unwinding the damaged DNA region before the dual incision step. Moreover, two isoforms of
yeast rad23 gene were identified in a carrot cDNA library when this library was used to complement
a UV-sensitive yeast mutant that is deficient in a single-stranded DNA binding protein associated
with DNA unwinding [12]. Although some infor- mation regarding NER proteins in higher plants
has been obtained, little is known about the nature of NER proteins in lower plants and the way they
interact with each other.
In vitro assays detecting damage-specific inci- sion of DNA the DNA incision assay and dam-
age-dependent DNA repair synthesis the repair synthesis assay developed in cell-free extracts
have been recognized as valuable tools for study- ing the biochemical mechanisms, including the
cutting site introduced by the 5 or 3 incision endonuclease, the size of the excised fragment, the
cofactors required for repair and the substrate specificity, of NER in E. coli and human cells
[13 – 17]. Unicellular algae are ideal model systems for exploring the biochemical mechanisms of NER
in lower plants, because they can be cultured and handled easily in the laboratory. We have devel-
oped an in vitro DNA incision assay in cell-free extracts of unicelluar alga C. pyrenoidosa, which
clearly revealed the cutting position introduced at the 3 side of a pyrimidine dimer by the extracts
[18]. Following the development of incision assay, a UV-damaged-DNA binding activity composed
of three polypeptides, p72, p80 and p90, was purified from C. pyrenoidosa extracts, and these
three polypeptides were found to bind directly to UV-damaged DNA as they could be extracted
from gel shift bands produced by the crude ex- tracts or more purified protein fractions [19]. This
binding activity may participate in the damage- recognition step of NER, since it recognized both
UV and cisplatin-damaged DNA in the absence of ATP. The goal of this research was to identify the
proteins involved in NER in C. pyrenoidosa by functional analysis. An in vitro DNA repair syn-
thesis assay monitoring NER was established and an affinity adsorption of binding proteins from the
algal extracts was performed with the same UV- damaged DNA probe [19] immobilized on a solid
phase. The effects of affinity adsorption on the capacity of NER were determined by the repair
synthesis assay and a polypeptide 72 kDa in molecular mass that preferentially binds to dam-
aged DNA is believed to play a crucial role in NER.
2. Materials and methods
2
.
1
. Plant materials and cell-free extracts C. pyrenoidosa kindly provided by Dr Jiunn-
Tzong Wu Institute of Botany, Academia Sinica, Taipei, Taiwan, ROC was grown in a synthetic
salt medium [20] at 25°C under illumination of a fluorescent light and gentle shaking. Algal growth
was monitored by absorbance at 685 nm. Algal cells 1000 ml at mid-log growth 5.5 – 7.0 × 10
7
cellsml were collected by centrifugation at 5000 × g and the pellet was washed twice with
ice-cold distilled water and once with extraction buffer 20 mM Tris – HCl pH 8.0, 1 mM EDTA,
1 mM DTT, 10 vv glycerol. Algal cells were then suspended in 4 ml hypotonic buffer 40 mM
Tris – HCl pH 8.0, 1 mM EDTA, 1 mM DTT containing protease inhibitors 2 mM PMSF, 4
m
gml leupeptin, 1 mgml pepstatin, and the swol- len cells were broken on ice by pulsed sonication.
After centrifugation at 14 000 × g for 10 min at 4°C, the supernatant was transferred to a beaker
on ice and nucleic acids were removed by stirring in the presence of 1.5 wv streptomycin sulfate
for 15 min. After centrifugation at 16 000 × g for 90 min, the supernatant was withdrawn and am-
monium sulfate was added to 55 saturation to fractionate repair proteins. The protein precipitate
was dissolved in 1 ml dialysis buffer 20 mM Tris – HCl pH 8.0, 1 mM EDTA, 2 mM DTT,
0.1 M KCl, 12 mM MgCl
2
, 17 vv glycerol, and dialyzed against the same buffer for 12 h at
4°C. The dialyzed protein solution was concen- trated with Centricon-10 Amicon, USA and used
as the cell-free extract for the repair synthesis assay. The protein concentration in the extract
was measured by a protein assay kit Bio-Rad, USA based on the method developed by Brad-
ford [21].
2
.
2
. Preparation of immobilized DNA ligands for affinity adsorption
A 27-mer oligonucleotide, 5-GAC CGA GCT GGG TTA CGA CGC GAC GCC-3 and its
complementary strand were synthesized and la-
beled with biotin at the 5 end by a commercial source. Both strands at equal concentrations 1
m gml were mixed and the mixture was placed in a
PCR thermocycler Perkin Elmer, Norwalk, CT, USA. The temperature was raised to 95°C and
decreased to 25°C at a rate of 1°Cmin. After filtering the mixture on a microcon-10 concentra-
tor Amicon, Beverly, MA, USA, the annealed 27 mer retained on the membrane was collected for
preparing UV-damaged DNA. The duplex 27 mer was pipetted onto a piece of parafilm, and UV
254 nm irradiation was performed in an XL-1500 UV
crosslinker Spectronics,
Westbury, NY,
USA. UV irradiation was expected to induce the formation of CPDs and 6-4PPs between adjacent
TT or CT on the 27 mer [22]. UV-irradiated or unirradiated 27 mer 2 mg was then incubated
with a suspension 20 ml of streptavidin-conju- gated agarose beads 1 – 3 mg streptavidinml,
Merck, Germany, and the oligonucleotide was immobilized on the beads through the strong
affinity between biotin and streptavidin.
2
.
3
. Affinity adsorption of repair proteins To pull down repair proteins from cell-free ex-
tracts of C. pyrenoidosa, the algal extract contain- ing 50 mg proteins in 20 ml dialysis buffer was
incubated at 30°C for 20 min with 20 ml agarose beads carrying 2 mg UV 27 kJ m
− 2
-irradiated 27 mer. The suspension was centrifuged at 5000 × g
at 4°C, and a small fraction of the extract superna- tant was taken for the determination of protein
concentration. An aliquot of the extract contain- ing 30-mg proteins was used for the repair synthe-
sis assay. The repair capacity in the extract incubated with beads carrying unirradiated 27 mer
or no 27 mer was also tested as a comparison.
2
.
4
. DNA substrates for the repair synthesis assay Plasmid DNA damaged by the alkylating agent
cisplatin or MMC was used as the substrate for the repair synthesis assay. pBR 322 4.3 kb and
pGEM IV 2.8 kb plasmid DNA were isolated from E. coli JM 109 by a plasmid DNA prepara-
tion kit Qiagen, Valencia, CA, USA, and the closed circular form DNA was purified by CsCl
gradient centrifugation. The concentration of DNA in TE buffer was determined by the ab-
sorbance at 260 nm. Alkylating agent-damaged DNA was prepared by incubating pGEM IV plas-
mid 0.1 mgml in TE buffer with the same volume of aqueous solution containing different amount
of cisplatin or mitomycin C at 37°C overnight in the dark. The damaged DNA was precipitated
with ethanol and solubilized in distilled water. Untreated pBR 322 was used as an internal con-
trol in each repair reaction mixture.
2
.
5
. In 6itro DNA repair synthesis assay The standard repair reaction mixture 50 ml
contained 45 mM Tris – HCl pH 7.8, 4 mM EDTA, 70 mM KCl, 4 mM MgCl
2
, 1 mM DTT, 40 mM creatine phosphate-Tris pH 7.7, 2.5 mg
phosphocreatine kinase, 2 mM ATP, 18 mg bovine serum albumin, 50 mM each of dGTP, dCTP,
dTTP, dATP, 2 mCi [a-
32
P]dATP 3000 Cimmol, 1 mg pBR 322 plasmid DNA, 1 mg control or
damaged pGEM IV plasmid DNA, and 30 mg algal extract proteins. After incubating the reac-
tion mixture at 30°C for 2 h, the repair reaction was terminated by the addition of EDTA to a final
concentration of 25 mM. The extract proteins were removed by proteinase K 200 mgml diges-
tion in the presence of 0.5 SDS. The mixture was then extracted with phenolchloroformisoamyl al-
cohol 25:24:1, and DNA products were precipi- tated with ethanol. Purified DNA products were
linearized with Pst I and electrophoresed on a 1.2 agarose gel. After ethidium bromide staining,
the gel was vacuum dried and exposed to a Kodak XAR-5 X-ray film with an intensifying screen.
Band intensities on the autoradiograph were quan- titated using a AmershamPharmacia Imagemaster
documentation system. Damage-stimulated repair synthesis was expressed as repair factor deter-
mined by dividing the band intensity of the dam- aged DNA by that of the internal control.
2
.
6
. SDS-PAGE analysis of proteins captured by affinity adsorption
To analyze the proteins that were captured on the beads, agarose beads were washed thoroughly
with 20 mM KH
2
PO
4
pH 7.5 containing 0.15 M NaCl to remove loosely bound proteins. The
beads were then boiled for 10 min in 2 × gel loading buffer to release the tightly bound
proteins. The proteins present in the boiled mix- tures were analyzed by 12.5 SDS-PAGE and
silver staining.
Non-specific binding
proteins washed from the beads were also analyzed on the
same gel for comparison.
2
.
7
. Chemicals PMSF, leupeptin, pepstatin, streptomycin sul-
fate, cisplatin and mitomycin C were purchased from Sigma St Louis, MO, USA.Other reagent
grade chemicals were obtained from J.T. Baker Phillipsburg, NJ, USA. DTT and streptavidin-
conjugated agarose beads were obtained from Boehringer Mannheim Mannheim, Germany. [a-
32
P]dATP 3000 Ci mmol
− 1
was a product of Amersham Little Chalfont, Amersham, Bucking-
hamshire, UK. Biotin-labeled oligonucleotides were synthesized by Perkin Elmer Norwalk, CT,
USA.
3. Results
