|
Items in cart [0] |
Questions? Call 888-624-8373 |
PAPERBACK Free Resources |
||||||||||||||||
|
||||||||||||||||
The following HTML text is provided to enhance online readability. Many aspects of typography translate only awkwardly to HTML. Please use the page image as the authoritative form to ensure accuracy. in the cell? Clearly, regression of the origin-proximal end of the replicated chromosome arm will provide little benefit for restarting a stalled fork. Currently there is no information extent that addresses the fate of the proteins at a stalled fork in vivo. If the half-life that we have measured in vitro reflects what happens in the cell, then it seems reasonable to suggest that there may be active mechanisms that act to displace the replisome from a stalled replication fork to allow regression. Perhaps there are helicases that target stalled forks to displace the bound proteins. Spontaneous nascent strand regression required that the ERI be positively supercoiled. The positive supercoiling is an impressive driving force for this reaction in that all of the ERI existed as topologically relaxed DNA as a result of nascent strand regression. If the ERI was negatively supercoiled by the action of gyrase, fork regression was severely inhibited. Can fork regression therefore occur spontaneously in vivo? This is a difficult question to answer. Previous studies on the decatenating activity of Topo IV and DNA gyrase in vitro (29) and in vivo (31) suggested that these enzymes lacked the catalytic turnover necessary to keep pace with advancing replication forks that generate nearly 200 excess positive windings per second. If this scenario holds, then the replicating chromosome would probably be positively supercoiled. However, recent single enzyme studies (32) with Topo IV show that the number average turnover values calculated previously are gross underestimates. If the same holds for DNA gyrase, then it is more reasonable to expect that the replicating chromosome is, in fact, negatively supercoiled. Thus, if nascent strand regression plays a significant role at stalled replication forks in vivo, it would seem that there is a need for enzymes that act to facilitate formation of the Holliday junction. We have shown here that RecG fulfills this requirement. RecG stimulated RusA cleavage of the ERI and was required to observe any significant cleavage when the ERI was negatively supercoiled. Thus, the RecG branch-migration helicase activity can overcome the inhibitory effect of negative supercoiling and cause nascent strands to pair and regress enough that the Holliday junction that forms can be recognized and cleaved by RusA. We do not know whether the extent of RecG-catalyzed strand regression is as extensive as the regression that occurs spontaneously in the ERI. These findings suggest that RecG plays a central role in processing of stalled replication forks in the cell. In an accompanying paper in this colloquium (33), McGlynn and Lloyd show that RecG unwinds forked DNA by translocating simultaneously along both the leading- and lagging-strand templates. This unique helicase activity explains how RecG may promote formation of a Holliday junction. Elucidating how this enzyme cooperates with all of the others that are competing for the various DNA ends and single-stranded gaps that are present at the site of fork stalling will likely keep many laboratories occupied for some time to come. We thank Ed Bolt for providing RusA protein. These studies were supported by National Institutes of Health Grant GM34557 (to K.J.M.), a Medical Research Council Career Establishment grant (to P.M.), and a Medical Research Council Program grant (to R.G.L.). P.M. is a Lister Institute-Jenner Research Fellow. 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. Kuzminov, A. (1995) Mol. Microbiol. 16, 373–384. 3. Kogoma, T. (1996) Cell 85, 625–627. 4. McGlynn, P., Al-Deib, A.A., Liu, J., Marians, K.J. & Lloyd, R.G. (1997) J. Mol. Biol. 270, 212–221. 5. Nurse, P., Liu, J. & Marians, K.J. (1999) J. Biol. Chem. 274, 25026–25032. 6. Liu, J., Xu, L., Sandler, S.J. & Marians, K.J. (1999) Proc. Natl. Acad. Sci. USA 96, 3552–3555. 7. Horiuchi, T. & Fujimura, Y. (1995) J. Bacteriol. 177, 783–791. 8. Sharma, B. & Hill, T.M. (1995) Mol. Microbiol. 18, 45–61. 9. Michel, B, Ehrlich, S.D. & Uzest, M. (1997) EMBO J. 16, 430–438. 10. Scott, J.F., Eisenberg, S., Bertsch, L.L. & Kornberg, A. (1977) Proc. Natl. Acad. Sci. USA 74, 193–197. 11. Yancey-Wrona, J.E. & Matson, S.W. (1992) Nucleic Acids Res. 20, 6713–6721. 12. Seigneur, M., Bidnenko, V., Ehrlich, S.D. & Michel, B. (1998) Cell 95, 419–430. 13. West, S.C. (1997) Annu. Rev. Genet. 31, 213–244. 14. McGlynn, P. & Lloyd, R.G. (2000) Cell 101, 35–45. 15. Lloyd, R.G. & Sharples, G.J. (1993) EMBO J. 12, 17–22. 16. Whitby, M.C., Vincent, S. & Lloyd, R.G. (1994) EMBO J. 13, 5220–5228. 17. Hiasa, H. & Marians, K.J. (1994) J. Biol. Chem. 269, 6058–6063. 18. Marians, K.J. (1995) Methods Enzymol. 262, 507–521. 19. Bolt, E.L., Sharples, G.J. & Lloyd, R.G. (1999) J. Mol. Biol. 286, 403–415. 20. Tsaneva, I.R., Illing, G.T., Lloyd, R.G. & West, S.C. (1992) Mol. Gen. Genet. 235, 1–10. 21. Dunderdale, H.J., Sharples, G.J., Lloyd, R.G. & West, S.C. (1994) J. Biol. Chem. 269, 5187–5194. 22. McGlynn, P. & Lloyd, R.G. (1999) Nucleic Acids Res. 27, 3049–3056. 23. Hiasa, H. & Marians, K.J. (1994) J. Biol. Chem. 269, 26959–26968. 24. Hiasa, H. & Marians, K.J. (1994) J. Biol. Chem. 269, 16371–16375. 25. Postow, L., Ulsperger, C., Keller, R.W., Bustamante, C., Vologodski, A.V. & Cozzarelli, N.R. (2001) J. Biol. Chem. 276, 2790–2796. 26. Marians, K.J., Hiasa, H., Kim, D.R. & McHenry, C.S. (1998) J. Biol. Chem. 273, 2452–2457. 27. Whitby, M.C., Bolt, E.L., Chan, S.N. & Lloyd, R.G. (1996) J. Mol. Biol. 264, 878–890. 28. Chan, S.N., Harris, L., Bolt, E.L., Whitby, M.C. & Lloyd, R.G. (1997) J. Biol. Chem. 272, 14873–14882. 29. Hiasa, H. & Marians, K.J. (1996) J. Biol. Chem. 271, 21529–21535. 30. Ulsperger, C. & Cozzarelli, N.R. (1996) J. Biol. Chem. 271, 31549–31555. 31. Zecheidrich, E.L. & Cozzarelli, N.R. (1995) Genes Dev. 9, 2859–2869. 32. Crisona, N.J., Strick, T.R., Bensimon, D., Croquette, V. & Cozzarelli, N.R. (2000) Genes Dev. 14, 2881–2892. 33. McGlynn, P. & Lloyd, R.G. (2001) Proc. Natl. Acad. Sci. USA 98, 8227–8234.
|
|
|||||||||||||||
