A Genetic Selection for DinB Mutants Reveals an Interaction Between DNA Polymerase IV and the Replicative Polymerase That Is Required for Translesion Synthesis

Overview

Journal PLoS Genet

Specialty Genetics

Date 2015 Sep 10

PMID 26352807

Citations 23

Authors

Michelle K Scotland

Justin M H Heltzel

James E Kath

Jung-Suk Choi

Anthony J Berdis

Joseph J Loparo

Mark D Sutton

Affiliations

Soon will be listed here.

Abstract

Translesion DNA synthesis (TLS) by specialized DNA polymerases (Pols) is a conserved mechanism for tolerating replication blocking DNA lesions. The actions of TLS Pols are managed in part by ring-shaped sliding clamp proteins. In addition to catalyzing TLS, altered expression of TLS Pols impedes cellular growth. The goal of this study was to define the relationship between the physiological function of Escherichia coli Pol IV in TLS and its ability to impede growth when overproduced. To this end, 13 novel Pol IV mutants were identified that failed to impede growth. Subsequent analysis of these mutants suggest that overproduced levels of Pol IV inhibit E. coli growth by gaining inappropriate access to the replication fork via a Pol III-Pol IV switch that is mechanistically similar to that used under physiological conditions to coordinate Pol IV-catalyzed TLS with Pol III-catalyzed replication. Detailed analysis of one mutant, Pol IV-T120P, and two previously described Pol IV mutants impaired for interaction with either the rim (Pol IVR) or the cleft (Pol IVC) of the β sliding clamp revealed novel insights into the mechanism of the Pol III-Pol IV switch. Specifically, Pol IV-T120P retained complete catalytic activity in vitro but, like Pol IVR and Pol IVC, failed to support Pol IV TLS function in vivo. Notably, the T120P mutation abrogated a biochemical interaction of Pol IV with Pol III that was required for Pol III-Pol IV switching. Taken together, these results support a model in which Pol III-Pol IV switching involves interaction of Pol IV with Pol III, as well as the β clamp rim and cleft. Moreover, they provide strong support for the view that Pol III-Pol IV switching represents a vitally important mechanism for regulating TLS in vivo by managing access of Pol IV to the DNA.

Citing Articles

A bipartite interaction with the processivity clamp potentiates Pol IV-mediated TLS.

Chang S, Laureti L, Thrall E, Kay M, Philippin G, Jergic S Proc Natl Acad Sci U S A. 2025; 122(9):e2421471122.

PMID: 39993197 PMC: 11892629. DOI: 10.1073/pnas.2421471122.

A bipartite interaction with the processivity clamp potentiates Pol IV-mediated TLS.

Chang S, Laureti L, Thrall E, Kay M, Philippin G, Jergic S bioRxiv. 2024; .

PMID: 38853898 PMC: 11160790. DOI: 10.1101/2024.05.30.596738.

Molecular insights into the prototypical single-stranded DNA-binding protein from .

Bonde N, Kozlov A, Cox M, Lohman T, Keck J Crit Rev Biochem Mol Biol. 2024; 59(1-2):99-127.

PMID: 38770626 PMC: 11209772. DOI: 10.1080/10409238.2024.2330372.

Observing protein dynamics during DNA-lesion bypass by the replisome.

Wilkinson E, Spenkelink L, van Oijen A Front Mol Biosci. 2022; 9:968424.

PMID: 36213113 PMC: 9534484. DOI: 10.3389/fmolb.2022.968424.

Compartmentalization of the replication fork by single-stranded DNA-binding protein regulates translesion synthesis.

Chang S, Thrall E, Laureti L, Piatt S, Pages V, Loparo J Nat Struct Mol Biol. 2022; 29(9):932-941.

PMID: 36127468 PMC: 9509481. DOI: 10.1038/s41594-022-00827-2.

References

Wagner J, Gruz P, Kim S, Yamada M, Matsui K, Fuchs R . The dinB gene encodes a novel E. coli DNA polymerase, DNA pol IV, involved in mutagenesis. Mol Cell. 1999; 4(2):281-6. DOI: 10.1016/s1097-2765(00)80376-7. View

Sutton M, Opperman T, Walker G . The Escherichia coli SOS mutagenesis proteins UmuD and UmuD' interact physically with the replicative DNA polymerase. Proc Natl Acad Sci U S A. 1999; 96(22):12373-8. PMC: 22924. DOI: 10.1073/pnas.96.22.12373. View

Datsenko K, Wanner B . One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A. 2000; 97(12):6640-5. PMC: 18686. DOI: 10.1073/pnas.120163297. View

Dalrymple B, Kongsuwan K, Wijffels G, Dixon N, Jennings P . A universal protein-protein interaction motif in the eubacterial DNA replication and repair systems. Proc Natl Acad Sci U S A. 2001; 98(20):11627-32. PMC: 58780. DOI: 10.1073/pnas.191384398. View

Ling H, Boudsocq F, Woodgate R, Yang W . Crystal structure of a Y-family DNA polymerase in action: a mechanism for error-prone and lesion-bypass replication. Cell. 2001; 107(1):91-102. DOI: 10.1016/s0092-8674(01)00515-3. View

Kim S, Matsui K, Yamada M, Gruz P, Nohmi T . Roles of chromosomal and episomal dinB genes encoding DNA pol IV in targeted and untargeted mutagenesis in Escherichia coli. Mol Genet Genomics. 2001; 266(2):207-15. DOI: 10.1007/s004380100541. View

Hamdan S, Bulloch E, Thompson P, Beck J, Yang J, Crowther J . Hydrolysis of the 5'-p-nitrophenyl ester of TMP by the proofreading exonuclease (epsilon) subunit of Escherichia coli DNA polymerase III. Biochemistry. 2002; 41(16):5266-75. DOI: 10.1021/bi0159480. View

Sutton M, Narumi I, Walker G . Posttranslational modification of the umuD-encoded subunit of Escherichia coli DNA polymerase V regulates its interactions with the beta processivity clamp. Proc Natl Acad Sci U S A. 2002; 99(8):5307-12. PMC: 122765. DOI: 10.1073/pnas.082322099. View

Kannouche P, de Henestrosa A, Coull B, Vidal A, Gray C, Zicha D . Localization of DNA polymerases eta and iota to the replication machinery is tightly co-ordinated in human cells. EMBO J. 2002; 21(22):6246-56. PMC: 137208. View

10.

Becherel O, Fuchs R, Wagner J . Pivotal role of the beta-clamp in translesion DNA synthesis and mutagenesis in E. coli cells. DNA Repair (Amst). 2003; 1(9):703-8. DOI: 10.1016/s1568-7864(02)00106-4. View

11.

Oakley A, Prosselkov P, Wijffels G, Beck J, Wilce M, Dixon N . Flexibility revealed by the 1.85 A crystal structure of the beta sliding-clamp subunit of Escherichia coli DNA polymerase III. Acta Crystallogr D Biol Crystallogr. 2003; 59(Pt 7):1192-9. DOI: 10.1107/s0907444903009958. View

12.

Capson T, Peliska J, Kaboord B, Frey M, LIVELY C, Dahlberg M . Kinetic characterization of the polymerase and exonuclease activities of the gene 43 protein of bacteriophage T4. Biochemistry. 1992; 31(45):10984-94. DOI: 10.1021/bi00160a007. View

13.

Bunting K, Roe S, Pearl L . Structural basis for recruitment of translesion DNA polymerase Pol IV/DinB to the beta-clamp. EMBO J. 2003; 22(21):5883-92. PMC: 275425. DOI: 10.1093/emboj/cdg568. View

14.

Lopez de Saro F, Georgescu R, Goodman M, ODonnell M . Competitive processivity-clamp usage by DNA polymerases during DNA replication and repair. EMBO J. 2003; 22(23):6408-18. PMC: 291844. DOI: 10.1093/emboj/cdg603. View

15.

Wijffels G, Dalrymple B, Prosselkov P, Kongsuwan K, Epa V, Lilley P . Inhibition of protein interactions with the beta 2 sliding clamp of Escherichia coli DNA polymerase III by peptides from beta 2-binding proteins. Biochemistry. 2004; 43(19):5661-71. DOI: 10.1021/bi036229j. View

16.

Kannouche P, Wing J, Lehmann A . Interaction of human DNA polymerase eta with monoubiquitinated PCNA: a possible mechanism for the polymerase switch in response to DNA damage. Mol Cell. 2004; 14(4):491-500. DOI: 10.1016/s1097-2765(04)00259-x. View

17.

Boudsocq F, Kokoska R, Plosky B, Vaisman A, Ling H, Kunkel T . Investigating the role of the little finger domain of Y-family DNA polymerases in low fidelity synthesis and translesion replication. J Biol Chem. 2004; 279(31):32932-40. DOI: 10.1074/jbc.M405249200. View

18.

Duzen J, Walker G, Sutton M . Identification of specific amino acid residues in the E. coli beta processivity clamp involved in interactions with DNA polymerase III, UmuD and UmuD'. DNA Repair (Amst). 2004; 3(3):301-12. DOI: 10.1016/j.dnarep.2003.11.008. View

19.

Sutton M . The Escherichia coli dnaN159 mutant displays altered DNA polymerase usage and chronic SOS induction. J Bacteriol. 2004; 186(20):6738-48. PMC: 522196. DOI: 10.1128/JB.186.20.6738-6748.2004. View

20.

Sutton M, Duzen J, Maul R . Mutant forms of the Escherichia colibeta sliding clamp that distinguish between its roles in replication and DNA polymerase V-dependent translesion DNA synthesis. Mol Microbiol. 2005; 55(6):1751-66. DOI: 10.1111/j.1365-2958.2005.04500.x. View