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Myron F Goodman - One of the best experts on this subject based on the ideXlab platform.
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Conformational regulation of Escherichia coli DNA Polymerase V by RecA and ATP
PLoS genetics, 2019Co-Authors: Malgorzata Jaszczur, Roger Woodgate, Jeffrey G. Bertram, Michael M. Cox, Ramunas Stanciauskas, Adhirath Sikand, Chi H. Mak, Fabien Pinaud, Myron F GoodmanAbstract:Mutagenic translesion DNA Polymerase V (UmuD′2C) is induced as part of the DNA damage-induced SOS response in Escherichia coli, and is subjected to multiple leVels of regulation. The UmuC subunit is sequestered on the cell membrane (spatial regulation) and enters the cytosol after forming a UmuD′2C complex, ~ 45 min post-SOS induction (temporal regulation). HoweVer, DNA binding and synthesis cannot occur until pol V interacts with a RecA nucleoprotein filament (RecA*) and ATP to form a mutasome complex, pol V Mut = UmuD′2C-RecA-ATP. The location of RecA relatiVe to UmuC determines whether pol V Mut is catalytically on or off (conformational regulation). Here, we present three interrelated experiments to address the biochemical basis of conformational regulation. We first inVestigate dynamic deactiVation during DNA synthesis and static deactiVation in the absence of DNA synthesis. Single-molecule (sm) TIRF-FRET microscopy is then used to explore multiple aspects of pol V mut dynamics. Binding of ATP/ATPγS triggers a conformational switch that reorients RecA relatiVe to UmuC to actiVate pol V Mut. This process is required for Polymerase-DNA binding and synthesis. Both dynamic and static deactiVation processes are goVerned by temperature and time, in which on → off switching is “rapid” at 37°C (~ 1 to 1.5 h), “slow” at 30°C (~ 3 to 4 h) and does not require ATP hydrolysis. Pol V Mut retains RecA in actiVated and deactiVated states, but binding to primer-template (p/t) DNA occurs only when actiVated. Studies are performed with two forms of the Polymerase, pol V Mut-RecA wt, and the constitutiVely induced and hypermutagenic pol V Mut-RecAE38K/ΔC17. We discuss conformational regulation of pol V Mut, determined from biochemical analysis in Vitro, in relation to the properties of pol V Mut in RecA wild-type and SOS constitutiVe genetic backgrounds in ViVo.
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Better liVing with hyper-mutation.
Environmental and molecular mutagenesis, 2016Co-Authors: Myron F GoodmanAbstract:The simplest forms of mutations, base substitutions, typically haVe negatiVe consequences, aside from their existential role in eVolution and fitness. Hypermutations, mutations on steroids, occurring at frequencies of 10(-2) -10(-4) per base pair, straddle a domain between fitness and death, depending on the presence or absence of regulatory constraints. Two facets of hypermutation, one in Escherichia coli inVolVing DNA Polymerase V (pol V), the other in humans, inVolVing actiVation-induced deoxycytidine deaminase (AID) are portrayed. Pol V is induced as part of the DNA-damage-induced SOS regulon, and is responsible for generating the lion's share of mutations when catalyzing translesion DNA synthesis (TLS). Four regulatory mechanisms, temporal, internal, conformational, and spatial, actiVate pol V to copy damaged DNA and then deactiVate it. On the flip side of the coin, SOS-induced pols V, IV, and II mutate undamaged DNA, thus proViding genetic diVersity heightening long-term surViVal and eVolutionary fitness. Fitness in humans is principally the domain of a remarkably Versatile immune system marked by somatic hypermutations (SHM) in immunoglobulin Variable (IgV) regions that ensure antibody (Ab) diVersity. AID initiates SHM by deaminating C → U, faVoring hot WRC (W = A/T, R = A/G) motifs. Since there are large numbers of trinucleotide motif targets throughout IgV, AID must exercise considerable catalytic restraint to aVoid attacking such sites repeatedly, which would otherwise compromise diVersity. ProcessiVe, random, and inefficient AID-catalyzed dC deamination simulates salient features of SHM, yet generates B-cell lymphomas when working at the wrong time in the wrong place. EnViron. Mol. Mutagen. 57:421-434, 2016. © 2016 Wiley Periodicals, Inc.
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Insights into the complex leVels of regulation imposed on Escherichia coli DNA Polymerase V.
DNA repair, 2016Co-Authors: Myron F Goodman, Malgorzata Jaszczur, John P. Mcdonald, Roger WoodgateAbstract:It is now close to 40 years since the isolation of non-mutable umu/uVm strains of Escherichia coli and the realization that damage induced mutagenesis in E.coli is not a passiVe process. Early models of mutagenesis enVisioned the Umu proteins as accessory factors to the cell's replicase that not only reduced its normally high fidelity, but also allowed the enzyme to traVerse otherwise replication-blocking lesions in the genome. HoweVer, these models underwent a radical reVision approximately 15 years ago, with the discoVery that the Umu proteins actually encode for a DNA Polymerase, E.coli pol V. The Polymerase lacks 3'→5' exonucleolytic proofreading actiVity and is inherently error-prone when replicating both undamaged and damage DNA. So as to limit any "gratuitous" mutagenesis, the actiVity of pol V is strictly regulated in the cell at multiple leVels. This reView will summarize our current understanding of the myriad leVels of regulation imposed on pol V including transcriptional control, posttranslational modification, targeted proteolysis, actiVation of the catalytic actiVity of pol V through protein-protein interactions and the Very recently described intracellular spatial regulation of pol V. Remarkably, despite the multiple leVels at which pol V is regulated, the enzyme is neVertheless able to contribute to the genetic diVersity and eVolutionary fitness of E.coli.
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Mutations for Worse or Better: Low-Fidelity DNA Synthesis by SOS DNA Polymerase V Is a Tightly Regulated Double-Edged Sword
Biochemistry, 2016Co-Authors: Malgorzata Jaszczur, Roger Woodgate, Jeffrey G. Bertram, Michael M. Cox, Andrew Robinson, Antoine M. Van Oijen, Myron F GoodmanAbstract:1953, the year of Watson and Crick, bore witness to a less acclaimed yet highly influential discoVery. Jean Weigle demonstrated that upon infection of Escherichia coli, λ phage deactiVated by UV radiation, and thus unable to form progeny, could be reactiVated by irradiation of the bacterial host. EVelyn Witkin and MiroslaV Radman later reVealed the presence of the SOS regulon. The more than 40 regulon genes are repressed by LexA protein and induced by the coproteolytic cleaVage of LexA, catalyzed by RecA protein bound to single-stranded DNA, the RecA* nucleoprotein filament. SeVeral SOS-induced proteins are engaged in repairing both cellular and extracellular damaged DNA. There's no "free lunch", howeVer, because error-free repair is accompanied by error-prone translesion DNA synthesis (TLS), inVolVing E. coli DNA Polymerase V (UmuD'2C) and RecA*. This reView describes the biochemical mechanisms of pol V-mediated TLS. pol V is actiVe only as a mutasomal complex, pol V Mut = UmuD'2C-RecA-ATP. RecA* donates a single RecA subunit to pol V. We highlight three recent insights. (1) pol V Mut has an intrinsic DNA-dependent ATPase actiVity that goVerns Polymerase binding and dissociation from DNA. (2) ActiVe and inactiVe states of pol V Mut are determined at least in part by the distinct interactions between RecA and UmuC. (3) pol V is actiVated by RecA*, not at a blocked replisome, but at the inner cell membrane.
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Regulation of Mutagenic DNA Polymerase V ActiVation in Space and Time.
PLoS genetics, 2015Co-Authors: Andrew Robinson, Roger Woodgate, Michael M. Cox, Meghna Patel, John P. Mcdonald, Victor E.a. Caldas, Elizabeth A. Wood, Christiaan M. Punter, Harshad Ghodke, Myron F GoodmanAbstract:Spatial regulation is often encountered as a component of multi-tiered regulatory systems in eukaryotes, where processes are readily segregated by organelle boundaries. Well-characterized examples of spatial regulation are less common in bacteria. Low-fidelity DNA Polymerase V (UmuD′2C) is produced in Escherichia coli as part of the bacterial SOS response to DNA damage. Due to the mutagenic potential of this enzyme, pol V actiVity is controlled by means of an elaborate regulatory system at transcriptional and posttranslational leVels. Using single-molecule fluorescence microscopy to Visualize UmuC inside liVing cells in space and time, we now show that pol V is also subject to a noVel form of spatial regulation. After an initial delay (~ 45 min) post UV irradiation, UmuC is synthesized, but is not immediately actiVated. Instead, it is sequestered at the inner cell membrane. The release of UmuC into the cytosol requires the RecA* nucleoprotein filament-mediated cleaVage of UmuD→UmuD′. Classic SOS damage response mutants either block [umuD(K97A)] or constitutiVely stimulate [recA(E38K)] UmuC release from the membrane. Foci of mutagenically actiVe pol V Mut (UmuD′2C-RecA-ATP) formed in the cytosol after UV irradiation do not co-localize with pol III replisomes, suggesting a capacity to promote translesion DNA synthesis at lesions skipped oVer by DNA Polymerase III. In effect, at least three molecular mechanisms limit the amount of time that pol V has to access DNA: (1) transcriptional and posttranslational regulation that initially keep the intracellular leVels of pol V to a minimum; (2) spatial regulation Via transient sequestration of UmuC at the membrane, which further delays pol V actiVation; and (3) the hydrolytic actiVity of a recently discoVered pol V Mut ATPase function that limits actiVe Polymerase time on the chromosomal template.
Roger Woodgate - One of the best experts on this subject based on the ideXlab platform.
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Conformational regulation of Escherichia coli DNA Polymerase V by RecA and ATP
PLoS genetics, 2019Co-Authors: Malgorzata Jaszczur, Roger Woodgate, Jeffrey G. Bertram, Michael M. Cox, Ramunas Stanciauskas, Adhirath Sikand, Chi H. Mak, Fabien Pinaud, Myron F GoodmanAbstract:Mutagenic translesion DNA Polymerase V (UmuD′2C) is induced as part of the DNA damage-induced SOS response in Escherichia coli, and is subjected to multiple leVels of regulation. The UmuC subunit is sequestered on the cell membrane (spatial regulation) and enters the cytosol after forming a UmuD′2C complex, ~ 45 min post-SOS induction (temporal regulation). HoweVer, DNA binding and synthesis cannot occur until pol V interacts with a RecA nucleoprotein filament (RecA*) and ATP to form a mutasome complex, pol V Mut = UmuD′2C-RecA-ATP. The location of RecA relatiVe to UmuC determines whether pol V Mut is catalytically on or off (conformational regulation). Here, we present three interrelated experiments to address the biochemical basis of conformational regulation. We first inVestigate dynamic deactiVation during DNA synthesis and static deactiVation in the absence of DNA synthesis. Single-molecule (sm) TIRF-FRET microscopy is then used to explore multiple aspects of pol V mut dynamics. Binding of ATP/ATPγS triggers a conformational switch that reorients RecA relatiVe to UmuC to actiVate pol V Mut. This process is required for Polymerase-DNA binding and synthesis. Both dynamic and static deactiVation processes are goVerned by temperature and time, in which on → off switching is “rapid” at 37°C (~ 1 to 1.5 h), “slow” at 30°C (~ 3 to 4 h) and does not require ATP hydrolysis. Pol V Mut retains RecA in actiVated and deactiVated states, but binding to primer-template (p/t) DNA occurs only when actiVated. Studies are performed with two forms of the Polymerase, pol V Mut-RecA wt, and the constitutiVely induced and hypermutagenic pol V Mut-RecAE38K/ΔC17. We discuss conformational regulation of pol V Mut, determined from biochemical analysis in Vitro, in relation to the properties of pol V Mut in RecA wild-type and SOS constitutiVe genetic backgrounds in ViVo.
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SetRICE391, a negatiVe transcriptional regulator of the integrating conjugatiVe element 391 mutagenic response.
DNA repair, 2018Co-Authors: Martín Gonzalez, John P. Mcdonald, Donald Huston, Mary P. Mclenigan, Audrey M. Garcia, Kylie S. Borden, Roger WoodgateAbstract:The integrating conjugatiVe element ICE391 (formerly known as IncJ R391) harbors an error-prone DNA Polymerase V ortholog, polVICE391, encoded by the ICE391 rumAB operon. polV and its orthologs haVe preViously been shown to be major contributors to spontaneous and DNA damage-induced mutagenesis in ViVo. As a result, multiple leVels of regulation are imposed on the Polymerases so as to aVoid aberrant mutagenesis. We report here, that the mutagenesis-promoting actiVity of polVICE391 is additionally regulated by a transcriptional repressor encoded by SetRICE391, since Escherichia coli expressing SetRICE391 demonstrated reduced leVels of polVICE391-mediated spontaneous mutagenesis relatiVe to cells lacking SetRICE391. SetRICE391 regulation was shown to be specific for the rumAB operon and in Vitro studies with highly purified SetRICE391 reVealed that under alkaline conditions, as well as in the presence of actiVated RecA, SetRICE391 undergoes a self-mediated cleaVage reaction that inactiVates repressor functions. ConVersely, a non-cleaVable SetRICE391 mutant capable of maintaining repressor actiVity, eVen in the presence of actiVated RecA, exhibited low leVels of polVICE391-dependent mutagenesis. Electrophoretic mobility shift assays reVealed that SetRICE391 acts as a transcriptional repressor by binding to a site oVerlapping the -35 region of the rumAB operon promoter. Our study therefore proVides eVidence indicating that SetRICE391 acts as a transcriptional repressor of the ICE391-encoded mutagenic response.
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Insights into the complex leVels of regulation imposed on Escherichia coli DNA Polymerase V.
DNA repair, 2016Co-Authors: Myron F Goodman, Malgorzata Jaszczur, John P. Mcdonald, Roger WoodgateAbstract:It is now close to 40 years since the isolation of non-mutable umu/uVm strains of Escherichia coli and the realization that damage induced mutagenesis in E.coli is not a passiVe process. Early models of mutagenesis enVisioned the Umu proteins as accessory factors to the cell's replicase that not only reduced its normally high fidelity, but also allowed the enzyme to traVerse otherwise replication-blocking lesions in the genome. HoweVer, these models underwent a radical reVision approximately 15 years ago, with the discoVery that the Umu proteins actually encode for a DNA Polymerase, E.coli pol V. The Polymerase lacks 3'→5' exonucleolytic proofreading actiVity and is inherently error-prone when replicating both undamaged and damage DNA. So as to limit any "gratuitous" mutagenesis, the actiVity of pol V is strictly regulated in the cell at multiple leVels. This reView will summarize our current understanding of the myriad leVels of regulation imposed on pol V including transcriptional control, posttranslational modification, targeted proteolysis, actiVation of the catalytic actiVity of pol V through protein-protein interactions and the Very recently described intracellular spatial regulation of pol V. Remarkably, despite the multiple leVels at which pol V is regulated, the enzyme is neVertheless able to contribute to the genetic diVersity and eVolutionary fitness of E.coli.
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Mutations for Worse or Better: Low-Fidelity DNA Synthesis by SOS DNA Polymerase V Is a Tightly Regulated Double-Edged Sword
Biochemistry, 2016Co-Authors: Malgorzata Jaszczur, Roger Woodgate, Jeffrey G. Bertram, Michael M. Cox, Andrew Robinson, Antoine M. Van Oijen, Myron F GoodmanAbstract:1953, the year of Watson and Crick, bore witness to a less acclaimed yet highly influential discoVery. Jean Weigle demonstrated that upon infection of Escherichia coli, λ phage deactiVated by UV radiation, and thus unable to form progeny, could be reactiVated by irradiation of the bacterial host. EVelyn Witkin and MiroslaV Radman later reVealed the presence of the SOS regulon. The more than 40 regulon genes are repressed by LexA protein and induced by the coproteolytic cleaVage of LexA, catalyzed by RecA protein bound to single-stranded DNA, the RecA* nucleoprotein filament. SeVeral SOS-induced proteins are engaged in repairing both cellular and extracellular damaged DNA. There's no "free lunch", howeVer, because error-free repair is accompanied by error-prone translesion DNA synthesis (TLS), inVolVing E. coli DNA Polymerase V (UmuD'2C) and RecA*. This reView describes the biochemical mechanisms of pol V-mediated TLS. pol V is actiVe only as a mutasomal complex, pol V Mut = UmuD'2C-RecA-ATP. RecA* donates a single RecA subunit to pol V. We highlight three recent insights. (1) pol V Mut has an intrinsic DNA-dependent ATPase actiVity that goVerns Polymerase binding and dissociation from DNA. (2) ActiVe and inactiVe states of pol V Mut are determined at least in part by the distinct interactions between RecA and UmuC. (3) pol V is actiVated by RecA*, not at a blocked replisome, but at the inner cell membrane.
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Regulation of Mutagenic DNA Polymerase V ActiVation in Space and Time.
PLoS genetics, 2015Co-Authors: Andrew Robinson, Roger Woodgate, Michael M. Cox, Meghna Patel, John P. Mcdonald, Victor E.a. Caldas, Elizabeth A. Wood, Christiaan M. Punter, Harshad Ghodke, Myron F GoodmanAbstract:Spatial regulation is often encountered as a component of multi-tiered regulatory systems in eukaryotes, where processes are readily segregated by organelle boundaries. Well-characterized examples of spatial regulation are less common in bacteria. Low-fidelity DNA Polymerase V (UmuD′2C) is produced in Escherichia coli as part of the bacterial SOS response to DNA damage. Due to the mutagenic potential of this enzyme, pol V actiVity is controlled by means of an elaborate regulatory system at transcriptional and posttranslational leVels. Using single-molecule fluorescence microscopy to Visualize UmuC inside liVing cells in space and time, we now show that pol V is also subject to a noVel form of spatial regulation. After an initial delay (~ 45 min) post UV irradiation, UmuC is synthesized, but is not immediately actiVated. Instead, it is sequestered at the inner cell membrane. The release of UmuC into the cytosol requires the RecA* nucleoprotein filament-mediated cleaVage of UmuD→UmuD′. Classic SOS damage response mutants either block [umuD(K97A)] or constitutiVely stimulate [recA(E38K)] UmuC release from the membrane. Foci of mutagenically actiVe pol V Mut (UmuD′2C-RecA-ATP) formed in the cytosol after UV irradiation do not co-localize with pol III replisomes, suggesting a capacity to promote translesion DNA synthesis at lesions skipped oVer by DNA Polymerase III. In effect, at least three molecular mechanisms limit the amount of time that pol V has to access DNA: (1) transcriptional and posttranslational regulation that initially keep the intracellular leVels of pol V to a minimum; (2) spatial regulation Via transient sequestration of UmuC at the membrane, which further delays pol V actiVation; and (3) the hydrolytic actiVity of a recently discoVered pol V Mut ATPase function that limits actiVe Polymerase time on the chromosomal template.
Zvi Livneh - One of the best experts on this subject based on the ideXlab platform.
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Single-stranded DNA-binding protein recruits DNA Polymerase V to primer termini on RecA-coated DNA.
The Journal of biological chemistry, 2008Co-Authors: Gali Arad, Ayal Hendel, Claus Urbanke, Ute Curth, Zvi LivnehAbstract:Translesion DNA synthesis (TLS) by DNA Polymerase V (polV) in Escherichia coli inVolVes accessory proteins, including RecA and single-stranded DNA-binding protein (SSB). To elucidate the role of SSB in TLS we used an in Vitro exonuclease protection assay and found that SSB increases the accessibility of 3' primer termini located at abasic sites in RecA-coated gapped DNA. The mutant SSB-113 protein, which is defectiVe in protein-protein interactions, but not in DNA binding, was as effectiVe as wild-type SSB in increasing primer termini accessibility, but deficient in supporting polV-catalyzed TLS. Consistently, the heterologous SSB proteins gp32, encoded by phage T4, and ICP8, encoded by herpes simplex Virus 1, could replace E. coli SSB in the TLS reaction, albeit with lower efficiency. Immunoprecipitation experiments indicated that polV directly interacts with SSB and that this interaction is disrupted by the SSB-113 mutation. Taken together our results suggest that SSB functions to recruit polV to primer termini on RecA-coated DNA, operating by two mechanisms: 1) increasing the accessibility of 3' primer termini caused by binding of SSB to DNA and 2) a direct SSB-polV interaction mediated by the C terminus of SSB.
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Lesion bypass DNA Polymerases replicate across non-DNA segments
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Ayelet Maor-shoshani, Vered Ben-ari, Zvi LivnehAbstract:A critical feature of the robustness of the DNA replication machinery is the ability to complete its task in the presence of interfering DNA damage. A key mechanism responsible for this task is translesion replication (also termed translesion synthesis), carried out by specialized lesion bypass DNA Polymerases of the Y superfamily. Here we show that in Escherichia coli, plasmids can be replicated across a segment of foreign non-DNA material, consisting of hydrocarbon chains of 3 or 12 methylene residues. This replication is carried out by DNA Polymerase V and proceeds by at least two mechanisms: (i) Editing out the foreign insert, by Polymerase "hopping" across it, which can be mediated by looping out of the insert, leading to its deletion, while preserVing the DNA sequence. (ii) DNA synthesis through the insert, which occurs by incorporating one or two nucleotides opposite the hydrocarbon chain, yielding a net increase in the length of the DNA sequence. The remarkable ability of DNA Polymerase V to insert nucleotides opposite a hydrocarbon chain shows that DNA synthesis can occur in a region of the template strand, which lacks all fundamental features of DNA, including its purine, pyrimidine, sugar, and phosphate moieties, and its hydrophilic and ionic nature. This bypass ability reflects a striking robustness of the translesion replication apparatus and is likely to contribute to its effectiVeness in maintaining genome stability.
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Analysis of the stimulation of DNA Polymerase V of Escherichia coli by processiVity proteins.
Biochemistry, 2002Co-Authors: Ayelet Maor-shoshani, Zvi LivnehAbstract:Bypass of replication-blocking lesions in Escherichia coli is carried out by DNA Polymerase V (UmuC) in a reaction that requires UmuD', RecA, and single-strand DNA-binding protein (SSB). The actiVity of this four-component basic bypass system is a low-fidelity and low-processiVity actiVity. Addition of the processiVity subunits of pol III, the beta subunit sliding DNA clamp, and the fiVe-subunit gamma complex clamp loader increased the rate of translesion replication approximately 3-fold. This stimulation was specific to the lesion bypass step, with no effect on the initiation of synthesis by pol V. The beta subunit and gamma complex increased the processiVity of pol V from 3 to approximately 14-18 nucleotides, proViding a mechanistic basis for their stimulatory effect. Stimulation of bypass was obserVed oVer a range of RecA and SSB concentrations. ATPgammaS, which strongly inhibits translesion replication by pol V, primarily Via inhibition of the initiation stage, caused the same inhibition also in the presence of the processiVity proteins. The in ViVo role of the processiVity proteins in translesion replication was examined by assaying UV mutagenesis. This was done in a strain carrying the DNAN59 allele, encoding a temperature-sensitiVe beta subunit. When assayed in an excision repair-defectiVe background, the DNAN59 mutant exhibited a leVel of UV mutagenesis reduced up to 3-fold compared to that of the isogenic DNAN(+) strain. This suggests that like in the in Vitro system, the beta subunit stimulates lesion bypass in ViVo.
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Lesion Bypass by the Escherichia coli DNA Polymerase V Requires Assembly of a RecA Nucleoprotein Filament
The Journal of biological chemistry, 2000Co-Authors: Nina Reuven, Gali Arad, Alicja Z. Stasiak, Zvi LivnehAbstract:Abstract Translesion replication is carried out inEscherichia coli by the SOS-inducible DNA Polymerase V (UmuC), an error-prone Polymerase, which is specialized for replicating through lesions in DNA, leading to the formation of mutations. Lesion bypass by pol V requires the SOS-regulated proteins UmuD′ and RecA and the single-strand DNA-binding protein (SSB). Using an in Vitro assay system for translesion replication based on a gapped plasmid carrying a site-specific synthetic abasic site, we show that the assembly of a RecA nucleoprotein filament is required for lesion bypass by pol V. This is based on the reaction requirements for stoichiometric amounts of RecA and for single-stranded gaps longer than 100 nucleotides and on direct Visualization of RecA-DNA filaments by electron microscopy. SSB is likely to facilitate the assembly of the RecA nucleoprotein filament; howeVer, it has at least one additional role in lesion bypass. ATPγS, which is known to strongly increase binding of RecA to DNA, caused a drastic inhibition of pol V actiVity. Lesion bypass does not require stoichiometric binding of UmuD′ along RecA filaments. In summary, the RecA nucleoprotein filament, preViously known to be required for SOS induction and homologous recombination, is also a critical intermediate in translesion replication.
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plasmid encoded mucb protein is a DNA Polymerase pol ri specialized for lesion bypass in the presence of muca reca and ssb
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Moshe Goldsmith, Lea Sarovblat, Zvi LivnehAbstract:Replication through damaged sites in DNA requires in Escherichia coli the SOS stress-inducible DNA Polymerase V (UmuC), which is specialized for lesion bypass. Homologs of the umuC gene were found on natiVe conjugatiVe plasmids, which often carry multiple antibiotic-resistant genes. MucB is a UmuC homolog present on plasmid R46, and its Variant plasmid pKM101 has been introduced into Salmonella strains for use in the Ames test for mutagens. Using a translesion replication assay based on a gapped plasmid carrying a site-specific synthetic abasic site in the single-stranded DNA region, we show that MucB is a DNA Polymerase, termed pol RI, which is specialized for lesion bypass. The actiVity of pol RI requires the plasmid-encoded MucA′ protein and the E. coli RecA and single-strand DNA binding proteins. Elimination of any of the proteins from the reaction abolished lesion bypass and Polymerase actiVity. The unprocessed MucA could not substitute for MucA′ in the bypass reaction. The presence of a lesion bypass DNA Polymerase on a natiVe conjugatiVe plasmid, which has a broad host range specificity and carries multiple antibiotic-resistant genes, raises the possibility that mutagenesis caused by pol RI plays a role in the spreading of antibiotic resistance among bacterial pathogens.
Graham C. Walker - One of the best experts on this subject based on the ideXlab platform.
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DNA Polymerase V allows bypass of toxic guanine oxidation products in ViVo.
The Journal of biological chemistry, 2007Co-Authors: William L. Neeley, Sarah Delaney, Yuriy O. Alekseyev, Daniel F. Jarosz, James C. Delaney, Graham C. Walker, John M. EssigmannAbstract:ReactiVe oxygen and nitrogen radicals produced during metabolic processes, such as respiration and inflammation, combine with DNA to form many lesions primarily at guanine sites. Understanding the roles of the Polymerases responsible for the processing of these products to mutations could illuminate molecular mechanisms that correlate oxidatiVe stress with cancer. Using M13 Viral genomes engineered to contain single DNA lesions and Escherichia coli strains with specific Polymerase (pol) knockouts, we show that pol V is required for efficient bypass of structurally diVerse, highly mutagenic guanine oxidation products in ViVo. We also find that pol IV participates in the bypass of two spiroiminodihydantoin lesions. Furthermore, we report that one lesion, 5-guanidino-4-nitroimidazole, is a substrate for multiple SOS Polymerases, whereby pol II is necessary for error-free replication and pol V for error-prone replication past this lesion. The results spotlight a major role for pol V and minor roles for pol II and pol IV in the mechanism of guanine oxidation mutagenesis.
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A single amino acid goVerns enhanced actiVity of DinB DNA Polymerases on damaged templates
Nature, 2006Co-Authors: Daniel F. Jarosz, James C. Delaney, John M. Essigmann, Veronica G. Godoy, Graham C. WalkerAbstract:Translesion synthesis (TLS) by Y-family DNA Polymerases is a chief mechanism of DNA damage tolerance^ 1 . Such TLS can be accurate or error-prone, as it is for bypass of a cyclobutane pyrimidine dimer by DNA Polymerase η (XP-V or Rad30) or bypass of a (6-4) TT photoproduct by DNA Polymerase V (UmuD′_2C), respectiVely^ 2 , 3 . Although DinB is the only Y-family DNA Polymerase conserVed among all domains of life, the biological rationale for this striking conserVation has remained enigmatic^ 4 . Here we report that the Escherichia coli dinB gene is required for resistance to some DNA-damaging agents that form adducts at the N ^2-position of deoxyguanosine (dG). We show that DinB (DNA Polymerase IV) catalyses accurate TLS oVer one such N ^2-dG adduct ( N ^2-furfuryl-dG), and that DinB and its mammalian orthologue, DNA Polymerase κ, insert deoxycytidine (dC) opposite N ^2-furfuryl-dG with 10–15-fold greater catalytic proficiency than opposite undamaged dG. We also show that mutating a single amino acid, the ‘steric gate’ residue of DinB (Phe13 → Val) and that of its archaeal homologue Dbh (Phe12 → Ala), separates the abilities of these enzymes to perform TLS oVer N ^2-dG adducts from their abilities to replicate an undamaged template. We propose that DinB and its orthologues are specialized to catalyse relatiVely accurate TLS oVer some N ^2-dG adducts that are ubiquitous in nature, that lesion bypass occurs more efficiently than synthesis on undamaged DNA, and that this specificity may be achieVed at least in part through a lesion-induced conformational change.
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Posttranslational modification of the umuD-encoded subunit of Escherichia coli DNA Polymerase V regulates its interactions with the β processiVity clamp
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Mark Sutton, Issay Narumi, Graham C. WalkerAbstract:The Escherichia coli umuDC (pol V) gene products participate in both a DNA damage checkpoint control and translesion DNA synthesis. Interactions of the two umuD gene products, the 139-aa UmuD and the 115-aa UmuD′ proteins, with components of the replicatiVe DNA Polymerase (pol III), are important for determining which biological role the umuDC gene products will play. Here we report our biochemical characterizations of the interactions of UmuD and UmuD′ with the pol III β processiVity clamp. These analyses demonstrate that UmuD possesses a higher affinity for β than does UmuD′ because of the N-terminal arm of UmuD (residues 1–39), much of which is missing in UmuD′. Furthermore, we haVe identified specific amino acid residues of UmuD that crosslink to β with p-azidoiodoacetanilide, defining the domain of UmuD important for the interaction. We haVe recently proposed a model for the solution structure of UmuD2 in which the N-terminal arm of each protomer makes extensiVe contacts with the C-terminal globular domain of its intradimer partner, masking part of each surface. Taken together, our findings suggest that UmuD2 has a higher affinity for the β-clamp than does UmuD′2 because of the structures of its N-terminal arms. Viewed in this way, posttranslational modification of UmuD, which entails the remoVal of its N-terminal 24 residues to yield UmuD′, acts in part to attenuate the affinity of the umuD gene product for the β-clamp. Implications of these structure–function analyses for the checkpoint and translesion DNA synthesis functions of the umuDC gene products are discussed.
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ConVerting a DNA damage checkpoint effector (UmuD2C) into a lesion bypass Polymerase (UmuD'2C).
The EMBO journal, 2001Co-Authors: Ann E. Ferentz, Graham C. Walker, Gerhard WagnerAbstract:During the SOS response of Escherichia coli to DNA damage, the umuDC operon is induced, producing the trimeric protein complexes UmuD2C, a DNA damage checkpoint effector, and UmuD′2C (DNA Polymerase V), which carries out translesion synthesis, the basis of ‘SOS mutagenesis’. UmuD′2, the homodimeric component of DNA pol V, is produced from UmuD by RecA‐facilitated self‐cleaVage, which remoVes the 24 N‐terminal residues of UmuD. We report the solution structure of UmuD′2 (PDB ID 1I4V) and interactions within UmuD′–UmuD, a heterodimer inactiVe in translesion synthesis. The oVerall shape of UmuD′2 in solution differs substantially from the preViously reported crystal structure, eVen though the topologies of the two structures are quite similar. Most significantly, the actiVe site residues S60 and K97 do not point directly at one another in solution as they do in the crystal, suggesting that self‐cleaVage of UmuD might require RecA to assemble the actiVe site. Structural differences between UmuD′2 and UmuD′–UmuD suggest that UmuD′2C and UmuD2C might achieVe their different biological actiVities through distinct interactions with RecA and DNA pol III.
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Genetic Interactions between the Escherichia coli umuDC Gene Products and the β ProcessiVity Clamp of the ReplicatiVe DNA Polymerase
Journal of bacteriology, 2001Co-Authors: Mark Sutton, Mary F. Farrow, Briana M. Burton, Graham C. WalkerAbstract:The umuDC genes encode a DNA Polymerase, DNA Polymerase V (Pol V), that has a remarkable ability to copy oVer abasic sites (43, 63), cyclobutane dimers (62), and pyrimidine-pyrimidone [6-4] photoproducts (62), a process referred to as translesion DNA synthesis (TLS). This ability, howeVer, comes at the cost of reduced fidelity. Thus, replication by Pol V is inherently less accurate, leading to the formation of mutations, eVen when replicating undamaged templates (29, 62). Therefore, to limit the ability of Pol V to introduce mutations into the host genome, expression of the umuDC genes is tightly regulated as part of the Escherichia coli stress-induced SOS response (12). This highly regulated response helps the cell maintain the integrity of its genome following treatments that lead either directly or indirectly to DNA damage (12). The E. coli SOS response consists of at least 30 unlinked genes (9, 12), collectiVely referred to as the SOS regulon. Expression of the Various SOS-regulated genes is coordinately regulated at the leVel of their transcription by the LexA and RecA proteins (28). In the absence of DNA damage, LexA acts to repress the expression of the members of the SOS regulon (27). RecA protein, the main bacterial recombinase required for essentially all homologous recombination (reViewed in reference 22), binds to single-stranded DNA (ssDNA) generated by the cell's failed attempts to replicate past lesions in its genome, thus forming RecA-ssDNA nucleoprotein filaments (47). These RecA-ssDNA filaments, in addition to acting in homologous recombination, also act to facilitate the latent capacity of LexA to autodigest (26). Autodigestion of LexA serVes to inactiVate it as a transcriptional repressor, leading to the concomitant increase in expression of LexA-regulated genes (12). The UmuD protein similarly undergoes a RecA-ssDNA-facilitated autodigestion that serVes to remoVe its first 24 residues to yield UmuD′ (3, 34, 48). The UmuD′2 homodimer then interacts with UmuC (18, 59, 68), which has an ability to catalyze the formation of phosphodiester bonds, in such a way that the UmuD′2C complex is able to participate in TLS (43). In addition to participating in TLS, UmuC together with the full-length UmuD2 homodimer participates in a DNA damage checkpoint control that acts to regulate DNA replication in response to DNA damage, thereby allowing additional time for nucleotide excision repair to accurately remoVe lesions in the DNA prior to continued replication (37). Thus, self-cleaVage of UmuD to UmuD′ can be regarded as a molecular switch that acts to temporally regulate these two distinct actiVities of the UmuD2C and UmuD′2C complexes (37, 57). In addition to participating in a DNA damage checkpoint control and enabling TLS, oVerexpression of the umuDC gene products confers a cold sensitiVity for growth (30, 38, 59). Our recent characterizations of umuDC-mediated cold sensitiVity indicated that (i) moderately eleVated leVels of the umuDC gene products confer a cold-sensitiVe growth phenotype, while similarly eleVated leVels of the umuD′C gene products do not (59), and (ii) the catalytic DNA Polymerase actiVity of UmuC is not required for this cold sensitiVity (59). These findings, together with others, suggest that the cold sensitiVity conferred by eleVated leVels of the umuDC gene products is a manifestation of the inappropriate expression of UmuD2C functions inVolVed in the DNA damage checkpoint control (37, 38, 59). Therefore, in an effort to better characterize the components of the UmuD2C-dependent checkpoint control, we haVe embarked on an analysis of the genetic requirements of umuDC-mediated cold sensitiVity. We haVe preViously suggested that interactions of the umuDC gene products with components of the E. coli replicatiVe DNA Polymerase, DNA Polymerase III holoenzyme (Pol III), could serVe as a conVenient mechanism for regulating the checkpoint and TLS roles of the umuDC gene products (37, 57). On the basis of this hypothesis, we reasoned that if interactions inVolVing the umuDC gene products and components of Pol III were important for the checkpoint role of UmuD2C, we might then be able to obserVe an effect on the extent of the cold sensitiVity conferred by umuDC by the simultaneous oVerexpression of certain (i.e., releVant) components of Pol III. Using such an approach, we haVe recently reported that oVerproduction of the ɛ proofreading subunit of Pol III or deletion of its structural gene (DNAQ) suppresses umuDC-mediated cold sensitiVity (56). A systematic analysis of the remaining nine Pol III subunits indicated that the homodimeric β processiVity clamp was the only other Pol III subunit that when oVerexpressed affected the extent of umuDC-mediated cold sensitiVity (56). In this report, we describe how oVerexpression of the β processiVity clamp (encoded by DNAN) strongly exacerbates umuDC-mediated cold sensitiVity. We haVe exploited this ability of the β clamp to confer a cold-sensitiVe growth phenotype upon a umuD′C-expressing E. coli strain (a strain that is not normally cold sensitiVe [59]) to identify noVel DNAN alleles unable to confer this phenotype. Our genetic characterizations of these noVel DNAN alleles indicate that they are also unable to exacerbate the cold sensitiVity conferred by eleVated leVels of the umuDC gene products. Our results described in this report, taken together with others (37, 38, 56, 57, 59), suggest that the UmuD2C-dependent DNA damage checkpoint control is a manifestation of protein-protein interactions inVolVing UmuD2C and the ɛ and the β subunits of Pol III.
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A DNAN Plasmid Shuffle Strain for Rapid In ViVo Analysis of Mutant Escherichia coli β Clamps ProVides Insight Into the Role of Clamp in umuDC-Mediated Cold SensitiVity
PloS one, 2014Co-Authors: Vignesh M. P. Babu, Mark SuttonAbstract:The E. coli umuDC gene products participate in two temporally distinct roles: UmuD2C acts in a DNA damage checkpoint control, while UmuD'2C, also known as DNA Polymerase V (Pol V), catalyzes replication past DNA lesions Via a process termed translesion DNA synthesis. These different roles of the umuDC gene products are managed in part by the DNAN-encoded β sliding clamp protein. Co-oVerexpression of the β clamp and Pol V seVerely blocked E. coli growth at 30°C. We preViously used a genetic assay that was independent of the ability of β clamp to support E. coli Viability to isolate 8 mutant clamp proteins (βQ61K, βS107L, βD150N, βG157S, βV170M, βE202K, βM204K and βP363S) that failed to block growth at 30°C when co-oVerexpressed with Pol V. It was unknown whether these mutant clamps were capable of supporting E. coli Viability and normal umuDC functions in ViVo. The goals of this study were to answer these questions. To this end, we deVeloped a noVel DNAN plasmid shuffle assay. Using this assay, βD150N and βP363S were unable to support E. coli Viability. The remaining 6 mutant clamps, each of which supported Viability, were indistinguishable from β+ with respect to umuDC functions in ViVo. In light of these findings, we analyzed phenotypes of strains oVerexpressing either β clamp or Pol V alone. The strain oVerexpressing β+, but not those expressing mutant β clamps, displayed slowed growth irrespectiVe of the incubation temperature. MoreoVer, growth of the Pol V-expressing strain was modestly slowed at 30°, but not 42°C. Taken together, these results suggest the mutant clamps were identified due to their inability to slow growth rather than an inability to interact with Pol V. They further suggest that cold sensitiVity is due, at least in part, to the combination of their indiVidual effects on growth at 30°C.
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Roles of the Escherichia coli RecA Protein and the Global SOS Response in Effecting DNA Polymerase Selection In ViVo
Journal of bacteriology, 2005Co-Authors: Robert W. Maul, Mark SuttonAbstract:The Escherichia coli β sliding clamp protein is proposed to play an important role in effecting switches between different DNA Polymerases during replication, repair, and translesion DNA synthesis. We recently described how strains bearing the DNAN159 allele, which encodes a mutant form of the β clamp (β159), display a UV-sensitiVe phenotype that is suppressed by inactiVation of DNA Polymerase IV (M. D. Sutton, J. Bacteriol. 186:6738-6748, 2004). As part of an ongoing effort to understand mechanisms of DNA Polymerase management in E. coli, we haVe further characterized effects of the DNAN159 allele on Polymerase usage. Three of the fiVe E.coli DNA Polymerases (II, IV, and V) are regulated as part of the global SOS response. Our results indicate that eleVated expression of the dinB-encoded Polymerase IV is sufficient to result in conditional lethality of the DNAN159 strain. In contrast, chronically actiVated RecA protein, expressed from the recA730 allele, is lethal to the DNAN159 strain, and this lethality is suppressed by mutations that either mitigate RecA730 actiVity (i.e., ΔrecR), or impair the actiVities of DNA Polymerase II or DNA Polymerase V (i.e., ΔpolB or ΔumuDC). Thus, we haVe identified distinct genetic requirements whereby each of the three different SOS-regulated DNA Polymerases are able to confer lethality upon the DNAN159 strain, suggesting the presence of multiple mechanisms by which the actions of the cell's different DNA Polymerases are managed in ViVo.
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Mutant forms of the Escherichia coliβ sliding clamp that distinguish between its roles in replication and DNA Polymerase V‐dependent translesion DNA synthesis
Molecular microbiology, 2005Co-Authors: Mark Sutton, Jill M. Duzen, Robert W. MaulAbstract:The Escherichia colibeta sliding clamp is proposed to play an important role in regulating DNA Polymerase traffic at the replication fork. As part of an ongoing effort to understand how organisms manage the actions of their multiple DNA Polymerases, we examined the ability of seVeral mutant forms of the beta clamp to function in DNA Polymerase V- (pol V-) dependent translesion DNA synthesis (TLS) in ViVo. Our results indicate that a DNAN159 strain, which expresses a temperature sensitiVe form of the beta clamp, was impaired for pol V-dependent TLS at the permissiVe temperature of 37 degrees C. This defect was complemented by a plasmid that expressed near-physiological leVels of the wild-type clamp. Using a DNAN159 mutant strain, together with Various plasmids expressing mutant forms of the clamp, we determined that residues H148 through R152, which comprise a portion of a solVent exposed loop, as well as position P363, which is located in the C-terminal tail of the beta clamp, are critically important for pol V-dependent TLS in ViVo. In contrast, these same residues appear to be less critical for pol III-dependent replication. Taken together, these findings indicate that: (i) the beta clamp plays an essential role in pol V-dependent TLS in ViVo and (ii) pol III and pol V interact with non-identical surfaces of the beta clamp.
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Posttranslational modification of the umuD-encoded subunit of Escherichia coli DNA Polymerase V regulates its interactions with the β processiVity clamp
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Mark Sutton, Issay Narumi, Graham C. WalkerAbstract:The Escherichia coli umuDC (pol V) gene products participate in both a DNA damage checkpoint control and translesion DNA synthesis. Interactions of the two umuD gene products, the 139-aa UmuD and the 115-aa UmuD′ proteins, with components of the replicatiVe DNA Polymerase (pol III), are important for determining which biological role the umuDC gene products will play. Here we report our biochemical characterizations of the interactions of UmuD and UmuD′ with the pol III β processiVity clamp. These analyses demonstrate that UmuD possesses a higher affinity for β than does UmuD′ because of the N-terminal arm of UmuD (residues 1–39), much of which is missing in UmuD′. Furthermore, we haVe identified specific amino acid residues of UmuD that crosslink to β with p-azidoiodoacetanilide, defining the domain of UmuD important for the interaction. We haVe recently proposed a model for the solution structure of UmuD2 in which the N-terminal arm of each protomer makes extensiVe contacts with the C-terminal globular domain of its intradimer partner, masking part of each surface. Taken together, our findings suggest that UmuD2 has a higher affinity for the β-clamp than does UmuD′2 because of the structures of its N-terminal arms. Viewed in this way, posttranslational modification of UmuD, which entails the remoVal of its N-terminal 24 residues to yield UmuD′, acts in part to attenuate the affinity of the umuD gene product for the β-clamp. Implications of these structure–function analyses for the checkpoint and translesion DNA synthesis functions of the umuDC gene products are discussed.
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Genetic Interactions between the Escherichia coli umuDC Gene Products and the β ProcessiVity Clamp of the ReplicatiVe DNA Polymerase
Journal of bacteriology, 2001Co-Authors: Mark Sutton, Mary F. Farrow, Briana M. Burton, Graham C. WalkerAbstract:The umuDC genes encode a DNA Polymerase, DNA Polymerase V (Pol V), that has a remarkable ability to copy oVer abasic sites (43, 63), cyclobutane dimers (62), and pyrimidine-pyrimidone [6-4] photoproducts (62), a process referred to as translesion DNA synthesis (TLS). This ability, howeVer, comes at the cost of reduced fidelity. Thus, replication by Pol V is inherently less accurate, leading to the formation of mutations, eVen when replicating undamaged templates (29, 62). Therefore, to limit the ability of Pol V to introduce mutations into the host genome, expression of the umuDC genes is tightly regulated as part of the Escherichia coli stress-induced SOS response (12). This highly regulated response helps the cell maintain the integrity of its genome following treatments that lead either directly or indirectly to DNA damage (12). The E. coli SOS response consists of at least 30 unlinked genes (9, 12), collectiVely referred to as the SOS regulon. Expression of the Various SOS-regulated genes is coordinately regulated at the leVel of their transcription by the LexA and RecA proteins (28). In the absence of DNA damage, LexA acts to repress the expression of the members of the SOS regulon (27). RecA protein, the main bacterial recombinase required for essentially all homologous recombination (reViewed in reference 22), binds to single-stranded DNA (ssDNA) generated by the cell's failed attempts to replicate past lesions in its genome, thus forming RecA-ssDNA nucleoprotein filaments (47). These RecA-ssDNA filaments, in addition to acting in homologous recombination, also act to facilitate the latent capacity of LexA to autodigest (26). Autodigestion of LexA serVes to inactiVate it as a transcriptional repressor, leading to the concomitant increase in expression of LexA-regulated genes (12). The UmuD protein similarly undergoes a RecA-ssDNA-facilitated autodigestion that serVes to remoVe its first 24 residues to yield UmuD′ (3, 34, 48). The UmuD′2 homodimer then interacts with UmuC (18, 59, 68), which has an ability to catalyze the formation of phosphodiester bonds, in such a way that the UmuD′2C complex is able to participate in TLS (43). In addition to participating in TLS, UmuC together with the full-length UmuD2 homodimer participates in a DNA damage checkpoint control that acts to regulate DNA replication in response to DNA damage, thereby allowing additional time for nucleotide excision repair to accurately remoVe lesions in the DNA prior to continued replication (37). Thus, self-cleaVage of UmuD to UmuD′ can be regarded as a molecular switch that acts to temporally regulate these two distinct actiVities of the UmuD2C and UmuD′2C complexes (37, 57). In addition to participating in a DNA damage checkpoint control and enabling TLS, oVerexpression of the umuDC gene products confers a cold sensitiVity for growth (30, 38, 59). Our recent characterizations of umuDC-mediated cold sensitiVity indicated that (i) moderately eleVated leVels of the umuDC gene products confer a cold-sensitiVe growth phenotype, while similarly eleVated leVels of the umuD′C gene products do not (59), and (ii) the catalytic DNA Polymerase actiVity of UmuC is not required for this cold sensitiVity (59). These findings, together with others, suggest that the cold sensitiVity conferred by eleVated leVels of the umuDC gene products is a manifestation of the inappropriate expression of UmuD2C functions inVolVed in the DNA damage checkpoint control (37, 38, 59). Therefore, in an effort to better characterize the components of the UmuD2C-dependent checkpoint control, we haVe embarked on an analysis of the genetic requirements of umuDC-mediated cold sensitiVity. We haVe preViously suggested that interactions of the umuDC gene products with components of the E. coli replicatiVe DNA Polymerase, DNA Polymerase III holoenzyme (Pol III), could serVe as a conVenient mechanism for regulating the checkpoint and TLS roles of the umuDC gene products (37, 57). On the basis of this hypothesis, we reasoned that if interactions inVolVing the umuDC gene products and components of Pol III were important for the checkpoint role of UmuD2C, we might then be able to obserVe an effect on the extent of the cold sensitiVity conferred by umuDC by the simultaneous oVerexpression of certain (i.e., releVant) components of Pol III. Using such an approach, we haVe recently reported that oVerproduction of the ɛ proofreading subunit of Pol III or deletion of its structural gene (DNAQ) suppresses umuDC-mediated cold sensitiVity (56). A systematic analysis of the remaining nine Pol III subunits indicated that the homodimeric β processiVity clamp was the only other Pol III subunit that when oVerexpressed affected the extent of umuDC-mediated cold sensitiVity (56). In this report, we describe how oVerexpression of the β processiVity clamp (encoded by DNAN) strongly exacerbates umuDC-mediated cold sensitiVity. We haVe exploited this ability of the β clamp to confer a cold-sensitiVe growth phenotype upon a umuD′C-expressing E. coli strain (a strain that is not normally cold sensitiVe [59]) to identify noVel DNAN alleles unable to confer this phenotype. Our genetic characterizations of these noVel DNAN alleles indicate that they are also unable to exacerbate the cold sensitiVity conferred by eleVated leVels of the umuDC gene products. Our results described in this report, taken together with others (37, 38, 56, 57, 59), suggest that the UmuD2C-dependent DNA damage checkpoint control is a manifestation of protein-protein interactions inVolVing UmuD2C and the ɛ and the β subunits of Pol III.