F. 1999 Proc. Natl. Acad. Sci. USA 96, 8919–8924. Iwai, S. Hanaoka, F. 1999 EMBO J. 18, 3491–3501. C., Ohmori, H. Kunkel, T. A. 2000 J. Biol. Chem. 275, 39678–39684. Biol. Chem. 276, 92–98.

with additional factors involved in DNA replication, DNA repair, and possibly recombination as well. Consistent with this notion, a report by Lo ´pez de Saro and O’Donnell in this issue 60 discusses interactions involving the b clamp with Pol I and DNA ligase, which play roles in the maturation of Okazaki fragments, as well as with MutS, which acts in methyl-directed mismatch repair. Although one can easily imagine how the association of UmuD 2 C with b could effectively block access of the b clamp to the a and g complex components of Pol III, it prompts the important question of how these various protein– protein interactions are coordinately regulated such that the correct protein complex is formed. As discussed above, the clamp loader complex of Pol III serves an important role in displacing primase and recycling b clamps during lagging strand replication 57. A poorly understood com- ponent of this process, however, relates to the release of the newly loaded b clamp from the clamp loader complex to allow for its association with the a catalytic subunit of Pol III. The factor or factors that governs this process clearly serves an important role helping to determine which DNA polymerase will gain access to the replication fork. Given that Pol III and the clamp loader complex can exist as part of the same multiprotein machine, this process is likely to be achieved via a coordinated set of protein–protein and protein–nucleic acid interactions. Our current challenge is to better understand these interactions at a molecular level. Genetic and Biochemical Evidence Suggesting a Need for Multiple DNA Polymerases for Bypassing Certain DNA Lesions. A growing body of evidence suggests that TLS is even more complicated than initially thought, and can involve multiple DNA polymerases and hence multiple polymerase-switching events. Regardless of how one views TLS, the simplest of models would assume that the replicative polymerase, being unable to bypass a lesion in the DNA, would either ‘‘fall off’’ of the DNA or simply translocate downstream of the lesion to continue replication. This would then allow for the loading of another DNA polymerase capable of replicating over the lesion. Eventually, the replicative poly- merase would regain control of the template until such time as replication was completed or the polymerase again encountered a replication-blocking DNA lesion. Even in this overly simplistic model, one must account for at least two events of polymerase switching; first from the replicative polymerase to the TLS polymerase, and then back to the replicative polymerase. Given its distributive nature, Goodman 71 has suggested that after Pol V has bypassed a lesion, it ‘‘falls off’’ of the DNA, thereby allowing the reassociation of Pol III. However, the various interactions between the umuDC gene products and Pol III summarized above 44 suggest that a set of protein–protein contacts may guide these switches. Recently, it has become evident that lesion bypass can be considerably more complicated than the above model would suggest. The notion of multiple DNA polymerases being re- quired for bypass of a single DNA lesion, although in the forefront of discussion today, was first suggested nearly 5 years ago by Nelson et al. 14, 115. These two reports represent the first direct biochemical demonstrations of two key aspects of TLS. First, they showed that S. cerevisiae Rev1 protein a member of the UmuC superfamily contains an intrinsic DNA polymerase activity 14, albeit with remarkably limited abilities [Rev1p contains a template-dependent but not template- directed deoxycytidyl transferase activity], and second, they demonstrated that the combined actions of S. cerevisiae REV1 and S. cerevisiae Pol z was more effective at enabling replication past a synthetic abasic site than either enzyme alone 115. As discussed above, it is now generally appreciated that proteins related to S. cerevisiae Rev1p also contain an intrinsic DNA polymerase activity, and more recently, that multiple specialized DNA polymerases are involved in bypassing various types of DNA lesions. Some specific examples of a need for multiple DNA poly- merases, in addition to the replicative DNA polymerase, for efficient bypass of certain types of lesions include i bypass of a synthetic abasic site in vitro by the combined actions of human Pol h followed by S. cerevisiae Pol z 116, ii bypass of highly distorting or noninstructive DNA lesions in vitro by the sequen- tial actions of human Pol i followed by S. cerevisiae Pol z 117, and iii the requirement of both Pol IV and Pol V of E. coli for bypass of a benzo[a]pyrene adduct in vivo 118. Perspectives and Conclusion. The E. coli umuDC-encoded Pol V-dependent SOS response represents the paradigm for the study of TLS and induced mutagenesis. Significant advances have been made in understanding how a set of elaborate regulatory controls and a sophisticated system of protein– protein contacts ensure that the umuDC gene products carry out their appropriate biological roles. However, as is so often the case in science, the discoveries of today are posing even more challenging questions for tomorrow. For example, some poorly understood general issues that lie at the heart of not only TLS, but also DNA replication itself, include i an understanding of the molecular mechanism of polymerase switching, ii the identity and mode of action of factors involved in coordinating DNA replication with DNA repair, including cell cycle and checkpoint regulation, and determining how they act, and iii the controls systems governing the relationship between DNA replication and DNA recombination. Furthermore, there is growing evidence that DNA replication takes place in stationary factories 119–122 so that understanding the events such as polymerase-switching or the recombinational initiation of DNA synthesis in the context of these factories will be a challenging but fascinating problem. We anticipate that the lessons learned from studying the coordinated regulation of the actions of the five E. coli DNA polymerases, together with their associated accessory factors, in the context of DNA replication, DNA repair, recom- bination, and cell cycle progression, will help to provide a framework for characterizing similar control networks in eu- karyotes, where both the number of DNA polymerases and the level of complexity of the events are far greater. We thank Justin Courcelle, Phil Hanawalt, and Mike O’Donnell for sharing unpublished data and Brad Smith and other members of our research group for their help and support. This work was supported by Public Health Service Grant CA21615 to G.C.W. from the National Cancer Institute. M.D.S. was supported by a fellowship 5 F32 CA79161- 03 from the National Cancer Institute. 1. Cox, M. M., Goodman, M. F., Kreuzer, K. N., Sherratt, D. J., Sandler, S. J. Marians, K. J. 2000 Nature London 404, 37–41. 2. Marians, K. J. 2000 Curr. Opin. Genet. Dev. 10, 151–156. 3. Friedberg, E. C., Walker, G. C. Siede, W. 1995 DNA Repair and Mutagenesis Am. Soc. Microbiol., Washington, DC. 4. Sutton, M. D., Smith, B. T., Godoy, V. G. Walker, G. C. 2000 Annu. Rev. Genet. 34, 479–497. 5. Walker, G. C. 2001 Cold Spring Harbor Symp. Quant. Biol. 65, in press. 6. Tang, M., Shen, X., Frank, E. G., O’Donnell, M., Woodgate, R. Goodman,

M. F. 1999 Proc. Natl. Acad. Sci. USA 96, 8919–8924.

7. Reuven, N. B., Arad, G., Maor-Shoshani, A. Livneh, Z. 1999 J. Biol. Chem. 274, 31763–31766. 8. Smith, B. T. Walker, G. C. 1998 Genetics 148, 1599–1610. 9. Steinborn, G. 1978 Mol. Gen. Genet. 165, 87–93. 10. Kato, T. Shinoura, Y. 1977 Mol. Gen. Genet. 156, 121–131. 11. Larimer, F. W., Perry, J. R. Hardigree, A. A. 1989 J. Bacteriol. 171, 230–237. 12. Lemontt, J. F. 1971 Mutat. Res. 13, 311–317. 13. Wagner, J., Gruz, P., Kim, S. R., Yamada, M., Matsui, K., Fuchs, R. P. Nohmi, T. 1999 Mol. Cell 4, 281–286. Sutton and Walker PNAS u July 17, 2001 u vol. 98 u no. 15 u 8347 COLLOQUIUM

14. Nelson, J. R., Lawrence, C. W. Hinkle, D. C. 1996 Nature London 382,

729–731. 15. Johnson, R. E., Prakash, S. Prakash, L. 1999 Science 283, 1001–1004. 16. Masutani, C., Araki, M., Yamada, A., Kusumoto, R., Nogimori, T., Maekawa,

T., Iwai, S. Hanaoka, F. 1999 EMBO J. 18, 3491–3501.

17. Tissier, A., McDonald, J. P., Frank, E. G. Woodgate, R. 2000 Genes Dev.

14, 1642–1650.

18. Ohashi, E., Bebenek, K., Matsuda, T., Feaver, W. J., Gerlach, V. L., Friedberg,

E. C., Ohmori, H. Kunkel, T. A. 2000 J. Biol. Chem. 275, 39678–39684.

19. Gerlach, V. L., Feaver, W. J., Fischhaber, P. L. Friedberg, E. C. 2001

J. Biol. Chem. 276, 92–98.

20. Ohashi, E., Ogi, T., Kusumoto, R., Iwai, S., Masutani, C., Hanaoka, F. Ohmori, H. 2000 Genes Dev. 14, 1589–1594. 21. Friedberg, E. C. Gerlach, V. L. 1999 Cell 98, 413–416. 22. Woodgate, R. 1999 Genes Dev. 13, 2191–2195. 23. Johnson, R. E., Washington, M. T., Prakash, S. Prakash, L. 1999 Proc. Natl. Acad. Sci. USA 96, 12224–12226. 24. Friedberg, E. C., Feaver, W. J. Gerlach, V. L. 2000 Proc. Natl. Acad. Sci. USA 97, 5681–5683. First Published May 16, 2000; 10.1073ypnas.120152397 25. Tang, M., Pham, P., Shen, X., Taylor, J.-S., O’Donnell, M., Woodgate, M. Goodman, M. F. 2000 Nature London 404, 1014–1018. 26. Gerlach, V. L., Aravind, L., Gotway, G., Schultz, R. A., Koonin, E. V. Friedberg, E. C. 1999 Proc. Natl. Acad. Sci. USA 96, 11922–11927. 27. Elledge, S. J. Walker, G. C. 1983 J. Mol. Biol. 164, 175–192. 28. Perry, K. L., Elledge, S. J., Mitchell, B. B., Marsh, L. Walker, G. C. 1985 Proc. Natl. Acad. Sci. USA 82, 4331–4335. 29. Johnson, R. E., Kondratick, C. M., Prakash, S. Prakash, L. 1999 Science

285, 263–265.

30. McDonald, J. P., Rapic-Otrin, V., Epstein, J. A., Broughton, B. C., Wang, X., Lehmann, A. R., Wolgemuth, D. J. Woodgate, R. 1999 Genomics 60, 20–30. 31. Lin, W., Xin, H., Zhang, Y., Wu, X., Yuan, F. Wang, Z. 1999 Nucleic Acids Res. 27, 4468–4475. 32. Cordonnier, A. M. Fuchs, R. P. 1999 Mutat. Res. 435, 111–119. 33. Lehmann, A. R. 2000 Gene 253, 1–12. 34. Berneburg, M. Lehmann, A. R. 2001 Adv. Genet. 43, 71–102. 35. Masutani, C., Kusumoto, R., Yamada, A., Dohmae, N., Yokoi, M., Yuasa, M., Araki, M., Iwai, S., Takio, K. Hanaoka, F. 1999 Nature London 399, 700–704. 36. Garcia-Diaz, M., Dominguez, O., Lopez-Fernandez, L. A., de Lera, L. T., Saniger, M. L., Ruiz, J. F., Parraga, M., Garcia-Ortiz, M. J., Kirchhoff, T., del Mazo, J., et al. 2000 J. Mol. Biol. 301, 851–867. 37. Aoufouchi, S., Flatter, E., Dahan, A., Faili, A., Bertocci, B., Storck, S., Delbos,

F., Cocea, L., Gupta, N., Weill, J. C., et al. 2000 Nucleic Acids Res. 28, 3684–3693.