Biochemical basis for the essential genetic requirements of RecA and the beta-clamp in Pol V activation.
In Escherichia coli, it is genetically well established that the beta-clamp and RecA are essential cofactors that endow DNA polymerase (Pol) V with lesion bypass activity. However, the biochemical basis for these requirements is still largely unknown. Because the process of translesion synthesis (TLS) requires that the specialized DNA polymerase synthesize in a single binding event a TLS patch that is long enough to resist external proofreading, it is critical to monitor Pol V burst synthesis. Here, we dissect the distinct roles that RecA and the beta-clamp perform during the Pol V activation process using physiologically relevant long single-stranded template DNA, similar to those used in genetic assays. Our data show that the beta-clamp endows the complex between Pol V and the template DNA with increased stability. Also, the RecA filament formed in cis on the single-stranded DNA produced downstream from the lesion stretches the template DNA to allow smooth elongation of the nascent strand by Pol V. The concurrent action of both cofactors is required for achieving productive TLS events. The present article presents an integrated view of TLS under physiologically relevant conditions in E. coli that may represent a paradigm for lesion bypass in other organisms.
Fujii
Shingo
S
Centre National de la Recherche Scientifique, Unité Propre de Recherche 3081, Genome Instability and Carcinogenesis, Conventionné par l'Université d'Aix-Marseille 2, 31, chemin Joseph Aiguier, 13402 Marseille cedex 20, France.
Fuchs
Robert P
RP
D004247:DNA / Q000235:genetics / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011938:Rec A Recombinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013698:Templates, Genetic
D029968:Escherichia coli Proteins; D004247:DNA; D011938:Rec A Recombinases; C120926:DNA polymerase V, E coli; D004259:DNA-Directed DNA Polymerase
DNA Repair (Amst). 2006 Dec 9;5(12):1495-8
16956796
J Mol Biol. 2004 Aug 6;341(2):405-17
15276832
J Mol Biol. 2000 Mar 31;297(3):585-97
10731413
Mol Microbiol. 2000 Mar;35(6):1443-53
10760145
EMBO J. 2003 Nov 3;22(21):5883-92
14592985
EMBO J. 2006 Dec 13;25(24):5754-63
17139245
Proc Natl Acad Sci U S A. 2001 Jul 17;98(15):8566-71
11447256
EMBO J. 2004 Oct 27;23(21):4342-52
15470496
Science. 2003 May 23;300(5623):1300-3
12764199
J Mol Biol. 1997 Jul 11;270(2):201-11
9236122
Chem Res Toxicol. 1997 Jun;10(6):667-71
9208173
Nature. 2009 Jul 16;460(7253):359-63
19606142
J Bacteriol. 1989 May;171(5):2415-23
2651400
DNA Repair (Amst). 2007 Jul 1;6(7):1032-41
17403618
Proc Natl Acad Sci U S A. 1979 Apr;76(4):1702-5
221903
Mol Microbiol. 1993 Dec;10(5):963-71
7934872
Crit Rev Microbiol. 1994;20(2):125-42
8080625
Mol Microbiol. 1998 Apr;28(2):281-91
9622353
J Mol Biol. 2007 Sep 28;372(4):883-893
17707403
Biochimie. 1982 Aug-Sep;64(8-9):633-6
6814511
J Bacteriol. 1990 Jun;172(6):3030-6
2188949
Mol Gen Genet. 1978 Sep 20;165(1):87-93
362169
Genes Cells. 2003 May;8(5):437-49
12694533
Mol Gen Genet. 1992 Nov;235(2-3):373-80
1465109
Nature. 2006 Aug 24;442(7105):883-7
16929290
J Biol Chem. 2004 May 14;279(20):21543-51
15014081
Nature. 2008 May 22;453(7194):489-4
18497818
Biochimie. 1991 Apr;73(4):479-84
1911948
Mol Cell. 2006 Jul 21;23(2):155-60
16857582
DNA Repair (Amst). 2002 Sep 4;1(9):703-8
12509274
Proc Natl Acad Sci U S A. 2001 Sep 25;98(20):11627-32
11573000
Mol Gen Genet. 1977 Nov 14;156(2):121-31
340898