Skip to main content
PMC home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2008 Mar 7.
Published in final edited form as: Mol Cell. 2007 Dec 28;28(6):1058–1070. doi: 10.1016/j.molcel.2007.10.025

UmuD and RecA Directly Modulate the Mutagenic Potential of the Y-family DNA Polymerase DinB

Veronica G Godoy 1,3,*, Daniel F Jarosz 2,*,‡, Sharotka M Simon 1, Alexej Abyzov 3, Valentin Ilyin 3, Graham C Walker 1
PMCID: PMC2265384  NIHMSID: NIHMS37593  PMID: 18158902

Summary

DinB is the only translesion Y-family DNA polymerase conserved among bacteria, archaea, and eukaryotes. DinB and its orthologs possess a specialized lesion bypass function but also display potentially deleterious -1 frameshift mutagenic phenotypes when overproduced. We show that the DNA damage inducible proteins UmuD2 and RecA act in concert to modulate this mutagenic activity. Structural modeling suggests that the relatively open active site of DinB is enclosed by interaction with these proteins thereby preventing the template bulging responsible for -1 frameshift mutagenesis. Intriguingly, residues that define the UmuD2 interacting surface on DinB statistically co-vary throughout evolution, suggesting a driving force for the maintenance of a regulatory protein-protein interaction at this site. Together, these observations indicate that proteins like RecA and UmuD2 may be responsible for managing the mutagenic potential of DinB orthologs throughout evolution.

Introduction

Decades after their discovery, the dinB (Kenyon and Walker, 1980) and umuDC (Elledge and Walker, 1983; Kato and Shinoura, 1977) genes of Escherichia coli were shown to encode specialized Y-family DNA polymerases, DNA pol IV (Wagner et al., 1999) and pol V (Reuven et al., 1999; Tang et al., 1999) respectively, that catalyze the insertion of deoxyribonucleoside triphosphates (dNTPs) opposite potentially lethal replication blocking lesions in a process termed translesion synthesis (TLS) (Goodman, 2002; Ohmori et al., 2001). TLS can proceed with a range of fidelities (Goodman, 2002), but in all cases Y-family polymerases replicate undamaged DNA with a reduced fidelity relative to the enzymes that replicate the majority of the genome (Friedberg et al., 2002). Therefore, Y-family polymerases must be excluded from improper access to replication intermediates to maintain genomic integrity (Sutton and Walker, 2001a).

Both dinB and umuDC are regulated transcriptionally by the SOS regulatory network (Friedberg et al., 2006), their expression being induced when LexA undergoes facilitated autocleavage upon interaction with the RecA::ssDNA nucleoprotein filament formed after DNA damage. The subsequent control of UmuC function is remarkably complex and involves the gene products of the co-transcribed umuD gene. UmuD2 undergoes an SOS-mediated posttranslational modification when it too is subject to facilitated autodigestion upon interaction with RecA::ssDNA nucleoprotein filaments to yield UmuD′2 (Burckhardt et al., 1988; Nohmi et al., 1988). The conversion of UmuD2 to UmuD′2 activates UmuC polymerase function (Reuven et al., 1999; Tang et al., 1999) and RecA::ssDNA filaments are required in trans for UmuD′2C-catalyzed translesion synthesis (Schlacher et al., 2006). In addition, RecF, RecO, and RecR cooperate to alleviate the inhibition of UmuD′2C mediated TLS that is brought about by DNA pol III (Fujii et al., 2006). Finally, UmuD′2C must interact with the β processivity clamp of DNA pol III to function in vivo (Becherel et al., 2002; Sutton et al., 2001).

DinB is the only Y-family DNA polymerases conserved among all domains of life (Ohmori et al., 2001) and under conditions of DNA damage it is the most abundant DNA polymerase in E. coli (Kim et al., 2001). Despite the remarkable evolutionary conservation of DinB, the details of its biochemical regulation are comparatively unknown (Fuchs et al., 2004). We recently showed that an important function of DinB and its orthologs is to carry out highly proficient and accurate TLS past a particular class of N2-deoxyguanosine adducts (Jarosz et al., 2006). On the surface, these observations seem incompatible with DinB’s role in the elevated frequency of -1 frameshift mutations observed during λ untargeted mutagenesis (Brotcorne-Lannoye and Maenhaut-Michel, 1986) and adaptive mutagenesis (McKenzie et al., 2001; Tompkins et al., 2003) or with the -1 frameshift mutator effect caused by DinB overproduction (Kim et al., 1997).

Previous studies of DinB and its orthologs, as well as of other Y-family DNA polymerases, have focused on the pivotal role of processivity clamps in regulation of TLS and mutagenesis (Becherel et al., 2002; Bergoglio et al., 2002; Beuning et al., 2006a; Bunting et al., 2003; Frampton et al., 2006; Lenne-Samuel et al., 2002; Ogi et al., 2005). However, given DinB’s documented potential to cause deleterious -1 frameshift mutations (Kim et al., 2001; Nohmi, 2006), we thought it possible that DinB might be subject to control beyond transcriptional induction and interaction with the processivity clamp. We therefore searched for additional regulatory factors that might manage this potentially problematic function. Remarkably, we found that UmuD, UmuD′, and RecA, previously known only to regulate UmuC function, regulate both the activity and mutagenic properties of DinB via protein-protein interactions that enclose its active site. Our findings suggest that mechanistic features of this regulation may be maintained in eukaryotes consistent with a common pattern of regulation for these DNA polymerases through evolution.

Results

DinB Interacts with Numerous Cellular Factors

To identify proteins that might regulate DinB function, we covalently coupled purified recombinant DinB to an affinity resin (Supplemental Figure 1A). Interacting proteins from lysates of constitutively SOS-induced E. coli were eluted and separated by SDS-PAGE. Those that bound in a DinB dependent fashion included the chaperones GroEL and DnaK and also the ribosomal protein L3 as well as lesser amounts of other ribosomal proteins (Supplemental Figure 1B). The presence of the chaperones was anticipated since GroEL has been shown to regulate the function of both DinB (Layton and Foster, 2005) and UmuD′2C (Donnelly and Walker, 1989) and DnaK regulates UmuC levels in vivo (Grudniak et al., 2005). The ribosomal proteins L3 and others seem less likely to be bona fide regulators of DinB function although this remains a formal possibility.

However, we also identified UmuD, UmuD′, and RecA as DinB interacting proteins. This was a complete surprise because, despite their intensively studied, highly nuanced roles in regulating UmuC function (Friedberg et al., 2006; Jarosz et al., 2007; Nohmi, 2006), none of these factors had previously been implicated in regulating DinB function aside from the indirect role of RecA in mediating DinB induction via the SOS regulatory network. Intriguingly, the levels of UmuD in vivo (180 molecules in non-SOS induced cells; 2400 molecules in SOS induced cells) parallel those of DinB (250 molecules in non-SOS induced cells; 2500 molecules in SOS induced cells) and greatly exceed what is required to interact with UmuC (17 molecules in non-SOS induced cells; 200 molecules in SOS-induced cells) (Kim et al., 1997; Woodgate and Ennis, 1991). We therefore investigated the ability of these proteins to affect DinB function in vivo and in vitro.

DinB Forms a Stable Interaction with UmuD2 and RecA

To ascertain whether the interactions we observed between DinB, RecA, UmuD, and UmuD′ were direct, we first performed a farwestern blot in which we probed membranes containing UmuD, UmuD′, and RecA with DinB. Each of the interactions appears to be direct in nature both in this experiment (Figure 1A) and in similar farwestern blots performed on eluates from the DinB affinity column (data not shown). To analyze the stoichiometry of the DinB·UmuD interaction, we crosslinked DinB and UmuD with formaldehyde and analyzed the products by immunoblot using an antibody against DinB. The crosslinked species corresponds to the molecular weight of a DinB·UmuD2 complex and the reaction appears to be inhibited by high concentrations of NaCl (Figure 1B), suggesting that the interface may partly involve ionic or polar interactions. The propensity of RecA to multimerize (McGrew and Knight, 2003) made it difficult to establish the stoichiometry of the DinB·RecA interaction.

Figure 1. DinB interacts directly with UmuD2 and RecA.

Figure 1

Open in a new tab

A Farwestern blot demonstrates that UmuD directly interacts with 32P-labeled (His)6HMK-DinB. Either 50 or 100 pmols of UmuD or UmuD′ were separated by 12% SDS-PAGE and transferred to a PDVF membrane. The HMK-DinB protein probe was radioactively labeled and incubated with the membrane for 15 min. after which the membrane was exposed to film. B. Crosslinking experiment suggests that DinB interacts with the UmuD2 homodimer. One hundred pmol of DinB and UmuD2 were mixed in a 10 μL volume in 50 mM Hepes pH 7.5, 25–500 mM NaCl, and 1 mM DTT and incubated for 10 min at 25°C. C. DinB forms a stable binary and ternary complexes with RecA and UmuD2. DinB(His)6 pulls down RecA alone and in combination with UmuD and UmuD′ on a Ni2+ affinity resin. UmuDD′ cannot be observed to similarly interact.

To test whether DinB, UmuD2 and/or UmuD′2, and RecA form a stable ternary complex in solution, a DinB variant with a hexahistidine affinity tag at its C-terminus was incubated with RecA – both alone and in combination with UmuD2, UmuD′2, and the heterodimeric species UmuDD′ (Battista et al., 1990). Complexes that formed with DinB were isolated using Ni2+ affinity resin. Using physiologically relevant concentrations (2.5 μM) of each protein, we observed the formation of a stable stoichiometric complex between DinB and RecA (Figure 1C); fluorescence anisotropy does not reveal such an association (Schlacher et al., 2005). Moreover, it appears that DinB’s interaction with RecA does not preclude association with UmuD or UmuD′. Unexpectedly, we did not observe an interaction between DinB and the UmuDD′ heterodimer alone (data not shown) or in combination with RecA (Figure 1C) in this experiment. Taken together, these data indicate that DinB, RecA, and UmuD2 (and to a lesser extent UmuD′2), can form ternary complexes under physiological conditions.

UmuD Suppresses DinB-dependent Mutagenic Phenomena in vivo

Since prior genetic studies had not implicated either the umuD or recA gene products in dinB+-dependent phenomena, we investigated whether these interactions were important in vivo. Because the cellular levels of UmuD are similar to those of DinB and are much higher than those of UmuC, we wondered whether the -1 frameshift mutator effect associated with overexpression of DinB might be a consequence of the number of molecules of DinB exceeding those of UmuD in the cell. Interestingly, that appears to be the case as we found that co-overproduction of UmuD eliminates much of the -1 frameshift mutagenesis caused by DinB overproduction (Figure 2A). Co-overproduction of a noncleavable UmuD variant, UmuD(S60A), completely eliminates DinB dependent frameshift mutagenesis (Figure 2A), indicating that full-length UmuD is sufficient for maximal inhibition. These plasmids result in a 3–4 fold increase in UmuD levels over those encoded by the chromosomal umuD gene as determined by immunoblot (Supplemental Figure 2).

Figure 2. UmuD2 regulates the -1 frameshift activity of DinB in vivo.

Figure 2

Open in a new tab

A. Lac+ reversion assay of the frameshift allele in CC108 demonstrates that UmuD2 modulates DinB’s -1 frameshift function. B. Deletion of umuD+ results in a significant increase in -1 frameshift mutagenesis. A histogram of Lac+ mutants obtained from umuD+ and ΔumuD strains reveals that even the modest chromosomally encoded levels of UmuD2 have a significant impact on -1 frameshift mutagenesis. Experiments were performed as indicated in materials and methods. Y-axis shows the frequency of plates with the number of Lac+ mutants indicated in the bins on the X-axis. C. UmuD2 also affects the number of Lac+ revertants in an adaptive mutagenesis experiment. FC40 with empty vector (closed circles); FC40 with pUmuD′ (closed triangles); FC40 with pUmuD (open circles). Error bars represent one standard deviation.

Strikingly, we observed that a precise deletion of the chromosomal umuD gene also results in an increased frequency of -1 frameshift mutagenesis. Indeed, deletion of umuD (Supplemental Table 1) leads to a considerable and unexpected increase in the frequency of -1 frameshift mutagenesis from 8.0 (± 2.5) × 10−8 (umuD+) to 41.5 (± 9.3) × 10−8 (ΔumuD) (P=3.96×10−24) (Figure 2B). Fluctuation analysis (Foster, 2006) reveals a similar increase in estimated mutation rates between the two strains. The so-calculated mutation rates (with 95% confidence limits in parenthesis) are 1.7×10−8 (1.3×10−8; 1.9×10−8) for umuD+ and 7.1×10−8 (5.7×10−8; 8.2×10−8). These results emphasize the ability of even the modest levels of chromosomally encoded UmuD to modulate DinB’s mutagenic function. Interestingly, the mutants do not appear to conform either to a classical Luria-Delbruck distribution or to others that we tested, an observation that may be of biological significance

We then investigated the possibility that the umuD gene products might similarly modulate the phenomenon of adaptive mutagenesis in the widely studied E. coli strain FC40 (Cairns and Foster, 1991), which is dependent on DinB-promoted -1 frameshifts (McKenzie et al., 2001; Tompkins et al., 2003). Under the conditions required to observe such mutagenesis, DinB levels are elevated by approximately 2–4-fold (Kim et al., 2001; Layton and Foster, 2003). Remarkably, overproduction of UmuD or UmuD′ strikingly reduced adaptive mutagenesis (Figure 2C). The 5-fold reduction in the frequency of adaptive mutagenesis caused by UmuD overexpression is equivalent to the decrease caused by dinB inactivation (McKenzie et al., 2001; Tompkins et al., 2003). These observations suggest that the umuD gene products are able to modulate the -1 frameshift activity of DinB both in exponential phase and also under the conditions of an adaptive mutagenesis experiment.

Initially, we were surprised that deletion of umuD has no effect on dinB+-dependent NFZ resistance but this point is discussed further below. Although ΔrecA strains do exhibit sensitivity to this agent (data not shown), the multitudinous physiological roles of RecA (Friedberg et al., 2006; McGrew and Knight, 2003) complicate the interpretation of this result.

To investigate whether DinB reciprocally affects UmuD2 function, we examined the effect of DinB overproduction on UV-induced mutagenesis, a phenomenon that is critically dependent on umuD+ as well as on umuC+ (Elledge and Walker, 1983; Kato and Shinoura, 1977). Expression of dinB+ from a low copy number plasmid suppressed UV-induced mutagenesis by approximately 7-fold (Supplemental table 2). It is a formal possibility that this effect reflects competition between DinB and UmuD′2C for the primer terminus, but it may also be related to DinB dependent inhibition of RecA-mediated UmuD2 autocleavage (Figure 5E), consistent with DinB and UmuD2 interacting in vivo.

Figure 5. UmuD2 and RecA directly modulate DinB -1 frameshift function.

Figure 5

Open in a new tab

A. Schematic of a mismatched DNA substrate that can be extended either by dGTP to generate a full length product or by dATP, thereby forming a dNTP stabilized misalignment and generating a -1 frameshift product. B. Plot of reaction velocity vs. dNTP substrate concentration for DinB in combination with RecA and UmuD2. Extension of the GG mismatch with dATP by DinB in combination with stoichiometric quantities of RecA (open squares) is detectable but weaker than extension of a GC basepair in the same sequence context (open circles). The addition of saturating UmuD2 (10 μM) profoundly inhibits DinB activity on a GG mismatch (closed squares) but stimulates DinB activity on a GC (closed circles) by more than 20-fold. Data for DinB alone (without RecA or UmuD2) on the GG mismatch substrate are shown in open triangles. C. recA+ is required for UmuD dependent inhibition of DinB promoted -1 frameshift mutagenesis in vivo. Overeproduction of DinB promotes -1 frameshift mutagenesis in a ΔrecA background but the co-overproduction of UmuD2 or a noncleavable UmuD(S60A) variant has little effect on mutation frequency. D. Plot of percent frameshift inhibition vs. UmuD variant concentration. The frameshift activity of DinB is efficiently inhibited by UmuD2 (closed circles) but the frameshift activity of DinB(F172A) is more inert to UmuD2 supression (open circles). The UmuD(D91A) variant is also very inefficient at inhibiting the -1 frameshift activity of wild-type DinB (open triangles). E. DinB efficiently inhibits UmuD2 autocleavage in vitro. Wild-type DinB (open circles) inhibits UmuD2 autocleavage far better than DinB(F172A) (closed circles). Error bars represent one standard deviation.

Identification of the Molecular Interface Between DinB and UmuD2

Our observation that UmuD suppresses the -1 frameshift activity of DinB in vivo was especially intriguing in light of structural studies of archaeal DinB homologs, which have been shown to possess remarkably open active sites (Ling et al., 2001; Silvian et al., 2001). We therefore analyzed the interaction between DinB and UmuD2 using cellulose filter peptide arrays (Frank, 2002). The membranes were probed with either DinB or UmuD2 (Supplemental figure 3), and interacting peptides were identified and mapped onto structural models of DinB (Jarosz et al., 2006) or UmuD2 (Beuning et al., 2006c; Sutton et al., 2002). Interestingly, the UmuD2 interacting peptides on DinB localize to a single face of the protein (Figure 3A). Further, the presence of an extended interacting surface on DinB suggests that its interaction with UmuD2 is qualitatively different from its interaction with the β processivity clamp, which depends on a highly conserved peptide motif (Bunting et al., 2003; Dalrymple et al., 2001). Most intriguingly, the interaction interface suggests that UmuD2 may suppress mutagenesis by helping to enclose the strikingly open active site of DinB, therby preventing the DNA template bulging necessary for -1 frameshift mutagenesis (Ling et al., 2001; Potapova et al., 2002).

Figure 3. Molecular characterization of the interaction between DinB and UmuD2.

Figure 3

Open in a new tab

A. Peptide array mapping of the UmuD binding interface on DinB reveals a surface composed of the thumb and finger domains of the polymerase. Several hydrophobic residues in the most strongly interacting peptide are conserved among DinB orthologs from organisms containing umuD. B. Peptide array mapping of the DinB binding interface on UmuD2 reveals a discontinuous interface on a structural model of trans-UmuD2 that is enlarged in an alternative isoenergetic trans-UmuD2 conformer. C. Alanine mutants of DinB Phe172 or UmuD Asp 91 result in a weakened interaction determined by fluorescence spectroscopy. D. A low-copy number plasmid encoding DinB(F172A) (pYG768-F172A; open squares) is fully able to rescue the NFZ sensitivity of a ΔdinB E. coli strain bearing the pWSK29 empty vector (open circles) to the levels of an isogenic dinB+ strain (closed circles), just as a plasmid encoding wild-type DinB (pYG768; closed squares). Error bars represent one standard deviation determined from three independent experiments.

The DinB interacting interface forms a somewhat less contiguous surface when mapped onto a specific UmuD2 model we had originally proposed (Sutton et al., 2002). However, the DinB binding interface forms a more extensive surface when mapped onto one of four isoenergetic models of UmuD2 (Beuning et al., 2006c), in which its N-terminus is raised to reveal an interacting surface across the side of the protein (Figure 3B). These observations suggest that our previous model of UmuD2 may represent only one of several biologically relevant UmuD2 conformers (Supplemental figure 4).

In an effort to design a DinB variant that is unable to interact with UmuD2, we identified a strongly interacting group of peptides from the DinB peptide array and examined conservation of this region in numerous umuD-containing organisms. Three residues, P166, F172, and L176 were strikingly conserved and we determined the effect of changing each residue to an alanine (Figure 3A). Although the mutant proteins DinB(P166A) and DinB(L176A) were insoluble (data not shown), we were able to express and purify DinB(F172A) in soluble form (Supplemental Figure 5). Moreover, we found that the dinB allele encoding DinB(F172A) complements the NFZ sensitivity of a ΔdinB strain (Figure 3D), indicating that this mutant is proficient for TLS in vivo.

A reciprocal approach was used to generate a UmuD2 mutant that might be impaired with respect to its ability to interact with DinB. The variant UmuD(D91A) is soluble and purifies as wild-type UmuD. It is also proficient for facilitated autoproteolysis (Supplemental Figure 6).

We determined that the dissociation constant between DinB and UmuD2 is 0.62 μM using fluorescence spectroscopy (Figure 3C; Supplemental Figure 7). Provocatively, the cellular concentration of UmuD2 rises from ca.0.35 μM under non-SOS-induced conditions to ca. 4.5 μM under conditions of SOS-induction (Kim et al., 2001), indicating that DinB and UmuD2 are capable of interaction within the range of physiologically relevant concentrations. Furthermore, the DinB(F172A) and UmuD(D91A)2 proteins were each greatly impaired with respect to their ability to bind their partners (Figure 3C), indicating that the interfaces we identified by peptide array mapping are functionally relevant.

Mutation of the Interface Between DinB and UmuD2 Impairs Function in vivo

To determine whether the physical interaction between DinB and UmuD2 we observed and analyzed in vitro is important for modulation of DinB dependent frameshift mutagenesis in vivo, we examined whether the -1 frameshifts produced by DinB(F172A) could be inhibited by UmuD. Overproduction of DinB(F172A) results in an increase in -1 frameshift mutagenesis by approximately 6-fold (Figure 4A). The precise reasons for the difference between this value and that obtained from wild-type DinB overproduction, which could include reduced -1 frameshift activity or affinity for frameshift intermediates in vivo, are unclear. However, the increase in -1 frameshift mutagenesis observed from DinB(F172A) overproduction is remarkably statistically significant (P=4×10−15). Co-overproduction of UmuD or UmuD′ does not substantially reduce the -1 frameshift mutation frequency due to DinB(F172A) overproduction (Figure 4A). These data suggest that a direct interaction of UmuD2 or UmuD′2 at the interface we have identified on DinB is important for modulation of -1 frameshift mutagenesis in vivo.

Figure 4. Single amino acid changes on the interface between DinB and UmuD perturb regulation of -1 frameshift activity.

Figure 4

Open in a new tab

A. The DinB(F172A) variant has a lower affinity for UmuD2 and is not as responsive as wild type DinB to regulation by UmuD2. B. The UmuD(D91A) variant has a lower affinity for DinB and does not regulate -1 frameshift activity as well as wild type UmuD. Error bars represent one standard deviation.

Reciprocally we examined whether UmuD(D91A) could suppress the -1 frameshift mutagenesis promoted by overproduction of wild-type DinB. Consistent with the weakened affinity of UmuD(D91A) for DinB, its overproduction only modestly suppresses -1 frameshift mutagenesis (Figure 4B). Moreover, even significant overproduction of DinB(F172A) was insufficient to impair UV-induced mutagenesis (Supplemental Table 2). These observations suggest that a direct interaction between DinB and UmuD2 is crucial for the ability of each protein to modulate the function of the other in vivo.

UmuD2 Inhibits DinB-dependent -1 Frameshift Activity in vitro

To gain more detailed insights into the mechanisms governing modulation of DinB function by UmuD in vivo, we reconstituted DinB dependent -1 frameshift activity in vitro with a substrate containing a G:G mispair that can be extended with either i) dGTP to generate a full-length product or ii) dATP to generate a -1 frameshift product that is one nucleotide shorter than the template (Figure 5A) (Kobayashi et al., 2002; Wagner et al., 1999). DinB is unable to extend from this mispair using dGTP under our experimental conditions, and although it can act on this substrate with modest efficiency using dATP, addition of UmuD2 alone did not alter its -1 frameshift activity (data not shown). However, we then discovered that, when RecA is added in a stoichiometric ratio with DinB, the addition of UmuD2 nearly completely inhibits the reaction (Figure 5B). In striking contrast, addition of UmuD2 and RecA results in a ca. 20-fold enhancement of the ability of DinB to extend from a correctly paired terminus in the same sequence context (Figure 5B). These observations indicate that UmuD2 and RecA act in concert to modulate DinB function in a highly sophisticated manner, promoting its ability to extend a properly paired primer terminus while suppressing its ability to extend a mismatched terminus.

Our discovery that RecA is required for UmuD2 to modulate DinB mutagenic function in vitro led us to examine whether recA+ is similarly required for UmuD-dependent suppression of -1 frameshift mutagenesis in vivo. It is, as co-overproduction of UmuD or UmuD(S60A) has little effect on the frequency of DinB-dependent -1 frameshift mutagenesis in a ΔrecA strain (Figure 5C). Intriguingly, the frequency of -1 frameshift mutagenesis caused by DinB overproduction is also modestly elevated in a ΔrecA strain. Taken together, these findings provide strong evidence that RecA is required for UmuD2 dependent modulation of DinB function.

In vitro, UmuD2 reduced the maximal -1 frameshift activity of wild-type DinB by one half at a concentration of 840 nM, while a concentration of 3.6 μM is needed to cause an equivalent effect on DinB(F172A) (Figure 5D). Relative to wild-type UmuD2, UmuD(D91A)2 also shows a marked 10-fold decrease in its ability to inhibit DinB dependent -1 frameshift activity in vitro. Additionally, we observed that DinB is able to inhibit the RecA-mediated autocleavage of UmuD2 in vitro, and moreover that the DinB(F172A) variant was unable to do so efficiently (Figure 5E). All of these data underscore the notion that a physical interaction between DinB and UmuD2 exists under physiological conditions and is required for UmuD2 dependent modulation of DinB function.

A TLS-deficient DinB Variant is Proficient for -1 Frameshift Function

It has been suggested that the -1 frameshift mutator signature of DinB is a direct consequence of structural features that enable it to act as a TLS DNA polymerase (Ling et al., 2001; Potapova et al., 2002). To ascertain whether DinB’s -1 frameshift mutator activity is separable from its function in TLS, we examined the DinB(F13V) variant, which is able to catalyze DNA synthesis on undamaged DNA but is virtually unable to perform TLS on certain adducted templates (Jarosz et al., 2006). We found that when overexpressed, this mutant is able to promote -1 frameshift mutagenesis in vivo (Figure 6A), indicating that DinB’s -1 frameshift mutator activity can be genetically separated from its ability to carry out proficient and accurate TLS over certain N2-dG adducts.

Figure 6. A TLS deficient variant of DinB is proficient for -1 frameshift function.

Figure 6

Open in a new tab

A. The DinB(F13V) variant can promote -1 frameshift mutagenesis but is not controlled by co-overproduction of UmuD. B. The -1 frameshift activity of DinB(F13V) is poorly inhibited by UmuD2 in vitro. Plot of frameshift activity vs. UmuD concentration indicates that DinB(F13V) (open squares) retains much of its frameshift activity at concentrations of UmuD that inhibit virtually all DinB frameshift activity (closed circles). All reactions contain RecA in stoichiometric ratios with DinB. Error bars represent one standard deviation.

Curiously, the mutation frequency induced by overexpression of DinB(F13V) is consistently greater than that produced by overexpression of wild-type DinB (Figure 2A) even though the levels of each protein are comparable in vivo (Supplemental Figure 8). This observation suggests either that DinB(F13V) has an increased -1 frameshift mutator activity or that some other mechanism is responsible for this phenomenon in vivo. Although the first explanation is formally possible, the -1 frameshift activity of DinB(F13V) in vitro is slightly reduced (ca. 3-fold) relative to wild-type DinB. This observation suggests that DinB(F13V) promotes increased mutagenesis by virtue of either its recruitment to, or association with, frameshift intermediates. Curiously, we were unable to observe UmuD2 dependent inhibition of DinB(F13V) -1 frameshift activity either in vivo or in vitro (Figures 6A–B). These observations may be at least in part due to the fact that DinB(F13V) has a reduced affinity for UmuD2 relative to the wild-type enzyme (Supplemental Figure 9).

RecA and UmuD2 May Modulate DinB Function by Restricting its Open Active Site

In an effort to explain how UmuD2 suppresses the intrinsic -1 frameshift mutator activity of DinB in a RecA-dependent manner, we generated a model of a ternary complex among DinB, RecA, and UmuD2. The structure of RecA (Story et al., 1992) and models of DinB (Jarosz et al., 2006) and UmuD2 (Beuning et al., 2006c) were docked using several constraints. First, UmuD2 was optimally positioned on DinB using our peptide array data (Figure 3A–B; Supplemental Figure 3). We then used distance constraints between RecA and UmuD2 from published monocysteine crosslinking studies (Lee and Walker, 1996) to orient RecA relative to UmuD2. Finally, we analyzed the RecA binding interface on DinB with an additional peptide array experiment (Supplemental Figure 10). Together, these data were used to generate the working model shown in Figure 7A–B, which suggests that RecA and UmuD2 act in concert to enclose the relatively open active site of DinB, perhaps thereby preventing the template bulging necessary for -1 frameshift mutagenesis. It is also provocative that in our model, RecA is positioned appropriately to interact with the end of a RecA-nucleoprotein filament, suggesting that this interaction may also play a pivotal role in targeting DinB to RecA-coated substrates, a concept that has been previously proposed for UmuD′2C (Bailone et al., 1991; Frank et al., 1993).

Figure 7. RecA and UmuD2 may enclose the open active site of DinB.

Figure 7

Open in a new tab

A–B. In silico modeling of a ternary complex of the proteins. The surface representation of DinB is shown in blue, UmuD2 in yellow, and RecA in orange. The DNA is relatively enclosed in the complex. C. Statistical covariance of DinB/pol κ residues across evolution. Residues that display statistical covariance with the UmuD2 binding interface on E. coli DinB define an interface in a similar position on pol κ (shown in red), suggesting a possible rationale for the maintenance of this interface as a site of regulatory protein-protein interactions.

Discussion

In this paper, we have used several in vitro methods to demonstrate unanticipated direct interactions among DinB, UmuD2, UmuD′2, and RecA (Figure 1A–C) and present evidence that these interactions are physiologically relevant. Indeed, we have been able to isolate a ternary complex of these three proteins that is stable on the minute timescale (Figure 1C). The dissociation constant between DinB and UmuD2 is 620 nM, a concentration that is squarely within the physiological range of UmuD2 concentrations (0.35 μM uninduced; 4.5 μM SOS-induced). The estimated Kd between DinB and RecA is ca. 1 μM and the RecA concentration under normal conditions is 2.5 μM. The cellular levels of all of these proteins rise at least 10-fold upon SOS induction (Kim et al., 2001; Salles and Paoletti, 1983; Woodgate and Ennis, 1991). Therefore, many molecules of DinB are likely to exist as at least a binary and perhaps ternary complex under physiological conditions. Furthermore, these findings provide provide a potential rationale for the observation that the physiological levels of the umuD gene products greatly exceed those of UmuC.

We find that DinB-dependent -1 frameshift mutagenesis can be suppressed by co-overproduction of UmuD (Figure 2A–B), and elevated by absence of umuD+ on the chromosome (Figure 2B). Moreover, a noncleavable UmuD variant completely suppresses -1 frameshift mutagenesis, indicating that only the function of full-length UmuD is required to control DinB’s mutator potential (Figure 2A). Reciprocally, the DinB-binding deficient UmuD(D91A) variant (Figure 3C), although perfectly proficient for RecA-mediated autoproteolysis (Supplemental Figure 6), does not efficiently reduce DinB dependent -1 frameshift mutagenesis (Figure 4B). Intriguingly, when mapped onto a structural model of UmuD2, the residue Asp91 in UmuD does not form a contiguous interface with the other residues identified as part of the DinB-binding interface as it is occluded by the curled N-terminus of UmuD (Figure 3B). However, recent computational studies have suggested that isoenergetic conformations of UmuD2 can be formed in which its N-terminus is raised (Beuning et al., 2006c), thereby potentially favoring interaction with DinB but also rendering it incompetent for autoproteolysis (Supplemental Figure 4). Such conformational flexibility is consistent with the recent discovery that UmuD2 and UmuD′2 are intrinsically disordered proteins (Simon and Walker, submitted). Our observation that DinB inhibits UmuD2 autoproteolysis in vitro and UV-induced mutagenesis in vivo is also consistent with this notion. Overproduction of UmuD, and to a lesser extent UmuD′, also inhibits adaptive mutagenesis (Figure 2C), which occurs via a -1 frameshift event (Cairns and Foster, 1991). Irrespective of the precise molecular mechanisms of adaptive mutagenesis, it is clear that the umuD gene products can play a role in modulating it.

Our efforts to reconstitute the UmuD2 modulation of DinB frameshift activity in vitro using a mismatched substrate (Kobayashi et al., 2002; Wagner et al., 1999) revealed that RecA needs to be present in stoichiometric quantities with DinB for this to occur (Figure 5B). Consistent with these observations, we found that recA+ is required for the suppression of DinB-dependent -1 frameshift mutator activity in vivo, as the co-overproduction of UmuD2 does not suppress this mutagenesis in a recA strain (Figure 5C). A noncleavable UmuD(S60A) variant is fully proficient for suppression of -1 frameshift mutagenesis (Figure 2A), indicating that RecA’s role in this phenomenon is distinct from its function in promoting UmuD autocleavage.

Strikingly, addition of RecA and UmuD2 to an assay in which DinB replicates a template with a properly paired terminus results in a remarkable increase in DinB catalytic proficiency (Figure 5B; open and closed circles). This observation suggests that RecA and UmuD2 may modulate DinB function on diverse DNA substrates.

Taken together, these discoveries significantly expand our view of how the fidelity of Y-family DNA polymerases is regulated in response to DNA damage or environmental stress. Under normal conditions cellular levels of UmuD2 are relatively low but comparable to those of DinB. Upon SOS induction, UmuD2 predominates for 30–40 minutes, a phase during which accurate repair and damage tolerance mechanisms operate (Sutton and Walker, 2001b). The subsequent phase, in which UmuD′2 predominates, is when potentially mutagenic TLS by UmuD′2C takes place. Our results suggest that in uninduced cells, as well as during the first phase of SOS induction, UmuD2 and RecA would act in concert to restrict the -1 frameshift mutagenic potential of DinB by closing in its active site and simultaneously stimulating its activity on templates with properly paired termini. This control would then be relaxed during the second more mutagenic phase of SOS induction. Our results further suggest that DinB would be mutagenic under conditions of chronic SOS induction.

We propose, as have others (Ling et al., 2001; Potapova et al., 2002), that the propensity of DinB and its orthologs for -1 frameshift mutagenesis may arise as a result of their unique active sites that are specialized for TLS function. DinB overproduction, whether synthetic or apparently natural as during adaptive mutagenesis, renders the cell vulnerable to the -1 frameshift mutator potential of DinB, which likely results at least in part from exceeding the capacity of its UmuD2 manager. Similarly, we observe a significant increase in -1 frameshift mutagenesis when the umuD+ gene is deleted (Figure 2B). Unexpectedly, this regulation cannot be fully explained by a simple analysis of the relative levels of DinB and UmuD2. The CC108 episome contains an additional copy of dinB+ that expresses approximately 750 molecules of DinB in addition to the 250 molecules expressed from the chromosomal dinB+ gene (Kim et al., 2001). In a ΔumuD CC108 strain (0 molecules UmuD2; 1,000 molecules DinB) we observe a five-fold increase in -1 frameshift mutagenesis relative to a umuD+ CC108 strain (90 molecules UmuD2; 1,000 molecules DinB). If all molecules of DinB participate equally in -1 frameshift mutagenesis under these conditions, this experiment should result in at most a 1.1-fold effect. Under the conditions of overproduction used in this study (Figure 2A) the number of molecules of DinB (Kim et al., 2001) likewise exceeds those of UmuD. These observations suggest that other limiting cellular factors may be required for DinB to promote -1 frameshift mutagenesis and moreover that these factors may also interact with UmuD2.

Our discovery that UmuD2 and RecA appear to modulate the mutagenic potential of DinB by enclosing its open active site may have implications for the control of DinB orthologs in other organisms. Overproduction of mammalian pol κ has similarly deleterious mutagenic consequences to DinB overproduction (Bavoux et al., 2005b), despite the fact that its active site appears to be comparatively closed (Lone et al., 2007). A comparison of the pol κstructure with our working model of the DinB·UmuD2·RecA complex suggests that part of the pol κ structure may play a role equivalent to RecA, but that its function might be further regulated by a partner protein interacting in a manner analogous to UmuD interacting with DinB.

Although the UmuD protein is only conserved among certain bacteria, we wondered whether the UmuD-binding interface on DinB might be maintained throughout evolution to interact either with a highly diverged UmuD or with a different but functionally equivalent partner protein. Despite the fact that residues on DinB that participate in this interface are not strongly conserved, we considered whether they would exhibit statistical covariance through evolution. We therefore assembled an alignment of numerous DinB and pol κ sequences from all domains of life and examined which positions showed statistically significant covariance (Afonnikov and Kolchanov, 2004) with at least two of three residues that comprise the UmuD2 interacting interface of DinB. Strikingly, these residues define an interface on pol κ that is similar to the one we identified experimentally on DinB (Figure 7C). These observations suggest that there may be an evolutionary driving force for the maintenance of this interface, perhaps as a site for regulatory protein-protein interactions.

We had anticipated that deletion of either umuD or recA would affect DinB TLS function, and were therefore initially surprised that deletion of umuD had no effect on dinB+-dependent resistance to NFZ in a wild-type E. coli strain. Deletion of recA dramatically increased sensitivity to NFZ but, because of the central role of RecA in coordinating numerous aspects of the DNA damage response (Friedberg et al., 2006), we were unable to infer that this results from a loss of DinB mediated TLS. This led us to wonder whether the -1 frameshift mutator activity of DinB is genetically separable from its ability to proficiently and accurately bypass certain N2-dG adducts. We have previously characterized a separation of function mutant of DinB’s steric gate residue, DinB(F13V), which is active as a conventional DNA polymerase but is virtually unable to catalyze TLS on N2-dG adducted templates (Jarosz et al., 2006). In vivo, DinB(F13V) is exceptionally proficient at promoting -1 frameshift mutagenesis, although its levels are comparable to wild type by immunoblot (Supplemental figure 8), indicating that certain mechanistic attributes of DinB that are required for its proficient and accurate N2-dG lesion bypass ability are not required for its -1 frameshift mutator activity (Figure 6A). This observation is consistent with the fact that the DinB(F172A) variant can confer NFZ resistance to a ΔdinB strain, despite the fact that it is somewhat compromised with respect to its -1 frameshift mutator activity.

Taken together our results indicate that DinB plays at least two separable roles in nature, one in which it promotes survival by accurately bypassing a common class of N2-dG adducts and another in which it promotes mutagenesis under stressful conditions. The additional levels of DinB regulation we have described suggest ways in which the balance of these two diverse roles could be tuned to the physiological conditions being experienced by the organism.

Experimental Procedures

Protein Expression and Purification

Native DinB, UmuD, UmuD′, and RecA and their variants were purified as described previously (Beuning et al., 2006b; Jarosz et al., 2006; Konola et al., 1995), but 50 mM Hepes pH 7.2 was used as a buffering agent. Plasmids expressing DinB(F172A) and UmuD(D91A) were constructed from pDFJ1 and pSG5 (Beuning et al., 2006b) using a Quikchange kit (Stratagene). Both DinB(F172A) and UmuD(D91A) behaved as wild-type DinB and UmuD during purification. A plasmid encoding (His)6HMK-DinB (Chen and Hai, 1994) was constructed in pET16B using standard cloning procedures. (His)6HMK-DinB was purified using Ni2+-NTA affinity resin (Qiagen) following the manufacturer’s instructions.

Affinity Chromatography

The details of the procedure are described in supplemental material accompanying this manuscript.

Crosslinking and Binding Measurements

Crosslinking reactions were initiated with formaldehyde as described (Sutton et al., 1999) and allowed to proceed for 10 min before quenching with SDS-PAGE loading buffer containing 5% β-mercaptoethanol. Fluorescence spectroscopy was performed as described (Beuning et al., 2006b).

Farwestern assays and Peptide Array Experiments

Farwestern blots, as well as kinase labeling of purified (His)6HMK-DinB with γ-32P-ATP by heart muscle kinase (Sigma), were performed according to standard procedures as previously described (Sutton et al., 1999). Cellulose filter peptide arrays were synthesized with overlapping 12-mer peptides offset by two residues (MIT CCR Core Facility). The arrays were probed with 100 nM UmuD2 or DinB, and washed and developed as described (Niehbuhr and Wehland, 1997). Control arrays were performed using DinB or UmuD2 antibodies alone.

Mutagenesis Assays

DinB dependent -1 frameshift mutagenesis experiments were performed as previously described (Kim et al., 1997) except that IPTG was not added to the media. Ampicillin (100 μg/mL) and spectinomycin (60 μg/mL) were used as necessary for plasmid maintenance. Adaptive mutagenesis was performed as previously described (Godoy et al., 2000). A table of the strains and plasmids used in this study is provided as supplemental material (Supplemental Table 1).

DinB and UmuD2 Activity Assays

DinB was assayed as described previously (Jarosz et al., 2006) except 50 nM enzyme and 10 nM primer/template was used. The oligonucleotides 5′-ATCCTAGTCCAGGCTGCTGACAACTCGGGAACGTGCTACATGAAT-3′, 5′-ATTCATGTAGCAGCGTTCCC-3′, and 5′ATTCATGTAGCAGCGTTCCG-3′ were designed based on those used previously (Wagner et al., 1999). Reactions were initiated with the appropriate dNTP, quenched after 20 min, and separated on a 16% denaturing polyacrylamide gel, which was quantified using a Typhoon phosphorimager (GE Healthcare). UmuD2 autocleavage reactions were performed as described (Beuning et al., 2006b).

Molecular Modeling and Statistical Covariance

A model of the DinB·UmuD2 complex was constructed using the application 3D-dock (http://www.bmm.icnet.uk/docking) based on the following constraints: E168 < 6 Å from either UmuD chain, and L176, P177, K180, and F172 < 8 Å from D91 of either UmuD chain. The UmuD2·RecA model was made using the following constraints: RecA S117 < 7 Å from either UmuD chain (Lee and Walker, 1996), UmuD residues L101, R102, V34, S81 are 6–12 Å from RecA, and UmuD residue E11 is 6–25 Å from RecA. Resulting complexes were filtered based the RecA residues T243 and R244 < 10 Å from DinB to generate a model of the DinB·RecA·UmuD2 ternary complex. We performed the same procedure by docking DinB to the UmuD2·RecA models using the constraints described above and obtained similar results. Statistical covariance was performed by aligning 84 DinB and pol κsequences from diverse organisms using ClustalW and analyzing significant pairwise correlation of alignment positions with the CRASP algorithm (Afonnikov and Kolchanov, 2004).

Supplementary Material

01

Supplemental Data

Supplemental Data includes 9 figures and 2 tables.

Acknowledgments

We wish to thank Dr. Steve Bell for use of the phosphorimager and members of the Walker and Godoy laboratories for discussions. We also wish to thank Dr. P. Joshi (RTI international) for assistance with statistical analysis. This work was supported by NIH grant CA021615 to G.C.W and NIEHS grant P30 ES002109 to the MIT Center for Environmental Health Sciences. G.C.W. is an American Cancer Society Professor. SMS was a Cleo and Paul Schimmel fellow.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  1. Afonnikov DA, Kolchanov NA. CRASP: a program for analysis of coordinated substitutions in multiple alignments of protein sequences. Nucleic Acids Res. 2004;32:W64–68. doi: 10.1093/nar/gkh451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bailone A, Sommer S, Knezevic J, Dutreix M, Devoret R. A RecA protein mutant deficient in its interaction with the UmuDC complex. Biochimie. 1991;73:479–484. doi: 10.1016/0300-9084(91)90115-h. [DOI] [PubMed] [Google Scholar]
  3. Battista JR, Ohta T, Nohmi T, Sun W, Walker GC. Dominant negative umuD mutations decreasing RecA-mediated cleavage suggest roles for intact UmuD in modulation of SOS mutagenesis. Proc Natl Acad Sci USA. 1990;87:7190–7194. doi: 10.1073/pnas.87.18.7190. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bavoux C, Hoffmann JS, Cazaux C. Adaptation to DNA damage and stimulation of genetic instability: the double-edged sword mammalian DNA polymerase kappa. Biochimie. 2005a;87:637–646. doi: 10.1016/j.biochi.2005.02.007. [DOI] [PubMed] [Google Scholar]
  5. Bavoux C, Leopoldino AM, Bergoglio V, J OW, Ogi T, Bieth A, Judde JG, Pena SD, Poupon MF, Helleday T, et al. Up-regulation of the error-prone DNA polymerase kappa promotes pleiotropic genetic alterations and tumorigenesis. Cancer Res. 2005b;65:325–330. [PubMed] [Google Scholar]
  6. Becherel OJ, Fuchs RP, Wagner J. Pivotal role of the beta-clamp in translesion DNA synthesis and mutagenesis in E. coli cells. DNA Repair (Amst) 2002;1:703–708. doi: 10.1016/s1568-7864(02)00106-4. [DOI] [PubMed] [Google Scholar]
  7. Bergoglio V, Bavoux C, Verbiest V, Hoffmann JS, Cazaux C. Localisation of human DNA polymerase kappa to replication foci. J Cell Sci. 2002;115:4413–4418. doi: 10.1242/jcs.00162. [DOI] [PubMed] [Google Scholar]
  8. Beuning PJ, Sawicka D, Barsky D, Walker GC. Two processivity clamp interactions differentially alter the dual activities of UmuC. Mol Microbiol. 2006a;59:460–474. doi: 10.1111/j.1365-2958.2005.04959.x. [DOI] [PubMed] [Google Scholar]
  9. Beuning PJ, Simon SM, Godoy VG, Jarosz DF, Walker GC. Characterization of Escherichia coli translesion synthesis polymerases and their accessory factors. Methods Enzymol. 2006b;408:318–340. doi: 10.1016/S0076-6879(06)08020-7. [DOI] [PubMed] [Google Scholar]
  10. Beuning PJ, Simon SM, Zemla A, Barsky D, Walker GC. A non-cleavable UmuD variant that acts as a UmuD′ mimic. J Biol Chem. 2006c;281:9633–9640. doi: 10.1074/jbc.M511101200. [DOI] [PubMed] [Google Scholar]
  11. Brotcorne-Lannoye A, Maenhaut-Michel G. Role of RecA protein in untargeted UV mutagenesis of bacteriophage lambda: evidence for the requirement for the dinB gene. Proc Natl Acad Sci USA. 1986;83:3904–3908. doi: 10.1073/pnas.83.11.3904. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Bunting KA, Roe SM, Pearl LH. Structural basis for recruitment of translesion DNA polymerase Pol IV/DinB to the beta-clamp. EMBO J. 2003;22:5883–5892. doi: 10.1093/emboj/cdg568. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Burckhardt SE, Woodgate R, Scheuermann RH, Echols H. UmuD mutagenesis protein of Escherichia coli: overproduction, purification, and cleavage by RecA. Proc Natl Acad Sci U S A. 1988;85:1811–1815. doi: 10.1073/pnas.85.6.1811. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Cairns J, Foster PL. Adaptive reversion of a frameshift mutation in Escherichia coli. Genetics. 1991;128:695–701. doi: 10.1093/genetics/128.4.695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Chen BP, Hai T. Expression vectors for affinity purification and radiolabeling of proteins using Escherichia coli as host. Gene. 1994;139:73–75. doi: 10.1016/0378-1119(94)90525-8. [DOI] [PubMed] [Google Scholar]
  16. Dalrymple BP, Kongsuwan K, Wijffels G, Dixon NE, Jennings PA. A universal protein-protein interaction motif in the eubacterial DNA replication and repair systems. Proc Natl Acad Sci U S A. 2001;98:11627–11632. doi: 10.1073/pnas.191384398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Donnelly CE, Walker GC. groE mutants of Escherichia coli are defective in umuDC-dependent UV mutagenesis. J Bacteriol. 1989;171:6117–6125. doi: 10.1128/jb.171.11.6117-6125.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Elledge SJ, Walker GC. Proteins required for ultraviolet light and chemical mutagenesis. Identification of the products of the umuC locus of Escherichia coli. J Mol Biol. 1983;164:175–192. doi: 10.1016/0022-2836(83)90074-8. [DOI] [PubMed] [Google Scholar]
  19. Foster PL. Methods for determining spontaneous mutation rates. Methods Enzymol. 2006;409:195–213. doi: 10.1016/S0076-6879(05)09012-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Frampton J, Irmisch A, Green CM, Neiss A, Trickey M, Ulrich HD, Furuya K, Watts FZ, Carr AM, Lehmann AR. Postreplication repair and PCNA modification in Schizosaccharomyces pombe. Mol Biol Cell. 2006;17:2976–2985. doi: 10.1091/mbc.E05-11-1008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Frank EG, Hauser J, Levine AS, Woodgate R. Targeting of the UmuD, UmuD′, and MucA′ mutagenesis proteins to DNA by RecA protein. Proc Natl Acad Sci U S A. 1993;90:8169–8173. doi: 10.1073/pnas.90.17.8169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Frank R. The SPOT-synthesis technique. Synthetic peptide arrays on membrane supports--principles and applications. J Immunol Methods. 2002;267:13–26. doi: 10.1016/s0022-1759(02)00137-0. [DOI] [PubMed] [Google Scholar]
  23. Friedberg EC, Wagner R, Radman M. Specialized DNA polymerases, cellular survival, and the genesis of mutations. Science. 2002;296:1627–1630. doi: 10.1126/science.1070236. [DOI] [PubMed] [Google Scholar]
  24. Friedberg EC, Walker GC, Siede W, Wood RD, Schultz RA, Ellenberger T. DNA Repair and Mutagenesis. 2. Washington, DC: ASM Press; 2006. [Google Scholar]
  25. Fuchs RP, Fujii S, Wagner J. Properties and functions of Escherichia coli: Pol IV and Pol V. Adv Protein Chem. 2004;69:229–264. doi: 10.1016/S0065-3233(04)69008-5. [DOI] [PubMed] [Google Scholar]
  26. Fujii S, Isogawa A, Fuchs RP. RecFOR proteins are essential for Pol V-mediated translesion synthesis and mutagenesis. EMBO J. 2006;25:5754–5763. doi: 10.1038/sj.emboj.7601474. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Godoy VG, Gizatullin FS, Fox MS. Some features of the mutability of bacteria during nonlethal selection. Genetics. 2000;154:49–59. doi: 10.1093/genetics/154.1.49. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Goodman MF. Error-prone repair DNA polymerases in prokaryotes and eukaryotes. Annu Rev Biochem. 2002;71:17–50. doi: 10.1146/annurev.biochem.71.083101.124707. [DOI] [PubMed] [Google Scholar]
  29. Grudniak AM, Kuc M, Wolska KI. Role of Escherichia coli DnaK and DnaJ chaperones in spontaneous and induced mutagenesis and their effect on UmuC stability. FEMS Microbiol Lett. 2005;242:361–366. doi: 10.1016/j.femsle.2004.11.037. [DOI] [PubMed] [Google Scholar]
  30. Jarosz DF, Beuning PJ, Cohen SE, Walker GC. Y-family DNA polymerases in Escherichia coli. Trends Microbiol. 2007 doi: 10.1016/j.tim.2006.12.004. [DOI] [PubMed] [Google Scholar]
  31. Jarosz DF, Godoy VG, Delaney JC, Essigmann JM, Walker GC. A single amino acid governs enhanced activity of DinB DNA polymerases on damaged templates. Nature. 2006;439:225–228. doi: 10.1038/nature04318. [DOI] [PubMed] [Google Scholar]
  32. Kato T, Shinoura Y. Isolation and characterization of mutants of Escherichia coli deficient in induction of mutations by ultraviolet light. Mol Gen Genet. 1977;156:121–131. doi: 10.1007/BF00283484. [DOI] [PubMed] [Google Scholar]
  33. Kenyon CJ, Walker GC. DNA-damaging agents stimulate gene expression at specific loci in Escherichia coli. Proc Natl Acad Sci U S A. 1980;77:2819–2823. doi: 10.1073/pnas.77.5.2819. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Kim SR, Maenhaut-Michel G, Yamada M, Yamamoto Y, Matsui K, Sofuni T, Nohmi T, Ohmori H. Multiple pathways for SOS-induced mutagenesis in Escherichia coli: an overexpression of dinB/dinP results in strongly enhancing mutagenesis in the absence of any exogenous treatment to damage DNA. Proc Natl Acad Sci U S A. 1997;94:13792–13797. doi: 10.1073/pnas.94.25.13792. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Kim SR, 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;266:207–215. doi: 10.1007/s004380100541. [DOI] [PubMed] [Google Scholar]
  36. Kobayashi S, Valentine MR, Pham P, O’Donnell M, Goodman MF. Fidelity of Escherichia coli DNA polymerase IV. Preferential generation of small deletion mutations by dNTP-stabilized misalignment. J Biol Chem. 2002;277:34198–34207. doi: 10.1074/jbc.M204826200. [DOI] [PubMed] [Google Scholar]
  37. Konola JT, Nastri HG, Logan KM, Knight KL. Mutations at Pro67 in the RecA protein P-loop motif differentially modify coprotease function and separate coprotease from recombination activities. J Biol Chem. 1995;270:8411–8419. doi: 10.1074/jbc.270.15.8411. [DOI] [PubMed] [Google Scholar]
  38. Layton JC, Foster PL. Error-prone DNA polymerase IV is controlled by the stress-response sigma factor, RpoS, in Escherichia coli. Mol Microbiol. 2003;50:549–561. doi: 10.1046/j.1365-2958.2003.03704.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Layton JC, Foster PL. Error-prone DNA polymerase IV is regulated by the heat shock chaperone GroE in Escherichia coli. J Bacteriol. 2005;187:449–457. doi: 10.1128/JB.187.2.449-457.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Lee MH, Walker GC. Interactions of Escherichia coli UmuD with activated RecA analyzed by cross-linking UmuD monocysteine derivatives. J Bacteriol. 1996;178:7285–7294. doi: 10.1128/jb.178.24.7285-7294.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Lenne-Samuel N, Wagner J, Etienne H, Fuchs RP. The processivity factor beta controls DNA polymerase IV traffic during spontaneous mutagenesis and translesion synthesis in vivo. EMBO Rep. 2002;3:45–49. doi: 10.1093/embo-reports/kvf007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. 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;107:91–102. doi: 10.1016/s0092-8674(01)00515-3. [DOI] [PubMed] [Google Scholar]
  43. Lone S, Townson SA, Uljon SN, Johnson RE, Brahma A, Nair DT, Prakash S, Prakash L, Aggarwal AK. Human DNA Polymerase κ Encircles DNA: Implications for Mismatch Extension and Lesion Bypass. Mol Cell. 2007;25:601–614. doi: 10.1016/j.molcel.2007.01.018. [DOI] [PubMed] [Google Scholar]
  44. McGrew DA, Knight KL. Molecular design and functional organization of the RecA protein. Crit Rev Biochem Mol Biol. 2003;38:385–432. doi: 10.1080/10409230390242489. [DOI] [PubMed] [Google Scholar]
  45. McKenzie GJ, Lee PL, Lombardo MJ, Hastings PJ, Rosenberg SM. SOS mutator DNA polymerase IV functions in adaptive mutation and not adaptive amplification. Mol Cell. 2001;7:571–579. doi: 10.1016/s1097-2765(01)00204-0. [DOI] [PubMed] [Google Scholar]
  46. Niehbuhr K, Wehland J. Screening of Antibody Epitopes and Regions of Protein-Protein Interaction Sites Using SPOT Peptides. In: Lefkovits I, editor. Immunology Methods Manual: The Comprehensive Sourcebook of Techniques. San Diego, CA: Academic Press; 1997. [Google Scholar]
  47. Nohmi T. Environmental stress and lesion-bypass DNA polymerases. Annu Rev Microbiol. 2006;60:231–253. doi: 10.1146/annurev.micro.60.080805.142238. [DOI] [PubMed] [Google Scholar]
  48. Nohmi T, Battista JR, Dodson LA, Walker GC. RecA-mediated cleavage activates UmuD for mutagenesis: mechanistic relationship between transcriptional derepression and posttranslational activation. Proc Natl Acad Sci U S A. 1988;85:1816–1820. doi: 10.1073/pnas.85.6.1816. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Ogi T, Kannouche P, Lehmann AR. Localisation of human Y-family DNA polymerase kappa: relationship to PCNA foci. J Cell Sci. 2005;118:129–136. doi: 10.1242/jcs.01603. [DOI] [PubMed] [Google Scholar]
  50. Ohmori H, Friedberg EC, Fuchs RP, Goodman MF, Hanaoka F, Hinkle D, Kunkel TA, Lawrence CW, Livneh Z, Nohmi T, et al. The Y-family of DNA polymerases. Mol Cell. 2001;8:7–8. doi: 10.1016/s1097-2765(01)00278-7. [DOI] [PubMed] [Google Scholar]
  51. Potapova O, Grindley ND, Joyce CM. The mutational specificity of the Dbh lesion bypass polymerase and its implications. J Biol Chem. 2002;277:28157–28166. doi: 10.1074/jbc.M202607200. [DOI] [PubMed] [Google Scholar]
  52. Reuven NB, Arad G, Maor-Shoshani A, Livneh Z. The mutagenesis protein UmuC is a DNA polymerase activated by UmuD′, RecA, and SSB and is specialized for translesion replication. J Biol Chem. 1999;274:31763–31766. doi: 10.1074/jbc.274.45.31763. [DOI] [PubMed] [Google Scholar]
  53. Salles B, Paoletti C. Control of UV induction of RecA protein. Proc Natl Acad Sci U S A. 1983;80:65–69. doi: 10.1073/pnas.80.1.65. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Schlacher K, Cox MM, Woodgate R, Goodman MF. RecA acts in trans to allow replication of damaged DNA by DNA polymerase V. Nature. 2006;442:883–887. doi: 10.1038/nature05042. [DOI] [PubMed] [Google Scholar]
  55. Schlacher K, Leslie K, Wyman C, Woodgate R, Cox MM, Goodman MF. DNA polymerase V and RecA protein, a minimal mutasome. Mol Cell. 2005;17:561–572. doi: 10.1016/j.molcel.2005.01.006. [DOI] [PubMed] [Google Scholar]
  56. Silvian LF, Toth EA, Pham P, Goodman MF, Ellenberger T. Crystal structure of a DinB family error-prone DNA polymerase from Sulfolobus solfataricus. Nat Struct Biol. 2001;8:984–989. doi: 10.1038/nsb1101-984. [DOI] [PubMed] [Google Scholar]
  57. Story RM, Weber IT, Steitz TA. The structure of the E. coli RecA protein monomer and polymer. Nature. 1992;355:318–325. doi: 10.1038/355318a0. [DOI] [PubMed] [Google Scholar]
  58. Sutton MD, Farrow MF, Burton BM, Walker GC. Genetic interactions between the Escherichia coli umuDC gene products and the beta processivity clamp of the replicative DNA polymerase. J Bacteriol. 2001;183:2897–2909. doi: 10.1128/JB.183.9.2897-2909.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Sutton MD, Guzzo A, Narumi I, Costanzo M, Altenbach C, Ferentz AE, Hubbell WL, Walker GC. A model for the structure of the Escherichia coli SOS-regulated UmuD2 protein. DNA Repair (Amst) 2002;1:77–93. doi: 10.1016/s1568-7864(01)00006-4. [DOI] [PubMed] [Google Scholar]
  60. Sutton MD, Opperman T, Walker GC. The Escherichia coli SOS mutagenesis proteins UmuD and UmuD′ interact physically with the replicative DNA polymerase. Proc Natl Acad Sci U S A. 1999;96:12373–12378. doi: 10.1073/pnas.96.22.12373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Sutton MD, Walker GC. Managing DNA polymerases: coordinating DNA replication, DNA repair, and DNA recombination. Proc Natl Acad Sci U S A. 2001a;98:8342–8349. doi: 10.1073/pnas.111036998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Sutton MD, Walker GC. umuDC-mediated cold sensitivity is a manifestation of functions of the UmuD(2)C complex involved in a DNA damage checkpoint control. J Bacteriol. 2001b;183:1215–1224. doi: 10.1128/JB.183.4.1215-1224.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Tang M, Shen X, Frank EG, O’Donnell M, Woodgate R, Goodman MF. UmuD′(2)C is an error-prone DNA polymerase, Escherichia coli pol V. Proc Natl Acad Sci U S A. 1999;96:8919–8924. doi: 10.1073/pnas.96.16.8919. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Tompkins JD, Nelson JL, Hazel JC, Leugers SL, Stumpf JD, Foster PL. Error-prone polymerase, DNA polymerase IV, is responsible for transient hypermutation during adaptive mutation in Escherichia coli. J Bacteriol. 2003;185:3469–3472. doi: 10.1128/JB.185.11.3469-3472.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Wagner J, Gruz P, Kim SR, Yamada M, Matsui K, Fuchs RP, Nohmi T. The dinB gene encodes a novel E. coli DNA polymerase, DNA pol IV, involved in mutagenesis. Mol Cell. 1999;4:281–286. doi: 10.1016/s1097-2765(00)80376-7. [DOI] [PubMed] [Google Scholar]
  66. Woodgate R, Ennis DG. Levels of chromosomally encoded Umu proteins and requirements for in vivo UmuD cleavage. Mol Gen Genet. 1991;229:10–16. doi: 10.1007/BF00264207. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

01

Supplemental Data

Supplemental Data includes 9 figures and 2 tables.

NIHMS37593-supplement-01.pdf (964KB, pdf)

RESOURCES