A Genetic Selection for dinB Mutants Reveals an Interaction between DNA Polymerase IV and the Replicative Polymerase That Is Required for Translesion Synthesis.
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.
Department of Biochemistry, School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, New York, United States of America; Witebsky Center for Microbial Pathogenesis and Immunology, School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, New York, United States of America.
Michelle K
MK
Scotland
Department of Biochemistry, School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, New York, United States of America; Witebsky Center for Microbial Pathogenesis and Immunology, School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, New York, United States of America.
Justin M H
JM
Heltzel
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, United States of America.
James E
JE
Kath
Department of Chemistry, Cleveland State University, Cleveland, Ohio, United States of America.
Jung-Suk
JS
Choi
Department of Chemistry, Cleveland State University, Cleveland, Ohio, United States of America; The Center for Gene Regulation in Health and Disease, Cleveland State University, Cleveland, Ohio, United States of America.
Anthony J
AJ
Berdis
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, United States of America.
Joseph J
JJ
Loparo
Department of Biochemistry, School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, New York, United States of America; Witebsky Center for Microbial Pathogenesis and Immunology, School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, New York, United States of America; Program in Genetics, Genomics and Bioinformatics, School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, New York, United States of America.
Mark D
MD
Sutton
D020134:Catalytic Domain; D004249:DNA Damage; D019951:DNA Polymerase beta / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair; D004261:DNA Replication; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics*; D011485:Protein Binding; D012641:Selection, Genetic
C094399:DinB protein, E coli; D029968:Escherichia coli Proteins; D019951:DNA Polymerase beta