNA repair processes have resulted in additional investigations into interesting and more basic areas including the mechanisms of mutagenesis, the control of the cell cycle by DNA damage,
and the developmental and environmental regulation of repair.
Recent developments in the field of DNA repair are described below, with particular emphasis on the molecular genetics of repair. In
order to be concise, some aspects of repair, and the entire field of
damage tolerance (the surface of which has hardly been scratched in
plants) have been omitted and the reader is referred to comprehensive reviews of this field2–4, and reviews of the physiological effects
of UV radiation on plants5,6.
Repair of UV-induced DNA damage
UV radiation induces a degree of oxidative damage (pyrimidine
hydrates) and crosslinks (both DNA–protein and DNA–DNA). However, the predominant, and probably most significant, lesions are
various types of pyrimidine dimers. Cyclobutane pyrimidine dimers
(CPDs) make up the bulk of the damage (perhaps 75%, depending
on the sequence context), and pyrimidine [6-4]pyrimidinone dimers
(known as 6-4 products) make up the rest (Fig. 1). Both classes of
dimers act as blocks to transcription in mammalian cells, and inhibit DNA replication in bacteria and eukaryotes. Pyrimidine dimers
are also premutagenic lesions: C-containing dimers are subject to
conversion to TT mutations via a process termed ‘dimer bypass’.
The existence of this process has yet to be established in plants,
and little is known about the spectrum of mutations induced by
UV radiation in plants.
Because of their effect on transcription, the persistence of pyrimidine dimers is highly toxic, and a mechanism for their efficient removal, even from nonreplicating, terminally differentiated somatic
cells, might be regarded as an essential function for any living organism that is exposed to sunlight. This is especially true for plants,
because as obligate phototrophs there is no natural environment in
which visible light is not accompanied by UV radiation, and no
UV-absorbing pigment can absorb 100% of the incident radiation.
There are two major categories of mechanisms for the repair of
DNA damage: the damage can be directly reversed, or the damage
can be excised from the genome, and the resulting gap repaired
using the undamaged strand as a template. In the case of pyrimidine dimers, both classes of mechanisms exist in most organisms,
and plants, not unexpectedly, are able to repair dimers via both
mechanisms.
Photolyases and photoreactivation (live by the sword, die by
the sword)
Just as there is no natural environment in which plants can enjoy
visible radiation without having to endure UV radiation, so UV radiation never naturally occurs without accompanying visible light.
It has long been observed (in microbial systems) that the toxic and
mutagenic effects of UV radiation can be reversed by subsequent
exposure to radiation in the 360–420 nm range (UV-A to blue). This
phenomenon is termed ‘photoreactivation’ and is due to the actions of one or more proteins termed ‘photolyases’. These enzymes
specifically recognize and bind to pyrimidine dimers. The enzymes
carry two chromophores. The first is a flavin cofactor (FADH2),
which acts as a transient electron donor to reverse the crosslink between the bases. The second chromophore, which acts as an antenna pigment to excite the electron donor, is of varying chemical
composition in various species, and largely determines the action
spectrum of the enzyme7. Through the action of photolyases, pyrimidine dimers can be directly reversed, in an error-free fashion, to
pyrimidine monomers without excision of the damaged bases.
Photolyases are one of the handful of examples of repair enzymes
that are at once very simple, efficient and error free, because of
their ability to reverse rather than excise damage. In addition,
photolyases (as with all damage-reversal enzymes) are extremely
specialized in terms of their substrate-specificity.
Until the early 1990s only a handful of microbial CPD-specific
photolyases had been identified and characterized, either genetically or biochemically. The last decade has seen the discovery of
several new classes of photolyases and related enzymes as a consequence of studies of this protein in a wider range of species. The
first higher eukaryotic photolyase, a CPD-specific enzyme, was
cloned from goldfish (Carassius auratus) and found to have a
sequence that is related to, but highly diverged from, the microbial
genes (Fig. 2). The two classes of CPD photolyases share only
10–15% sequence identity8. Homologs of this so-called ‘metazoan’,
or ‘class II’ CPD photolyase were subsequently identified in insects,
several vertebrates (including marsupials, although not placental
mammals), some archaebacteria, some eubacteria and Arabidopsis.
The Arabidopsis homolog of this class II photolyase sequence
(PHR1) corresponds to a gene (UVR2) that was identified via
classical genetic analysis as being required for photoreactivation
of CPDs in vivo9,10.
At the same time, there was the unexpected discovery of a class
of photolyases specific for 6-4 photoproducts. First observed biochemically in extracts from Drosophila11, the repair activity was
also detected in Arabidopsis in vivo12. Arabidopsis mutants defective in this activity (uvr3 mutants) were isolated, and the UVR3
gene was shown to correspond to the Arabidopsis homolog of
sequences encoding a 6-4 photolyase in other species, including
January 1999, Vol. 4, No. 1
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‘Dark’ repair of pyrimidine dimers
Most organisms possess both substratespecific repair mechanisms and a more
Class I CPD
general pathway, termed nucleotide excision
Ec CPD
photolyase
repair (NER). This mechanism involves the
Hh CPD
recognition, with varying efficiencies, of a
very broad range of DNA damage prodCr CRY
ucts, including pyrimidine dimers (Fig. 3).
At CRY2/PHH1
The damaged DNA strand is then nicked 59
At CRY1/HY4
and 39 of the damage product, the oligonuAt 6-4/UVR3
cleotide removed, and the undamaged strand
6-4 photolyase
used as a template to restore the original
Dm 6-4
sequence. This pathway is fairly error-free,
Xl 6-4
although it is of course subject to the same
Hs CRY2
sort of errors inherent in any DNA repliHs CRY1
cation process. In human cells, NER is an
essential repair process (the existence of a
Mx CPD
CPD photolyase in human cells is still a
Mt CPD
subject of debate), as shown by the exAt CPD/UVR2
Class II CPD
treme UV-sensitivity of individuals afflicted
Dm CPD
photolyase
with Xeroderma pigmentosum (XP), a heritable disease affecting nucleotide excision
Ca CPD
repair. In contrast, NER probably only acts
Pt CPD
as a minor pathway for dimer repair in organisms that possess functional photolyases
Fig. 2. Phylogenetic analysis of photolyase homologs. The data presented is from Ref. 13:
(although it may be essential for the repair
their tree was generated by the ODEN software package (National Institute of Genetics,
of rare DNA damage products that lack a
Japan). Labeling of genes as ‘photolyases’ is based either on their ability to encode a funcdedicated repair pathway). Most of the genes
tional photolyase on expression in E. coli, or genetic analysis of their role in vivo. Genes
(approximately a dozen) required for NER
labeled ‘CRY’ (crytochrome) are known (via genetic analysis) or suspected to encode
in mammals and yeast have been identiphotoreceptors. Abbreviations: Sc, Saccharomyces cerevisiae; Ec, Escherichia coli; Hh,
Halobacterium halobium; Cr, Chlamydomonas reinhartii; At, Arabidopsis thaliana; Dm,
fied via classical genetics, cloned and seDrosophila melanogaster; Xl, Xenopus laevis; Hs, Homo sapiens; Mx, Myxococcus
quenced18. There is clear homology between
xanthus; Mt, Methanobacterium thermoautotrophicum; Ca, Carassius auratus; Pt, Potorous
the fungal and animal genes. Although bactridactylus.
teria possess a functionally similar, but less
complex, repair pathway, the homology
does not extend to the DNA sequence level.
Drosophila and Xenopus13. The 6-4 photolyase is clearly related
Plants are capable of repairing UV-induced dimers in the dark,
in sequence to the CPD photolyases, and in fact is more closely indicating that some sort of repair pathway exists in addition to
related to the class I ‘microbial’ CPD photolyase sequence than photolyases3,19,20. Classical genetic analysis has resulted in the
the class II CPD photolyase (Fig. 2).
identification of at least four complementation groups required for
The genomes of Chlamydomonas and Arabidopsis encode yet this repair in Arabidopsis (UVR1, UVR5, UVR7, and UVH1)21,22, and
another type of class I photolyase homolog. These homologs many more UV-sensitive mutants await further genetic and pheno(CRY1/HY4 and CRY2/PHH1) are not involved in DNA repair but typic characterization2. The classical genetics of the dark repair of
instead play a role in blue-light perception14. Interestingly, the hu- dimers remains unsaturated (i.e. few representatives of each compleman and mouse genomes also encode two 6-4 photolyase homo- mentation group have been identified), suggesting that additional
logs15. However, these proteins lack any repair activity in vitro, complementation groups exist. Some of these mutations result in a
and expression studies suggest that the genes, hCRY1 and hCRY2, gamma-radiation sensitive phenotype, as well as UV-sensitivity,
are involved in circadian rhythm entrainment16.
indicating that this repair pathway recognizes a variety of substrates.
In summary, the family of photolyase-related proteins is a very All of this is consistent with the existence of some sort of NER pathancient one. The duplication of the CPD photolyase to produce way in plants. A search of GenBank, using the human XP genes as
both class I and class II CPD photolyases may have occurred be- probes, reveals several very convincing homologs of these excision
fore the archaebacterial/eubacterial split (examples of both classes repair genes (Table 1). Furthermore, a homolog of the NER endoof photolyases can be observed in both kingdoms of bacteria, nuclease, ERCC1, was recently cloned from Lilium longiflorum.
although this might also be an example of horizontal gene trans- When expressed in ERCC1 deficient CHO cells, this gene was able
fer). Similarly, the 6-4 photolyase is not a recent development, but to enhance the cells’ resistance to the crosslinking agent mitois observed in both the plant and animal kingdoms. The early evo- mycin C (Ref. 23). Thus the gene almost certainly plays a similar
lution of repair activity is not particularly surprising, if one bears role in DNA repair in plants.
in mind that the Earth’s early atmosphere had a very low oxygen
Homologs of a second gene, RAD23, which influences the rate
content17, resulting in a much higher level of UV radiation at the of NER in yeast but is not absolutely required for repair24, have
Earth’s surface. In this remarkably harsh environment, nucleic- been identified in several plant species, and in one case shown to
acid based life was probably only possible in well-protected en- partially complement the UV-sensitive phenotype of yeast rad23
vironments (i.e. under a murky water column). The evolution mutants25. It remains to be seen whether any of these excision reof photolyases may have been an important step on the path to pair gene homologs are able to complement the repair deficiency of
phototrophy.
the uvr/uvh mutants isolated in Arabidopsis, but it appears likely that
Sc CPD
22
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the ‘dark’ repair of UV-induced dimers observed in plants is driven by a mechanism
directly related to NER in other eukaryotes.
Mismatch repair
Mismatched bases, regularly produced
through errors of replication or through
homologous recombination, pose a unique
problem to the repair systems of the cell4.
Both bases are legitimate and neither is a recognizable DNA damage product. Although
it is easy to conceive of enzymes that can
recognize mispaired bases (and the enzymes
involved in NER can do this to some extent)
the ‘trick’ to mismatch repair is the discrimination between the correct and incorrect base. In E. coli this problem is solved by
methylating specific sequences (G5mATC)
in its genome at some time after DNA
replication. The original template strands
(presumably correct in sequence) can then,
for a brief interval, be distinguished from
newly synthesized (error-prone) unmethylated strands. Once a mismatch is recognized
by the MutS protein, the MutL protein binds
to the complex and helps to promote an
interaction between the complex and the
MutH protein, an endonuclease which locates the nearest unmethylated GATC and
guides the degradation of the unmethylated
strand from the GATC to the mismatched
base. The original template strand can then
be replicated to fill the gap.
Eukaryotes possess multiple copies of
obvious mutS and mutL homologs. No mutH
homologs have been identified, and the
mechanism by which strand-specificity is
generated is unclear. The budding yeast S.
cerevisiae possesses at least six different
mutS homolog (MSH) genes, each with a
specialized function extending well beyond
the original function of MutS as a mismatch
repair protein. Several mammalian versions of MSH also exist. Similarities between the human and yeast MSH2 homologs
are greater than the similarities between
the various yeast MSH homologs, suggesting that the duplication of this locus occurred before the evolution of multicellular
organisms26.
Both mutS homologs and mutL homologs
have been found to play an important role in
the prevention of somatic mutagenesis; humans defective in some of these genes have
a heritable predisposition to certain types
of cancers. Other MSH and MLH genes are
required for meiosis. In addition, mutS,
mutL and some of their eukaryotic homologs have been found to have a powerful
anti-recombinogenic effect on homeologous
(similar, but not identical) sequences in bacteria, yeast, and mammals, without affecting
recombination between identical stretches of
DNA27. It is possible that mismatch repair
Recognition and incision enzymes
Helicases
DNA polymerase, ligase
Fig. 3. Nucleotide excision repair. DNA damage is excised as an oligonucleotide. In yeast
and mammals damage recognition and incision requires the coordinate actions of approximately 11 gene products and the excision tracts range in size from 23 to 32 nucleotides. In
E. coli, damage recognition and incision is catalyzed by the uvrA, uvrB, and uvrC gene
products, and the excised oligonucleotide is 12–13 bp in length.
Table 1. Human and yeast genes required for the excision repair of
pyrimidine dimers; identification of plant homologs
Human
S. cerevisiae
complementation homologa
group
XP-A
XP-B
RAD14
RAD25/SSL2
XP-C
RAD4
XP-D
XP-F
RAD3
RAD1
XP-G
CS-A
RAD2
RAD28
CS-B
RAD26
ERCC-1
RAD10
Arabidopsis
homolog
Two44, ~56% identity
over 260 amino acids.
21% identity over 290
amino acids.
39% identity over 1100
amino acids.
Lily homolog23,
53% identity over 216
amino acids.
Remarks/function
Damage recognition.
TFIIH subunit.
Binds ssDNA, damage
recognition, required for
global repair only.
TFIIH subunit.
5′ endonuclease.
3′ endonuclease.
Required for transcriptioncoupled repair only.
Required for transcriptioncoupled repair only.
5′ endonuclease.
a
Based on both sequence and functional analysis. Unless otherwise cited, sequence identity values were
calculated using the Gap Alignment program of the Genetics Computer Group (GCG) sequence
analysis package.
January 1999, Vol. 4, No. 1
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breaks via homologous recombination, provided sufficient homology is available. Organisms with larger genomes will virtually
A
B
C
D
E
F
always repair breaks via NHEJ, regardless
Degradation
of how much homology is provided. There
5′
are notable exceptions to this rule. For ex3′
A
B
C
D
E
F
ample, mouse embryonic stem cells will
integrate homologous transgenes at their
+ nonhomologous DNA
+ homologous DNA
homologous chromosomal locus with good
5′
5′
efficiency; this capacity for gene replace3′
3′
ment has spawned a minor industry for the
L
M
N
O
P
Q
A′
B′
C′
D′
E′
F′
generation of ‘knockout mice’.
Homologous
End-to-end joining
Analysis of the products of double strand
recombination
break repair can provide clues as to the
5′
5′
mechanism of repair. In plants, these ‘prod3′
3′
A
B
C:N O
P
Q
A′
B′
C′ D′/D E
F
ucts’ have largely been characterized as exIllegitimate recombinant product
Gene replacement event
cision products of transposable elements,
or insertion products of T-DNAs29–31. The
Fig. 4. Double strand break repair. Repair can occur via two independent pathways.
classification of these molecules as the
Homologous recombination restores the original sequence, whereas nonhomologous end
products of DSB repair involves certain asjoining (NHEJ) produces chromosomal deletions, translocations, and inversions. Evidence
sumptions and inherent logical circularities
from transformed plants suggests that the majority of transformed DNAs are integrated via
NHEJ. However, plants are capable of repairing double strand breaks via either pathway.
(i.e. transposable element excision products
look like mammalian DSB repair products,
therefore they are the products of DSB rehomologs might determine the degree of sequence identity required pair, therefore plant DSB repair is similar to that in mammals).
for meiotic recombination: there is evidence to this effect in MSH2 More recently, experiments involving the creation and healing, via
and PMS1 (a mutL homolog) mutants of yeast28. If mismatch repair NHEJ, of true DSBs at predetermined chromosomal loci (using
proteins play a similar role in inhibiting recombination between unique restriction sites, with transient introduction of the restricdiverged sequences in plants and animals, the study of mismatch tion enzyme32,33, or by sequencing of repaired lineared plasmids34)
repair proteins in plants has obvious applications to plant breed- have confirmed that the kinds of products observed as a result of
ing, as well as research into speciation.
T-DNA integration or TE excision do indeed resemble genuine
Although several research groups have informally reported the plant DSB repair products in many ways:
cloning of plant mismatch repair gene homologs, to date only one • Some degradation of the broken ends usually occurs before
sequence has been published, an MSH homolog from Arabidopsis26.
ligation, resulting in a loss of information.
Sequence analysis indicates that this is an MSH2 homolog. In both • Microhomologies are generally utilized during NHEJ.
humans and yeast, the MSH2 gene product acts as a heterodimer • ‘Filler’ DNAs are often integrated into the break. These may be
with MSH3 or MSH6 gene products to recognize and correct misrecognizable sequences from elsewhere in the genome, duplimatched bases. It also serves to inhibit both meiotic and mitotic
cations or inversions of local sequences, or simple sequences
recombination between diverged sequences in yeast. The function
that seem to be generated at random.
of this gene in plants has yet to be determined.
An interesting and important question is whether the majority of
genuine chromosomal DSBs are repaired via NHEJ, rather than
Repair of double strand breaks
through homologous recombination. There is some difficulty in
Double strand breaks (DBSs) can occur spontaneously in the cell comparing the actual rate of homologous versus nonhomologous reas a result of a nick in a single stranded region, due to mechanical pair of DSBs, because, unfortunately, experiments are set up to resstress of a chromosome, or as part of the initiation of crossing over cue only those events that regenerate a selectable marker (through
during meiosis4. Artificial sources of DSBs include ionizing radi- homologous recombination) or only those events that destroy a
ation, certain radiomimetic chemicals, and transgenic DNAs. There counterselectable marker (through NHEJ). Experiments have not
are two basic classes of pathways for the repair of double strand been performed that directly compare the relative rates of the two
breaks (DSBs; Fig. 4). The first, repair via homologous recombi- kinds of events. Surveys of T-DNA integration events and TE excination, uses a gene conversion mechanism to replace the break with sion products certainly suggest that in higher plants, as in mammals,
intact sequence from a sister chromatid, homolog or other identical breaks are repaired by nonhomologous recombination far more
(or nearly identical) sequence. The second mechanism, nonhomolo- frequently than via homologous recombination. In Arabidopsis,
gous end-joining (NHEJ), involves the resealing of two broken ends. T-DNAs integrate at random positions: the ratio of gene replaceAlthough there are often ‘microhomologies’ of two to five bases at ment events to random insertion events is approximately 1:1000
the resealed joint, these sequences probably only act to stabilize the (Ref. 35); in tobacco this ratio is closer to 1:10 000 (Ref. 36). Surligation reaction, and do not reflect the kind of true homology prisingly, even in experimental designs in which the repair of one
search involved in homologous recombination. Because there is no end of a linear transgene is ‘forced’ to occur via homologous recommechanism to ensure the pairing of the two original chromosome bination (through selection for the reconstitution of a fragmented
ends, NHEJ can produce chromosomal inversions, deletions, trans- drug resistance marker), the remaining end is often (in about one out
locations, and partial duplications. Although all organisms in which of four cases) repaired nonhomologously, in spite of the proximDSB repair has been studied have been found to possess both repair ity of homologous sequences32. This suggests that even in a case
mechanisms, the rate at which one or the other mechanism is actually where the search for homology is greatly simplified, the enzymes
employed varies widely. As a general rule, organisms with relatively involved in NHEJ often ‘out compete’ those required for homolocompact genomes (bacteria and yeast) will virtually always repair gous recombination in the race to process the double strand break.
5′
3′
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Very few of the plant genes involved in DSB repair have been
identified. Homologs of the recA genes (DMC1/LIM15, RAD51)37–39
have been cloned from plants, and the L. longiflorum homologs localized to meiotic prophase chromosomes40, but their activity has
not been determined through biochemical or mutational analysis.
Mutants sensitive to ionizing radiation41,42 have been isolated, but
the genes involved have not yet been cloned. Some of these mutants exhibit altered meiotic and extrachromosomal recombination
rates, suggesting that the genes are involved in either homologous
recombination or NHEJ (Ref. 43).
The rate of meiotic recombination, and the relative rates of gene
replacement versus gene addition events, are governed by the
enzymology of the pathways that are involved in these processes.
An understanding of these mechanisms, and the identification of
the genes involved, would enable us to truly ‘engineer’ the plant
genome, rather than mutating or transforming plants in the essentially random manner employed today.
Acknowledgements
Work cited from the author’s lab was supported by USDA NRICGP
grant 94-37301-0564 and NSF grant 90-19159.
References
1 Lawley, P. (1974) Some chemical aspects of dose-response relationships in
alkylation mutagenesis, Mutat. Res. 23, 283–295
2 Vonarx, E.J. et al. (1998) DNA repair in higher plants, Mutat. Res. 400, 187–200
3 Britt, A. (1998) DNA repair in higher plants, in DNA Damage and Repair
(Vol. 1) (Nickoloff, J. and Hoekstra, M., eds), pp. 577–595, Humana Press
4 Friedberg, E.C., Walker, G.C. and Siede, W. (1995) DNA Repair and
Mutagenesis, ASM Press
5 Rozema, J. et al. (1997) UV-B as an environmental factor in plant life: stress
and regulation, Trends Ecol. Evol. 12, 22–28
6 Jansen, M., Gaba, V. and Greenberg, B. (1998) Higher plants and UV-B
radiation: balancing damage, repair and acclimation, Trends Plant Sci. 3,
131–135
7 Sancar, A. (1994) Structure and function of DNA photolyase,Biochemistry 33, 2–9
8 Yasui, A. et al. (1994) A new class of DNA photolyases present in various
organisms including aplacental mammals, EMBO J. 13, 6143–6151
9 Ahmad, M. et al. (1997) An enzyme similar to animal type II photolyases
mediates photoreactivation in Arabidopsis, Plant Cell 9, 199–207
10 Jiang, C-Z. et al. (1997) Photorepair mutants of Arabidopsis, Proc. Natl. Acad.
Sci. U. S. A. 94, 7441–7445
11 Todo, T. et al. (1993) A new photoreactivating enzyme that specifically
repairs ultraviolet light-induced (6-4) photoproducts, Nature 361, 371–374
12 Chen, J-J., Mitchell, D. and Britt, A.B. (1994) A light-dependent pathway for
the elimination of UV-induced pyrimidine (6-4) pyrimidinone photoproducts
in Arabidopsis thaliana, Plant Cell 6, 1311–1317
13 Nakajima, S. et al. (1998) Cloning and characterization of a gene (UVR3)
required for photorepair of 6-4 products in Arabidopsis thaliana, Nucleic
Acids Res. 26, 638–644
14 Cashmore, A. (1997) The cryptochrome family of photoreceptors, Plant Cell
Environ. 20, 764–767
15 Hsu, D.S. et al. (1996) Putative human blue-light photoreceptors hCRY1 and
hCRY2 are flavoproteins, Biochemistry 35, 13871–13877
16 Miyamoto, Y. and Sancar, A. (1998) Vitamin B-2-based blue-light photoreceptors in the retinohypothalamic tract as the photoactive pigments for setting
the circadian clock in mammals, Proc. Natl. Acad. Sci. U. S. A. 95, 6097–6102
17 Caldwell, M. (1979) Plant life and ultraviolet radiation: some perspective in
the history of the earth’s UV climate, BioScience 29, 520–525
18 Sancar, A. (1996) DNA excision repair, Annu. Rev. Biochem. 65, 43–81
19 Britt, A.B. et al. (1993) A UV-sensitive mutant of Arabidopsis defective in the
repair of pyrimidine-pyrimidinone (6-4) dimers, Science 261, 1571–1574
20 Quaite, F.E. et al. (1994) DNA damage levels determine cyclobutyl pyrimidine
dimer repair mechanisms in alfalfa seedlings, Plant Cell 6, 1635–1641
21 Jenkins, M.E. et al. (1995) Radiation-sensitive mutants of Arabidopsis
thaliana, Genetics 140, 725–732
22 Jiang, C-Z. et al. (1997) UV- and gamma-radiation sensitive mutants of
Arabidopsis, Genetics 147, 1401–1409
23 Xu, H. et al. (1998) Plant homologue of human excision repair gene ERCC1
points to conservation of DNA repair mechanisms, Plant J. 13, 823–829
24 Mueller, J.P. and Smerdon, M.J. (1996) Rad23 is required for transcriptioncoupled repair and efficient overall repair in Saccharomyces cerevisiae, Mol.
Cell. Biol. 16, 2361–2368
25 Sturm, A. and Lienhard, S. (1998) Two isoforms of plant RAD23 complement
a UV-sensitive rad23 mutant in yeast, Plant J. 13, 815–821
26 Culligan, K.M. and Hays, J.B. (1997) DNA mismatch repair in plants. An
Arabidopsis thaliana gene that predicts a protein belonging to the MSH2
subfamily of eukaryotic mutS homologs, Plant Physiol. 115, 833–839
27 Modrich, P. and Lahue, R. (1996) Mismatch repair in replication fidelity,
genetic recombination, and cancer biology, Annu. Rev. Biochem. 65, 101–133
28 Hunter, N. et al. (1996) The mismatch repair system contributes to meiotic
sterility in an interspecific yeast hybrid, EMBO J. 15, 1726–1733
29 Scott, L., LaFoe, D. and Weil, C.F. (1996) Adjacent sequences influence DNA
repair accompanying transposon excision in maize, Genetics 142, 237–246
30 Ohba, T. et al. (1995) DNA rearrangement associated with the integration of
T-DNA in tobacco: an example for multiple duplications of DNA around the
integration target, Plant J. 7, 157–164
31 Takano, M. et al. (1997) The structures of integration sites in transgenic rice,
Plant J. 11, 353–361
32 Puchta, H., Dujon, B. and Hohn, B. (1996) Two different but related
mechanisms are used in plants for the repair of genomic double-strand breaks
by homologous recombination, Proc. Natl. Acad. Sci. U. S. A. 93, 5055–5060
33 Salomon, S. and Puchta, H. (1998) Capture of genomic and T-DNA sequences
during double-strand break repair in somatic plant cells, EMBO J. 17,
6086–6095
34 Gorbunova, V. and Levy, A.A. (1997) Non-homologous DNA end joining in
plant cells is associated with deletions and filler DNA insertions, Nucleic
Acids Res. 25, 4650–4657
35 Kempin, S. et al. (1997) Targeted disruption in Arabidopsis, Nature 389, 802–803
36 Paszkowski, J. et al. (1988) Gene targeting in plants, EMBO J. 7, 4021–4026
37 Kobayashi, T., Hotta, Y. and Tabata, S. (1993) Isolation and characterization
of a yeast gene that is homologous with a meiosis-specific cDNA from a plant,
Mol. Gen. Genet. 237, 225–232
38 Klimyuk, V. and Jones, J. (1996) AtDMC1, the Arabidopsis homologue of the
yeast DMC1 gene: characterisation, transposon-induced allelic variation and
meiosis-specific expression of a pAtDMC1:GUS fusion, Plant J. 11, 1–14
39 Doutriaux, M-P. et al. (1998) Isolation and characterisation of the RAD51 and
DMC1 homologs from Arabidopsis thaliana, Mol. Gen. Genet. 257, 283–291
40 Terasawa, M. et al. (1995) Localization of RecA-like recombination proteins
on chromosomes of the lily at various meiotic stages, Genes Dev. 9, 925–934
41 Davies, C. et al. (1994) Isolation of Arabidopsis thaliana mutants
hypersensitive to gamma radiation, Mol. Gen. Genet. 243, 660–665
42 Masson, J., King, P. and Paszkowski, J. (1997) Mutants of Arabidopsis
thaliana hypersensitive to DNA damaging treatments, Genetics 146, 401–407
43 Masson, J. and Paszkowski, J. (1997) Arabidopsis thaliana mutants altered in
homologous recombination, Proc. Natl. Acad. Sci. U. S. A. 94, 11731–11735
44 Ribeiro, D.T. et al. (1998) Cloning of a cDNA from Arabidopsis thaliana
homologous to the human XPB gene, Gene 208, 207–213
Anne B. Britt is at the Section of Plant Biology, University of
California, Davis, CA 95616, USA (tel 11 530 752 0699;
fax 11 530 752 5410; e-mail abbritt@ucdavis.edu).
Congratulations!
Our warmest congratulations to Anne Britt, who in the closing
stages of submitting her article also found time to give birth to
twin girls.
January 1999, Vol. 4, No. 1
25
reviews
19 Kempton, J.H. (1920) Heritable characters of maize V. Adherence, J. Hered.
11, 317–322
20 Becraft, P.W., Stinard, P.S. and McCarty, D.R. (1996) CRINKLY4: a TNFRlike receptor kinase involved in maize epidermal differentiation, Science 273,
1406–1409
21 Jenks, M.A. et al. (1996) Mutants in Arabidopsis thaliana altered in
epicuticular wax and leaf morphology, Plant Physiol. 110, 377–385
22 Lolle, S.J., Hsu, W. and Pruitt, R.E. (1998) Genetic analysis of organ fusion in
Arabidopsis thaliana, Genetics 149, 607–619
23 Sinha, N. (1998) Organ and cell fusions in the adherent1 mutant in maize, Int.
J. Plant Sci. 159, 702–715
24 Sinha, N. and Lynch, M. (1998) Fused organs in the adherent1 mutation in
maize show altered epidermal walls with no perturbations in tissue identities,
Planta 206, 184–195
25 Lolle, S.J., Cheung, A.Y. and Sussex, I.M. (1992) Fiddlehead: An Arabidopsis
mutant constitutively expressing an organ fusion program that involves
interactions between epidermal cells, Dev. Biol. 152, 383–392
26 Lolle, S.J. and Cheung, A.Y. (1993) Promiscuous germination and growth of
wild-type pollen from Arabidopsis and related species on the shoot of the
Arabidopsis mutant, fiddlehead, Dev. Biol. 155, 250–258
27 Lolle, S.J. et al. (1997) Developmental regulation of cell interactions in the
Arabidopsis fiddlehead-1 mutant: a role for the epidermal cell wall and cuticle,
Dev. Biol. 189, 311–321
28 Aida, M. et al. (1997) Genes involved in organ separation in Arabidopsis: an
analysis of the cup-shaped cotyledon mutant, Plant Cell 9, 841–857
29 Koornneef, M., Hanhart, C.J. and Thiel, F. (1989) A genetic and phenotypic
description of eceriferum (cer) mutants in Arabidopsis thaliana, J. Hered. 80,
118–122
30 Levin, J.Z. et al. (1998) A genetic screen for modifiers of UFO meristem
activity identifies three novel FUSED FLORAL ORGANS genes required for
early flower development in Arabidopsis, Genetics 149, 579–595
31 Neuffer, M.G., Coe, E.H. and Wessler, S.R. (1997) Mutants of Maize, Cold
Spring Harbor Laboratory Press
32 Becraft, P.W. (1998) Receptor kinases in plant development, Trends Plant Sci.
3, 384–388
33 Hülskamp, M. et al. (1995) Identification of genes required for pollen–stigma
recognition in Arabidopsis thaliana, Plant J. 8, 703–714
34 Heslop-Harrison, J., Knox, R.B. and Heslop-Harrison, Y. (1974) Pollen-wall
proteins: exine–held fractions associated with the incompatibility response in
Cruciferae, Theor. Appl. Genet. 44, 133–137
35 Verbeke, J.A. (1989) Stereological analysis of ultrastructural changes during
induced epidermal cell redifferentiation in developing flowers of
Catharanthus roseus (Apocynaceae), Am. J. Bot. 76, 952–957
36 Preuss, D. et al. (1993) A conditional sterile mutation eliminates surface
components from Arabidopsis pollen and disrupts cell signaling during
fertilization, Genes Dev. 7, 974–985
37 Taylor, L.P. and Hepler, P.K. (1997) Pollen germination and tube growth,
Annu. Rev. Plant Physiol. Plant Mol. Biol. 48, 461–491
38 Wolters-Arts, M., Lush, W.M. and Mariani, C. (1998) Lipids are required for
directional pollen-tube growth, Nature 392, 818–821
39 Bell, P.R. (1995) Incompatibility in flowering plants: adaptation of an ancient
response, Plant Cell 7, 5–6
40 Conner, J.A. et al. (1998) Comparative mapping of the Brassica S locus
region and its homeolog in Arabidopsis: implications for the evolution of
mating systems in the Brassicaceae, Plant Cell 10, 801–812
Susan J. Lolle* and Robert E. Pruitt are at the Dept of Molecular
and Cellular Biology, Harvard University, 16 Divinity Avenue,
Cambridge, MA 02138, USA.
*Author for correspondence (tel 11 617 495 0568;
fax 11 617 496 6702; e-mail lolle@billie.harvard.edu).
Molecular genetics of DNA repair in
higher plants Anne B. Britt
Damage to DNA occurs in all living things, and the toxicity and/or mutagenicity of the damage
products are reduced through the activities of one or more DNA repair pathways. The mechanisms of DNA repair are best understood in microorganisms and mammals, but the field has
recently expanded to include both plants and lower animals. These recent advances in our
understanding of the molecular and classical genetics of DNA repair in higher plants include
such aspects as the repair of UV-induced pyrimidine dimers, the correction of mismatched
bases, and the rejoining of double strand breaks.
T
he genomes of all living things are constantly subject to damage and decay. Some of the damage occurs as an inevitable
consequence of the chemical nature of DNA and its aqueous
environment, or as a result of errors of metabolism (i.e. the donation
of a methyl group to DNA rather than its intended target, or the
presence of stray radicals mistakenly formed during respiration or
photosynthesis). This endogenously generated damage is often
termed ‘spontaneous’ DNA damage to distinguish it from damage
due to exogenous sources, such as UV radiation and chemical DNA
damaging agents.
The wide variety of DNA damaging agents results in the formation
of an equally wide variety of damage products. Although DNA
damage is often associated with mutagenesis, the actual biological
consequences of these damaged products depends on the chemical
nature of the lesion. For example, the widely used artificial mutagen,
20
January 1999, Vol. 4, No. 1
ethyl methane sulfonate (EMS) can donate its alkyl group with varying efficiencies to almost all of the nitrogen and oxygen atoms
present on all of the bases1. Its most frequently induced product,
N7-alkylguanine, is also its most innocuous; this lesion apparently
has no biological consequences, and behaves as a proper guanine
both in terms of its ability to form base pairs and its accuracy in
being read as a ‘G’. In contrast, the second most frequently induced damage product, N3-alkyladenine, is highly toxic; because it
is incapable of being recognized at all by DNA polymerase it acts as
a block to DNA replication. This is in contrast to yet another alkylated base, O6-alkylguanine, which base pairs very efficiently during
replication, but with poor accuracy; DNA polymerase is as likely to
insert a T as a C opposite this lesion. The consequence of this tendency of O6-alkylguanine to mispair is readily visible in the dozen
or so EMS-induced alleles that have been sequenced from plants:
1360 - 1385/99/$ – see front matter © 1999 Elsevier Science. All rights reserved. PII: S1360-1385(98)01355-7
trends in plant science
reviews
O
O
O
HN
CH3
CH3
HN
CH3
NH2
HN
O
O
N
O
N
N
H
H 3C
N
O
N
Fig. 1. Two common UV-induced photoproducts: a cyclobutane
pyrimidine (in this example T-T) dimer and a pyrimidine [6-4]
pyrimidinone (in this example T-C) dimer. The dimers are formed
between adjacent (59 and 39) bases on the same DNA strand.
all of these mutations are point mutations involving the transition of
G to A. Thus, a single DNA damaging agent can produce lesions
that are inconsequential, mutagenic (due to mispairing), or cytotoxic (due to the blockage of transcription or DNA replication).
Of course, the toxicity or mutagenicity of any particular lesion also
depends on the efficiency with which it is eliminated by the cell.
All organisms employ a wide variety of strategies to either reverse,
excise, or tolerate the persistence of DNA damage products. Even the
extremely compact genomes of bacteriophage, where space is at a
premium, often encode a repair enzyme. Some of these damage
resistance mechanisms are ancient and almost ubiquitous, whereas
others seem to be of more recent origin. Although repair and damage
tolerance mechanisms have been thoroughly described in E. coli,
Saccharomyces cerevisiae, humans and rodents, remarkably little is
known about these processes in plants. However, in recent years, increasing interest in the effects of enhanced UV-B radiation has provided a lot of information on the repair of UV-induced pyrimidine
dimers. Similarly, interest in the development of genetic engineering
techniques with which to manipulate the plant genome, has resulted
in promising advances in research into the repair of double strand
breaks and homologous recombination. The potential ‘applications’
of these DNA repair processes have resulted in additional investigations into interesting and more basic areas including the mechanisms of mutagenesis, the control of the cell cycle by DNA damage,
and the developmental and environmental regulation of repair.
Recent developments in the field of DNA repair are described below, with particular emphasis on the molecular genetics of repair. In
order to be concise, some aspects of repair, and the entire field of
damage tolerance (the surface of which has hardly been scratched in
plants) have been omitted and the reader is referred to comprehensive reviews of this field2–4, and reviews of the physiological effects
of UV radiation on plants5,6.
Repair of UV-induced DNA damage
UV radiation induces a degree of oxidative damage (pyrimidine
hydrates) and crosslinks (both DNA–protein and DNA–DNA). However, the predominant, and probably most significant, lesions are
various types of pyrimidine dimers. Cyclobutane pyrimidine dimers
(CPDs) make up the bulk of the damage (perhaps 75%, depending
on the sequence context), and pyrimidine [6-4]pyrimidinone dimers
(known as 6-4 products) make up the rest (Fig. 1). Both classes of
dimers act as blocks to transcription in mammalian cells, and inhibit DNA replication in bacteria and eukaryotes. Pyrimidine dimers
are also premutagenic lesions: C-containing dimers are subject to
conversion to TT mutations via a process termed ‘dimer bypass’.
The existence of this process has yet to be established in plants,
and little is known about the spectrum of mutations induced by
UV radiation in plants.
Because of their effect on transcription, the persistence of pyrimidine dimers is highly toxic, and a mechanism for their efficient removal, even from nonreplicating, terminally differentiated somatic
cells, might be regarded as an essential function for any living organism that is exposed to sunlight. This is especially true for plants,
because as obligate phototrophs there is no natural environment in
which visible light is not accompanied by UV radiation, and no
UV-absorbing pigment can absorb 100% of the incident radiation.
There are two major categories of mechanisms for the repair of
DNA damage: the damage can be directly reversed, or the damage
can be excised from the genome, and the resulting gap repaired
using the undamaged strand as a template. In the case of pyrimidine dimers, both classes of mechanisms exist in most organisms,
and plants, not unexpectedly, are able to repair dimers via both
mechanisms.
Photolyases and photoreactivation (live by the sword, die by
the sword)
Just as there is no natural environment in which plants can enjoy
visible radiation without having to endure UV radiation, so UV radiation never naturally occurs without accompanying visible light.
It has long been observed (in microbial systems) that the toxic and
mutagenic effects of UV radiation can be reversed by subsequent
exposure to radiation in the 360–420 nm range (UV-A to blue). This
phenomenon is termed ‘photoreactivation’ and is due to the actions of one or more proteins termed ‘photolyases’. These enzymes
specifically recognize and bind to pyrimidine dimers. The enzymes
carry two chromophores. The first is a flavin cofactor (FADH2),
which acts as a transient electron donor to reverse the crosslink between the bases. The second chromophore, which acts as an antenna pigment to excite the electron donor, is of varying chemical
composition in various species, and largely determines the action
spectrum of the enzyme7. Through the action of photolyases, pyrimidine dimers can be directly reversed, in an error-free fashion, to
pyrimidine monomers without excision of the damaged bases.
Photolyases are one of the handful of examples of repair enzymes
that are at once very simple, efficient and error free, because of
their ability to reverse rather than excise damage. In addition,
photolyases (as with all damage-reversal enzymes) are extremely
specialized in terms of their substrate-specificity.
Until the early 1990s only a handful of microbial CPD-specific
photolyases had been identified and characterized, either genetically or biochemically. The last decade has seen the discovery of
several new classes of photolyases and related enzymes as a consequence of studies of this protein in a wider range of species. The
first higher eukaryotic photolyase, a CPD-specific enzyme, was
cloned from goldfish (Carassius auratus) and found to have a
sequence that is related to, but highly diverged from, the microbial
genes (Fig. 2). The two classes of CPD photolyases share only
10–15% sequence identity8. Homologs of this so-called ‘metazoan’,
or ‘class II’ CPD photolyase were subsequently identified in insects,
several vertebrates (including marsupials, although not placental
mammals), some archaebacteria, some eubacteria and Arabidopsis.
The Arabidopsis homolog of this class II photolyase sequence
(PHR1) corresponds to a gene (UVR2) that was identified via
classical genetic analysis as being required for photoreactivation
of CPDs in vivo9,10.
At the same time, there was the unexpected discovery of a class
of photolyases specific for 6-4 photoproducts. First observed biochemically in extracts from Drosophila11, the repair activity was
also detected in Arabidopsis in vivo12. Arabidopsis mutants defective in this activity (uvr3 mutants) were isolated, and the UVR3
gene was shown to correspond to the Arabidopsis homolog of
sequences encoding a 6-4 photolyase in other species, including
January 1999, Vol. 4, No. 1
21
trends in plant science
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‘Dark’ repair of pyrimidine dimers
Most organisms possess both substratespecific repair mechanisms and a more
Class I CPD
general pathway, termed nucleotide excision
Ec CPD
photolyase
repair (NER). This mechanism involves the
Hh CPD
recognition, with varying efficiencies, of a
very broad range of DNA damage prodCr CRY
ucts, including pyrimidine dimers (Fig. 3).
At CRY2/PHH1
The damaged DNA strand is then nicked 59
At CRY1/HY4
and 39 of the damage product, the oligonuAt 6-4/UVR3
cleotide removed, and the undamaged strand
6-4 photolyase
used as a template to restore the original
Dm 6-4
sequence. This pathway is fairly error-free,
Xl 6-4
although it is of course subject to the same
Hs CRY2
sort of errors inherent in any DNA repliHs CRY1
cation process. In human cells, NER is an
essential repair process (the existence of a
Mx CPD
CPD photolyase in human cells is still a
Mt CPD
subject of debate), as shown by the exAt CPD/UVR2
Class II CPD
treme UV-sensitivity of individuals afflicted
Dm CPD
photolyase
with Xeroderma pigmentosum (XP), a heritable disease affecting nucleotide excision
Ca CPD
repair. In contrast, NER probably only acts
Pt CPD
as a minor pathway for dimer repair in organisms that possess functional photolyases
Fig. 2. Phylogenetic analysis of photolyase homologs. The data presented is from Ref. 13:
(although it may be essential for the repair
their tree was generated by the ODEN software package (National Institute of Genetics,
of rare DNA damage products that lack a
Japan). Labeling of genes as ‘photolyases’ is based either on their ability to encode a funcdedicated repair pathway). Most of the genes
tional photolyase on expression in E. coli, or genetic analysis of their role in vivo. Genes
(approximately a dozen) required for NER
labeled ‘CRY’ (crytochrome) are known (via genetic analysis) or suspected to encode
in mammals and yeast have been identiphotoreceptors. Abbreviations: Sc, Saccharomyces cerevisiae; Ec, Escherichia coli; Hh,
Halobacterium halobium; Cr, Chlamydomonas reinhartii; At, Arabidopsis thaliana; Dm,
fied via classical genetics, cloned and seDrosophila melanogaster; Xl, Xenopus laevis; Hs, Homo sapiens; Mx, Myxococcus
quenced18. There is clear homology between
xanthus; Mt, Methanobacterium thermoautotrophicum; Ca, Carassius auratus; Pt, Potorous
the fungal and animal genes. Although bactridactylus.
teria possess a functionally similar, but less
complex, repair pathway, the homology
does not extend to the DNA sequence level.
Drosophila and Xenopus13. The 6-4 photolyase is clearly related
Plants are capable of repairing UV-induced dimers in the dark,
in sequence to the CPD photolyases, and in fact is more closely indicating that some sort of repair pathway exists in addition to
related to the class I ‘microbial’ CPD photolyase sequence than photolyases3,19,20. Classical genetic analysis has resulted in the
the class II CPD photolyase (Fig. 2).
identification of at least four complementation groups required for
The genomes of Chlamydomonas and Arabidopsis encode yet this repair in Arabidopsis (UVR1, UVR5, UVR7, and UVH1)21,22, and
another type of class I photolyase homolog. These homologs many more UV-sensitive mutants await further genetic and pheno(CRY1/HY4 and CRY2/PHH1) are not involved in DNA repair but typic characterization2. The classical genetics of the dark repair of
instead play a role in blue-light perception14. Interestingly, the hu- dimers remains unsaturated (i.e. few representatives of each compleman and mouse genomes also encode two 6-4 photolyase homo- mentation group have been identified), suggesting that additional
logs15. However, these proteins lack any repair activity in vitro, complementation groups exist. Some of these mutations result in a
and expression studies suggest that the genes, hCRY1 and hCRY2, gamma-radiation sensitive phenotype, as well as UV-sensitivity,
are involved in circadian rhythm entrainment16.
indicating that this repair pathway recognizes a variety of substrates.
In summary, the family of photolyase-related proteins is a very All of this is consistent with the existence of some sort of NER pathancient one. The duplication of the CPD photolyase to produce way in plants. A search of GenBank, using the human XP genes as
both class I and class II CPD photolyases may have occurred be- probes, reveals several very convincing homologs of these excision
fore the archaebacterial/eubacterial split (examples of both classes repair genes (Table 1). Furthermore, a homolog of the NER endoof photolyases can be observed in both kingdoms of bacteria, nuclease, ERCC1, was recently cloned from Lilium longiflorum.
although this might also be an example of horizontal gene trans- When expressed in ERCC1 deficient CHO cells, this gene was able
fer). Similarly, the 6-4 photolyase is not a recent development, but to enhance the cells’ resistance to the crosslinking agent mitois observed in both the plant and animal kingdoms. The early evo- mycin C (Ref. 23). Thus the gene almost certainly plays a similar
lution of repair activity is not particularly surprising, if one bears role in DNA repair in plants.
in mind that the Earth’s early atmosphere had a very low oxygen
Homologs of a second gene, RAD23, which influences the rate
content17, resulting in a much higher level of UV radiation at the of NER in yeast but is not absolutely required for repair24, have
Earth’s surface. In this remarkably harsh environment, nucleic- been identified in several plant species, and in one case shown to
acid based life was probably only possible in well-protected en- partially complement the UV-sensitive phenotype of yeast rad23
vironments (i.e. under a murky water column). The evolution mutants25. It remains to be seen whether any of these excision reof photolyases may have been an important step on the path to pair gene homologs are able to complement the repair deficiency of
phototrophy.
the uvr/uvh mutants isolated in Arabidopsis, but it appears likely that
Sc CPD
22
January 1999, Vol. 4, No. 1
trends in plant science
reviews
the ‘dark’ repair of UV-induced dimers observed in plants is driven by a mechanism
directly related to NER in other eukaryotes.
Mismatch repair
Mismatched bases, regularly produced
through errors of replication or through
homologous recombination, pose a unique
problem to the repair systems of the cell4.
Both bases are legitimate and neither is a recognizable DNA damage product. Although
it is easy to conceive of enzymes that can
recognize mispaired bases (and the enzymes
involved in NER can do this to some extent)
the ‘trick’ to mismatch repair is the discrimination between the correct and incorrect base. In E. coli this problem is solved by
methylating specific sequences (G5mATC)
in its genome at some time after DNA
replication. The original template strands
(presumably correct in sequence) can then,
for a brief interval, be distinguished from
newly synthesized (error-prone) unmethylated strands. Once a mismatch is recognized
by the MutS protein, the MutL protein binds
to the complex and helps to promote an
interaction between the complex and the
MutH protein, an endonuclease which locates the nearest unmethylated GATC and
guides the degradation of the unmethylated
strand from the GATC to the mismatched
base. The original template strand can then
be replicated to fill the gap.
Eukaryotes possess multiple copies of
obvious mutS and mutL homologs. No mutH
homologs have been identified, and the
mechanism by which strand-specificity is
generated is unclear. The budding yeast S.
cerevisiae possesses at least six different
mutS homolog (MSH) genes, each with a
specialized function extending well beyond
the original function of MutS as a mismatch
repair protein. Several mammalian versions of MSH also exist. Similarities between the human and yeast MSH2 homologs
are greater than the similarities between
the various yeast MSH homologs, suggesting that the duplication of this locus occurred before the evolution of multicellular
organisms26.
Both mutS homologs and mutL homologs
have been found to play an important role in
the prevention of somatic mutagenesis; humans defective in some of these genes have
a heritable predisposition to certain types
of cancers. Other MSH and MLH genes are
required for meiosis. In addition, mutS,
mutL and some of their eukaryotic homologs have been found to have a powerful
anti-recombinogenic effect on homeologous
(similar, but not identical) sequences in bacteria, yeast, and mammals, without affecting
recombination between identical stretches of
DNA27. It is possible that mismatch repair
Recognition and incision enzymes
Helicases
DNA polymerase, ligase
Fig. 3. Nucleotide excision repair. DNA damage is excised as an oligonucleotide. In yeast
and mammals damage recognition and incision requires the coordinate actions of approximately 11 gene products and the excision tracts range in size from 23 to 32 nucleotides. In
E. coli, damage recognition and incision is catalyzed by the uvrA, uvrB, and uvrC gene
products, and the excised oligonucleotide is 12–13 bp in length.
Table 1. Human and yeast genes required for the excision repair of
pyrimidine dimers; identification of plant homologs
Human
S. cerevisiae
complementation homologa
group
XP-A
XP-B
RAD14
RAD25/SSL2
XP-C
RAD4
XP-D
XP-F
RAD3
RAD1
XP-G
CS-A
RAD2
RAD28
CS-B
RAD26
ERCC-1
RAD10
Arabidopsis
homolog
Two44, ~56% identity
over 260 amino acids.
21% identity over 290
amino acids.
39% identity over 1100
amino acids.
Lily homolog23,
53% identity over 216
amino acids.
Remarks/function
Damage recognition.
TFIIH subunit.
Binds ssDNA, damage
recognition, required for
global repair only.
TFIIH subunit.
5′ endonuclease.
3′ endonuclease.
Required for transcriptioncoupled repair only.
Required for transcriptioncoupled repair only.
5′ endonuclease.
a
Based on both sequence and functional analysis. Unless otherwise cited, sequence identity values were
calculated using the Gap Alignment program of the Genetics Computer Group (GCG) sequence
analysis package.
January 1999, Vol. 4, No. 1
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trends in plant science
reviews
breaks via homologous recombination, provided sufficient homology is available. Organisms with larger genomes will virtually
A
B
C
D
E
F
always repair breaks via NHEJ, regardless
Degradation
of how much homology is provided. There
5′
are notable exceptions to this rule. For ex3′
A
B
C
D
E
F
ample, mouse embryonic stem cells will
integrate homologous transgenes at their
+ nonhomologous DNA
+ homologous DNA
homologous chromosomal locus with good
5′
5′
efficiency; this capacity for gene replace3′
3′
ment has spawned a minor industry for the
L
M
N
O
P
Q
A′
B′
C′
D′
E′
F′
generation of ‘knockout mice’.
Homologous
End-to-end joining
Analysis of the products of double strand
recombination
break repair can provide clues as to the
5′
5′
mechanism of repair. In plants, these ‘prod3′
3′
A
B
C:N O
P
Q
A′
B′
C′ D′/D E
F
ucts’ have largely been characterized as exIllegitimate recombinant product
Gene replacement event
cision products of transposable elements,
or insertion products of T-DNAs29–31. The
Fig. 4. Double strand break repair. Repair can occur via two independent pathways.
classification of these molecules as the
Homologous recombination restores the original sequence, whereas nonhomologous end
products of DSB repair involves certain asjoining (NHEJ) produces chromosomal deletions, translocations, and inversions. Evidence
sumptions and inherent logical circularities
from transformed plants suggests that the majority of transformed DNAs are integrated via
NHEJ. However, plants are capable of repairing double strand breaks via either pathway.
(i.e. transposable element excision products
look like mammalian DSB repair products,
therefore they are the products of DSB rehomologs might determine the degree of sequence identity required pair, therefore plant DSB repair is similar to that in mammals).
for meiotic recombination: there is evidence to this effect in MSH2 More recently, experiments involving the creation and healing, via
and PMS1 (a mutL homolog) mutants of yeast28. If mismatch repair NHEJ, of true DSBs at predetermined chromosomal loci (using
proteins play a similar role in inhibiting recombination between unique restriction sites, with transient introduction of the restricdiverged sequences in plants and animals, the study of mismatch tion enzyme32,33, or by sequencing of repaired lineared plasmids34)
repair proteins in plants has obvious applications to plant breed- have confirmed that the kinds of products observed as a result of
ing, as well as research into speciation.
T-DNA integration or TE excision do indeed resemble genuine
Although several research groups have informally reported the plant DSB repair products in many ways:
cloning of plant mismatch repair gene homologs, to date only one • Some degradation of the broken ends usually occurs before
sequence has been published, an MSH homolog from Arabidopsis26.
ligation, resulting in a loss of information.
Sequence analysis indicates that this is an MSH2 homolog. In both • Microhomologies are generally utilized during NHEJ.
humans and yeast, the MSH2 gene product acts as a heterodimer • ‘Filler’ DNAs are often integrated into the break. These may be
with MSH3 or MSH6 gene products to recognize and correct misrecognizable sequences from elsewhere in the genome, duplimatched bases. It also serves to inhibit both meiotic and mitotic
cations or inversions of local sequences, or simple sequences
recombination between diverged sequences in yeast. The function
that seem to be generated at random.
of this gene in plants has yet to be determined.
An interesting and important question is whether the majority of
genuine chromosomal DSBs are repaired via NHEJ, rather than
Repair of double strand breaks
through homologous recombination. There is some difficulty in
Double strand breaks (DBSs) can occur spontaneously in the cell comparing the actual rate of homologous versus nonhomologous reas a result of a nick in a single stranded region, due to mechanical pair of DSBs, because, unfortunately, experiments are set up to resstress of a chromosome, or as part of the initiation of crossing over cue only those events that regenerate a selectable marker (through
during meiosis4. Artificial sources of DSBs include ionizing radi- homologous recombination) or only those events that destroy a
ation, certain radiomimetic chemicals, and transgenic DNAs. There counterselectable marker (through NHEJ). Experiments have not
are two basic classes of pathways for the repair of double strand been performed that directly compare the relative rates of the two
breaks (DSBs; Fig. 4). The first, repair via homologous recombi- kinds of events. Surveys of T-DNA integration events and TE excination, uses a gene conversion mechanism to replace the break with sion products certainly suggest that in higher plants, as in mammals,
intact sequence from a sister chromatid, homolog or other identical breaks are repaired by nonhomologous recombination far more
(or nearly identical) sequence. The second mechanism, nonhomolo- frequently than via homologous recombination. In Arabidopsis,
gous end-joining (NHEJ), involves the resealing of two broken ends. T-DNAs integrate at random positions: the ratio of gene replaceAlthough there are often ‘microhomologies’ of two to five bases at ment events to random insertion events is approximately 1:1000
the resealed joint, these sequences probably only act to stabilize the (Ref. 35); in tobacco this ratio is closer to 1:10 000 (Ref. 36). Surligation reaction, and do not reflect the kind of true homology prisingly, even in experimental designs in which the repair of one
search involved in homologous recombination. Because there is no end of a linear transgene is ‘forced’ to occur via homologous recommechanism to ensure the pairing of the two original chromosome bination (through selection for the reconstitution of a fragmented
ends, NHEJ can produce chromosomal inversions, deletions, trans- drug resistance marker), the remaining end is often (in about one out
locations, and partial duplications. Although all organisms in which of four cases) repaired nonhomologously, in spite of the proximDSB repair has been studied have been found to possess both repair ity of homologous sequences32. This suggests that even in a case
mechanisms, the rate at which one or the other mechanism is actually where the search for homology is greatly simplified, the enzymes
employed varies widely. As a general rule, organisms with relatively involved in NHEJ often ‘out compete’ those required for homolocompact genomes (bacteria and yeast) will virtually always repair gous recombination in the race to process the double strand break.
5′
3′
24
January 1999, Vol. 4, No. 1
trends in plant science
reviews
Very few of the plant genes involved in DSB repair have been
identified. Homologs of the recA genes (DMC1/LIM15, RAD51)37–39
have been cloned from plants, and the L. longiflorum homologs localized to meiotic prophase chromosomes40, but their activity has
not been determined through biochemical or mutational analysis.
Mutants sensitive to ionizing radiation41,42 have been isolated, but
the genes involved have not yet been cloned. Some of these mutants exhibit altered meiotic and extrachromosomal recombination
rates, suggesting that the genes are involved in either homologous
recombination or NHEJ (Ref. 43).
The rate of meiotic recombination, and the relative rates of gene
replacement versus gene addition events, are governed by the
enzymology of the pathways that are involved in these processes.
An understanding of these mechanisms, and the identification of
the genes involved, would enable us to truly ‘engineer’ the plant
genome, rather than mutating or transforming plants in the essentially random manner employed today.
Acknowledgements
Work cited from the author’s lab was supported by USDA NRICGP
grant 94-37301-0564 and NSF grant 90-19159.
References
1 Lawley, P. (1974) Some chemical aspects of dose-response relationships in
alkylation mutagenesis, Mutat. Res. 23, 283–295
2 Vonarx, E.J. et al. (1998) DNA repair in higher plants, Mutat. Res. 400, 187–200
3 Britt, A. (1998) DNA repair in higher plants, in DNA Damage and Repair
(Vol. 1) (Nickoloff, J. and Hoekstra, M., eds), pp. 577–595, Humana Press
4 Friedberg, E.C., Walker, G.C. and Siede, W. (1995) DNA Repair and
Mutagenesis, ASM Press
5 Rozema, J. et al. (1997) UV-B as an environmental factor in plant life: stress
and regulation, Trends Ecol. Evol. 12, 22–28
6 Jansen, M., Gaba, V. and Greenberg, B. (1998) Higher plants and UV-B
radiation: balancing damage, repair and acclimation, Trends Plant Sci. 3,
131–135
7 Sancar, A. (1994) Structure and function of DNA photolyase,Biochemistry 33, 2–9
8 Yasui, A. et al. (1994) A new class of DNA photolyases present in various
organisms including aplacental mammals, EMBO J. 13, 6143–6151
9 Ahmad, M. et al. (1997) An enzyme similar to animal type II photolyases
mediates photoreactivation in Arabidopsis, Plant Cell 9, 199–207
10 Jiang, C-Z. et al. (1997) Photorepair mutants of Arabidopsis, Proc. Natl. Acad.
Sci. U. S. A. 94, 7441–7445
11 Todo, T. et al. (1993) A new photoreactivating enzyme that specifically
repairs ultraviolet light-induced (6-4) photoproducts, Nature 361, 371–374
12 Chen, J-J., Mitchell, D. and Britt, A.B. (1994) A light-dependent pathway for
the elimination of UV-induced pyrimidine (6-4) pyrimidinone photoproducts
in Arabidopsis thaliana, Plant Cell 6, 1311–1317
13 Nakajima, S. et al. (1998) Cloning and characterization of a gene (UVR3)
required for photorepair of 6-4 products in Arabidopsis thaliana, Nucleic
Acids Res. 26, 638–644
14 Cashmore, A. (1997) The cryptochrome family of photoreceptors, Plant Cell
Environ. 20, 764–767
15 Hsu, D.S. et al. (1996) Putative human blue-light photoreceptors hCRY1 and
hCRY2 are flavoproteins, Biochemistry 35, 13871–13877
16 Miyamoto, Y. and Sancar, A. (1998) Vitamin B-2-based blue-light photoreceptors in the retinohypothalamic tract as the photoactive pigments for setting
the circadian clock in mammals, Proc. Natl. Acad. Sci. U. S. A. 95, 6097–6102
17 Caldwell, M. (1979) Plant life and ultraviolet radiation: some perspective in
the history of the earth’s UV climate, BioScience 29, 520–525
18 Sancar, A. (1996) DNA excision repair, Annu. Rev. Biochem. 65, 43–81
19 Britt, A.B. et al. (1993) A UV-sensitive mutant of Arabidopsis defective in the
repair of pyrimidine-pyrimidinone (6-4) dimers, Science 261, 1571–1574
20 Quaite, F.E. et al. (1994) DNA damage levels determine cyclobutyl pyrimidine
dimer repair mechanisms in alfalfa seedlings, Plant Cell 6, 1635–1641
21 Jenkins, M.E. et al. (1995) Radiation-sensitive mutants of Arabidopsis
thaliana, Genetics 140, 725–732
22 Jiang, C-Z. et al. (1997) UV- and gamma-radiation sensitive mutants of
Arabidopsis, Genetics 147, 1401–1409
23 Xu, H. et al. (1998) Plant homologue of human excision repair gene ERCC1
points to conservation of DNA repair mechanisms, Plant J. 13, 823–829
24 Mueller, J.P. and Smerdon, M.J. (1996) Rad23 is required for transcriptioncoupled repair and efficient overall repair in Saccharomyces cerevisiae, Mol.
Cell. Biol. 16, 2361–2368
25 Sturm, A. and Lienhard, S. (1998) Two isoforms of plant RAD23 complement
a UV-sensitive rad23 mutant in yeast, Plant J. 13, 815–821
26 Culligan, K.M. and Hays, J.B. (1997) DNA mismatch repair in plants. An
Arabidopsis thaliana gene that predicts a protein belonging to the MSH2
subfamily of eukaryotic mutS homologs, Plant Physiol. 115, 833–839
27 Modrich, P. and Lahue, R. (1996) Mismatch repair in replication fidelity,
genetic recombination, and cancer biology, Annu. Rev. Biochem. 65, 101–133
28 Hunter, N. et al. (1996) The mismatch repair system contributes to meiotic
sterility in an interspecific yeast hybrid, EMBO J. 15, 1726–1733
29 Scott, L., LaFoe, D. and Weil, C.F. (1996) Adjacent sequences influence DNA
repair accompanying transposon excision in maize, Genetics 142, 237–246
30 Ohba, T. et al. (1995) DNA rearrangement associated with the integration of
T-DNA in tobacco: an example for multiple duplications of DNA around the
integration target, Plant J. 7, 157–164
31 Takano, M. et al. (1997) The structures of integration sites in transgenic rice,
Plant J. 11, 353–361
32 Puchta, H., Dujon, B. and Hohn, B. (1996) Two different but related
mechanisms are used in plants for the repair of genomic double-strand breaks
by homologous recombination, Proc. Natl. Acad. Sci. U. S. A. 93, 5055–5060
33 Salomon, S. and Puchta, H. (1998) Capture of genomic and T-DNA sequences
during double-strand break repair in somatic plant cells, EMBO J. 17,
6086–6095
34 Gorbunova, V. and Levy, A.A. (1997) Non-homologous DNA end joining in
plant cells is associated with deletions and filler DNA insertions, Nucleic
Acids Res. 25, 4650–4657
35 Kempin, S. et al. (1997) Targeted disruption in Arabidopsis, Nature 389, 802–803
36 Paszkowski, J. et al. (1988) Gene targeting in plants, EMBO J. 7, 4021–4026
37 Kobayashi, T., Hotta, Y. and Tabata, S. (1993) Isolation and characterization
of a yeast gene that is homologous with a meiosis-specific cDNA from a plant,
Mol. Gen. Genet. 237, 225–232
38 Klimyuk, V. and Jones, J. (1996) AtDMC1, the Arabidopsis homologue of the
yeast DMC1 gene: characterisation, transposon-induced allelic variation and
meiosis-specific expression of a pAtDMC1:GUS fusion, Plant J. 11, 1–14
39 Doutriaux, M-P. et al. (1998) Isolation and characterisation of the RAD51 and
DMC1 homologs from Arabidopsis thaliana, Mol. Gen. Genet. 257, 283–291
40 Terasawa, M. et al. (1995) Localization of RecA-like recombination proteins
on chromosomes of the lily at various meiotic stages, Genes Dev. 9, 925–934
41 Davies, C. et al. (1994) Isolation of Arabidopsis thaliana mutants
hypersensitive to gamma radiation, Mol. Gen. Genet. 243, 660–665
42 Masson, J., King, P. and Paszkowski, J. (1997) Mutants of Arabidopsis
thaliana hypersensitive to DNA damaging treatments, Genetics 146, 401–407
43 Masson, J. and Paszkowski, J. (1997) Arabidopsis thaliana mutants altered in
homologous recombination, Proc. Natl. Acad. Sci. U. S. A. 94, 11731–11735
44 Ribeiro, D.T. et al. (1998) Cloning of a cDNA from Arabidopsis thaliana
homologous to the human XPB gene, Gene 208, 207–213
Anne B. Britt is at the Section of Plant Biology, University of
California, Davis, CA 95616, USA (tel 11 530 752 0699;
fax 11 530 752 5410; e-mail abbritt@ucdavis.edu).
Congratulations!
Our warmest congratulations to Anne Britt, who in the closing
stages of submitting her article also found time to give birth to
twin girls.
January 1999, Vol. 4, No. 1
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