DNA polymerase switching: effects on spontaneous mutagenesis in Escherichia coli

Escherichia coli possesses five known DNA polymerases (pols). Pol III holoenzyme is the cell's main replicase, while pol I is responsible for the maturation of Okazaki fragments and filling gaps generated during nucleotide excision repair. Pols II, IV and V are significantly upregulated as part of the cell's global SOS response to DNA damage and under these conditions, may alter the fidelity of DNA replication by potentially interfering with the ability of pols I and III to complete their cellular functions. To test this hypothesis, we determined the spectrum of rpoB mutations arising in an isogenic set of mutL strains differentially expressing the chromosomally encoded pols. Interestingly, mutagenic hot spots in rpoB were identified that are susceptible to the actions of pols I–V. For example, in a recA730 lexA (Def) mutL background most transversions were dependent upon pols IV and V. In contrast, transitions were largely dependent upon pol I and to a lesser extent, pol III. Furthermore, the extent of pol I-dependent mutagenesis at one particular site was modulated by pols II and IV. Our observations suggest that there is considerable interplay among all five E. coli polymerases that either reduces or enhances the mutagenic load on the E. coli chromosome.     

Lyase activities intrinsic to Escherichia coli polymerases IV and V

Escherichia coli DNA polymerase IV and V (pol IV and pol V) are error-prone DNA polymerases that are induced as part of the SOS regulon in response to DNA damage. Both are members of the Y-family of DNA polymerases. Their principal biological roles appear to involve translesion synthesis (TLS) and the generation of mutational diversity to cope with stress. Although neither enzyme is known to be involved in base excision repair (BER), we have nevertheless observed apurinic/apyrimidinic 5'-deoxyribose phosphate (AP/5'-dRP) lyase activities intrinsic to each polymerase. Pols IV and V catalyze cleavage of the phosphodiester backbone at the 3'-side of an apurinic/apyrimidinic (AP) site as well as the removal of a 5'-deoxyribose phosphate (dRP) at a preincised AP site. The specific activities of the two error-prone polymerase-associated lyases are approximately 80-fold less than the associated lyase activity of human DNA polymerase beta, which is a key enzyme used in short patch BER. Pol IV forms a covalent Schiff's base intermediate with substrate DNA that is trapped by sodium borohydride, as proscribed by a beta-elimination mechanism. In contrast, a NaBH(4) trapped intermediate is not observed for pol V, even though the lyase specific activity of pol V is slightly higher than that of pol IV. Incubation of pol V (UmuD'(2)C) with a molar excess of UmuD drives an exchange of subunits to form UmuD'D+insoluble UmuC causing inactivation of polymerase and lyase activities. The concomitant loss of both activities is strong evidence that pol V contains a bona fide lyase activity.     

Replication slippage of different DNA polymerases is inversely related to their strand displacement efficiency.

Replication slippage is a particular type of error caused by DNA polymerases believed to occur both in bacterial and eukaryotic cells. Previous studies have shown that deletion events can occur in Escherichia coli by replication slippage between short duplications and that the main E. coli polymerase, DNA polymerase III holoenzyme is prone to such slippage. In this work, we present evidence that the two other DNA polymerases of E. coli, DNA polymerase I and DNA polymerase II, as well as polymerases of two phages, T4 (T4 pol) and T7 (T7 pol), undergo slippage in vitro, whereas DNA polymerase from another phage, Phi29, does not. Furthermore, we have measured the strand displacement activity of the different polymerases tested for slippage in the absence and in the presence of the E. coli single-stranded DNA-binding protein (SSB), and we show that: (i) polymerases having a strong strand displacement activity cannot slip (DNA polymerase from Phi29); (ii) polymerases devoid of any strand displacement activity slip very efficiently (DNA polymerase II and T4 pol); and (iii) stimulation of the strand displacement activity by E. coli SSB (DNA polymerase I and T7 pol), by phagic SSB (T4 pol), or by a mutation that affects the 3' --> 5' exonuclease domain (DNA polymerase II exo(-) and T7 pol exo(-)) is correlated with the inhibition of slippage. We propose that these observations can be interpreted in terms of a model, for which we have shown that high strand displacement activity of a polymerase diminishes its propensity to slip.     

Two processivity clamp interactions differentially alter the dual activities of UmuC

DNA polymerases of the Y family promote survival by their ability to synthesize past lesions in the DNA template. One Escherichia coli member of this family, DNA pol V (UmuC), which is primarily responsible for UV-induced and chemically induced mutagenesis, possesses a canonical beta processivity clamp-binding motif. A detailed analysis of this motif in DNA pol V (UmuC) showed that mutation of only two residues in UmuC is sufficient to result in a loss of UV-induced mutagenesis. Increased levels of wild-type beta can partially rescue this loss of mutagenesis. Alterations in this motif of UmuC also cause loss of the cold-sensitive and beta-dependent synthetic lethal phenotypes associated with increased levels of UmuD and UmuC that are thought to represent an exaggeration of a DNA damage checkpoint. By designing compensatory mutations in the cleft between domains II and III in beta, we restored UV-induced mutagenesis by a UmuC beta-binding motif variant. A recent co-crystal structure of the 'little finger' domain of E. coli pol IV (DinB) with beta suggests that, in addition to the canonical beta-binding motif, a second site of pol IV ((303)VWP(305)) interacts with beta at the outer rim of the dimer interface. Mutational analysis of the corresponding motif in UmuC showed that it is dispensable for induced mutagenesis, but that alterations in this motif result in loss of the cold-sensitive phenotype. These two beta interaction sites of UmuC affect the dual functions of UmuC differentially and indicate subtle and sophisticated polymerase management by the beta clamp.     

Simple and efficient purification of Escherichia coli DNA polymerase V: cofactor requirements for optimal activity and processivity in vitro

Most damage induced mutagenesis in Escherichia coli is dependent upon the UmuD'(2)C protein complex, which comprises DNA polymerase V (pol V). Biochemical characterization of pol V has been hindered by the fact that the enzyme is notoriously difficult to purify, largely because overproduced UmuC is insoluble. Here, we report a simple and efficient protocol for the rapid purification of milligram quantities of pol V from just 4 L of bacterial culture. Rather than over producing the UmuC protein, it was expressed at low basal levels, while UmuD'(2)C was expressed in trans from a high copy-number plasmid with an inducible promoter. We have also developed strategies to purify the β-clamp and γ-clamp loader free from contaminating polymerases. Using these highly purified proteins, we determined the cofactor requirements for optimal activity of pol V in vitro and found that pol V shows robust activity on an SSB-coated circular DNA template in the presence of the β/γ-complex and a RecA nucleoprotein filament (RecA*) formed in trans. This strong activity was attributed to the unexpectedly high processivity of pol V Mut (UmuD'(2)C · RecA · ATP), which was efficiently recruited to a primer terminus by SSB.     

Purification and characterization of an inducible Escherichia coli DNA polymerase capable of insertion and bypass at abasic lesions in DNA

We have investigated the ability of DNA polymerases from SOS-induced and uninduced Escherichia coli to incorporate nucleotides at a well-defined abasic (apurinic/apyrimidinic) DNA template site and to extend these chains from this unpaired 3' terminus. A DNA polymerase activity has been purified from E. coli, deleted for DNA polymerase I, that appears to be induced 7-fold in cells following treatment with nalidixic acid. Induction of this polymerase (designated DNA polymerase X) appears to be part of the SOS response of E. coli since it cannot be induced in strains containing a noncleavable form of the LexA repressor (Ind-). The enzyme is able to incorporate nucleotides efficiently opposite the abasic template lesion and to continue DNA synthesis. Although we observe an approximate 2-fold induction of DNA polymerase III in cells treated with nalidixic acid, several lines of evidence argue that DNA polymerase X is unrelated to DNA polymerase III (pol III). In contrast to pol X, pol III shows almost no detectable ability to incorporate at or extend beyond the abasic site; incorporation efficiency at the abasic lesion is at least 100-fold larger for pol X compared to pol III holoenzyme, pol III core, or pol III* (the polymerase III holoenzyme subassembly lacking the beta subunit). Pol X does not cross-react with polyclonal antibody directed against pol III holoenzyme complex or with monoclonal antibody prepared to the alpha subunit of pol III. Despite these structural and biochemical differences, pol X appears to interact specifically with the beta subunit of the pol III holoenzyme in the presence of single-stranded binding protein. Pol X has a molecular mass of 84 kDa. Our results indicate that this novel activity is likely to be identical to DNA polymerase II of E. coli.     

Identification of specific amino acid residues in the E. coli beta processivity clamp involved in interactions with DNA polymerase III, UmuD and UmuD'

Variants of a pentapeptide sequence (QL[S/F]LF), referred to as the eubacterial clamp-binding motif, appear to be required for certain proteins to bind specifically to the Escherichia coli beta sliding clamp, apparently by making contact with a hydrophobic pocket located at the base of the C-terminal tail of each beta protomer. Although both UmuC (DNA pol V) and the alpha catalytic subunit of DNA polymerase III (pol III) each bear a reasonable match to this motif, which appears to be required for their respective interactions with the clamp, neither UmuD not UmuD' do. As part of an ongoing effort to understand how interactions involving the different E. coli umuDC gene products and components of DNA polymerase III help to coordinate DNA replication with a DNA damage checkpoint control and translesion DNA synthesis (TLS) following DNA damage, we characterized the surfaces on beta important for its interactions with the two forms of the umuD gene product. We also characterized the surface of beta important for its interaction with the alpha catalytic subunit of pol III. Our results indicate that although UmuD, UmuD' and alpha share some common contacts with beta, each also makes unique contacts with the clamp. These findings suggest that differential interactions of UmuD and UmuD' with beta impose a DNA damage-responsive conditionality on how beta interacts with the translesion DNA polymerase UmuC. This is formally analogous to how post-translational modification of the eukaryotic PCNA clamp influences mutagenesis. We discuss the implications of our findings in terms of how E. coli might coordinate the actions of the umuDC gene products with those of pol III, as well as for how organisms in general might manage the actions of their multiple DNA polymerases.     

DNA polymerase V and RecA protein, a minimal mutasome

A hallmark of the Escherichia coli SOS response is the large increase in mutations caused by translesion synthesis (TLS). TLS requires DNA polymerase V (UmuD'2C) and RecA. Here, we show that pol V and RecA interact by two distinct mechanisms. First, pol V binds to RecA in the absence of DNA and ATP and second, through its UmuD' subunit, requiring DNA and ATP without ATP hydrolysis. TLS occurs in the absence of a RecA nucleoprotein filament but is inhibited in its presence. Therefore, a RecA nucleoprotein filament is unlikely to be required for SOS mutagenesis. Pol V activity is severely diminished in the absence of RecA or in the presence of RecA1730, a mutant defective for pol V mutagenesis in vivo. Pol V activity is strongly enhanced with RecA mutants constitutive for mutagenesis in vivo, suggesting that RecA is an obligate accessory factor that activates pol V for SOS mutagenesis.     

(5'S)-8,5'-cyclo-2'-deoxyguanosine is a strong block to replication, a potent pol V-dependent mutagenic lesion, and is inefficiently repaired in Escherichia coli

8,5'-Cyclopurines, making up an important class of ionizing radiation-induced tandem DNA damage, are repaired only by nucleotide excision repair (NER). They accumulate in NER-impaired cells, as in Cockayne syndrome group B and certain Xeroderma Pigmentosum patients. A plasmid containing (5'S)-8,5'-cyclo-2'-deoxyguanosine (S-cdG) was replicated in Escherichia coli with specific DNA polymerase knockouts. Viability was <1% in the wild-type strain, which increased to 5.5% with SOS. Viability decreased further in a pol II(-) strain, whereas it increased considerably in a pol IV(-) strain. Remarkably, no progeny was recovered from a pol V(-) strain, indicating that pol V is absolutely required for bypassing S-cdG. Progeny analyses indicated that S-cdG is significantly mutagenic, inducing ~34% mutation with SOS. Most mutations were S-cdG → A mutations, though S-cdG → T mutation and deletion of 5'C also occurred. Incisions of purified UvrABC nuclease on S-cdG, S-cdA, and C8-dG-AP on a duplex 51-mer showed that the incision rates are C8-dG-AP > S-cdA > S-cdG. In summary, S-cdG is a major block to DNA replication, highly mutagenic, and repaired slowly in E. coli.     

Evolution of the two-step model for UV-mutagenesis

It is quite remarkable how our understanding of translesion DNA synthesis (TLS) has changed so dramatically in the past 2 years. Until very recently, little was known about the molecular mechanisms of TLS in higher eukaryotes and what we did know, was largely based upon Escherichia coli and Saccharomyces cerevisiae model systems. The paradigm, proposed by Bryn Bridges and I [Mutat. Res. 150 (1985) 133] in 1985, was that error-prone TLS occurred in two steps; namely a misinsertion event opposite a lesion, followed by extension of the mispair so as to facilitate complete bypass of the lesion. The initial concept was that at least for E. coli, the misinsertion event was performed by the cell's main replicase, DNA polymerase III holoenzyme, and that elongation was achieved through the actions of specialized polymerase accessory proteins, such as UmuD and UmuC. Some 15 years later, we now know that this view is likely to be incorrect in that both misinsertion and bypass are performed by the Umu proteins (now called pol V). As pol V is normally a distributive enzyme, pol III may only be required to "fix" the misincorporation as a mutation by completing chromosome duplication. However, while the role of the E. coli proteins involved in TLS have changed, the initial concept of misincorporation followed by extension/bypass remains valid. Indeed, recent evidence suggests that it can equally be applied to TLS in eukaryotic cells where there are many more DNA polymerases to choose from. The aim of this review is, therefore, to provide a historical perspective to the "two-step" model for UV-mutagenesis, how it has recently evolved, and in particular, to highlight the seminal contributions made to it by Bryn Bridges.     

Critical amino acids in Escherichia coli UmuC responsible for sugar discrimination and base-substitution fidelity

The active form of Escherichia coli DNA polymerase V responsible for damage-induced mutagenesis is a multiprotein complex (UmuD'(2)C-RecA-ATP), called pol V Mut. Optimal activity of pol V Mut in vitro is observed on an SSB-coated single-stranded circular DNA template in the presence of the β/γ complex and a transactivated RecA nucleoprotein filament, RecA*. Remarkably, under these conditions, wild-type pol V Mut efficiently incorporates ribonucleotides into DNA. A Y11A substitution in the 'steric gate' of UmuC further reduces pol V sugar selectivity and converts pol V Mut into a primer-dependent RNA polymerase that is capable of synthesizing long RNAs with a processivity comparable to that of DNA synthesis. Despite such properties, Y11A only promotes low levels of spontaneous mutagenesis in vivo. While the Y11F substitution has a minimal effect on sugar selectivity, it results in an increase in spontaneous mutagenesis. In comparison, an F10L substitution increases sugar selectivity and the overall fidelity of pol V Mut. Molecular modeling analysis reveals that the branched side-chain of L10 impinges on the benzene ring of Y11 so as to constrict its movement and as a consequence, firmly closes the steric gate, which in wild-type enzyme fails to guard against ribonucleoside triphosphates incorporation with sufficient stringency.     

Rat DNA polymerase beta gene can join in excision repair of Escherichia coli.

Though DNA polymerase I (poll) of Escherichia (E.) coli is understood to play a role in repair synthesis of excision repair, it is still obscure whether DNA polymerase beta (pol beta) plays a similar role in eukaryotic cells. To estimate the role of pol beta in excision repair processes, we inserted the rat pol beta gene into several mutant E. coli defective in a diverse set of enzymatic activities of poll. UV resistance was seen only when the 5'----3' exonuclease (exo) activity of poll molecules remained. Therefore it is suggested that 5'----3' exo activity as well as pol beta activity are essential for repair synthesis of excision repair in eukaryotic cells.     

Specific amino acid residues in the beta sliding clamp establish a DNA polymerase usage hierarchy in Escherichia coli

Escherichia coli dnaN159 strains encode a mutant form of the beta sliding clamp (beta159), causing them to display altered DNA polymerase (pol) usage. In order to better understand mechanisms of pol selection/switching in E. coli, we have further characterized pol usage in the dnaN159 strain. The dnaN159 allele contains two amino acid substitutions: G66E (glycine-66 to glutamic acid) and G174A (glycine-174 to alanine). Our results indicated that the G174A substitution impaired interaction of the beta clamp with the alpha catalytic subunit of pol III. In light of this finding, we designed two additional dnaN alleles. One of these dnaN alleles contained a G174A substitution (beta-G174A), while the other contained D173A, G174A and H175A substitutions (beta-173-175). Examination of strains bearing these different dnaN alleles indicated that each conferred a distinct UV sensitive phenotype that was dependent upon a unique combination of Delta polB (pol II), Delta dinB (pol IV) and/or Delta umuDC (pol V) alleles. Taken together, these findings indicate that mutations in the beta clamp differentially affect the functions of these three pols, and suggest that pol II, pol IV and pol V are capable of influencing each others' abilities to gain access to the replication fork. These findings are discussed in terms of a model whereby amino acid residues in the vicinity of those mutated in beta159 (G66 and G174) help to define a DNA polymerase usage hierarchy in E. coli following UV irradiation.     

Efficiency and accuracy of SOS-induced DNA polymerases replicating benzo[a]pyrene-7,8-diol 9,10-epoxide A and G adducts.

Nucleotide incorporation fidelity, mismatch extension, and translesion DNA synthesis efficiencies were determined using SOS-induced Escherichia coli DNA polymerases (pol) II, IV, and V to copy 10R and 10S isomers of trans-opened benzo[a]pyrene-7,8-diol 9,10-epoxide (BaP DE) A and G adducts. A-BaP DE adducts were bypassed by pol V with moderate accuracy and considerably higher efficiency than by pol II or IV. Error-prone pol V copied G-BaP DE-adducted DNA poorly, forming A*G-BaP DE-S and -R mismatches over C*G-BaP DE-S and -R correct matches by factors of approximately 350- and 130-fold, respectively, even favoring G*G-BaP DE mismatches over correct matches by factors of 2-4-fold. In contrast, pol IV bypassed G-BaP DE adducts with the highest efficiency and fidelity, making misincorporations with a frequency of 10(-2) to 10(-4) depending on sequence context. G-BaP DE-S-adducted M13 DNA yielded 4-fold fewer plaques when transfected into SOS-induced DeltadinB (pol IV-deficient) mutant cells compared with the isogenic wild-type E. coli strain, consistent with the in vitro data showing that pol IV was most effective by far at copying the G-BaP DE-S adduct. SOS polymerases are adept at copying a variety of lesions, but the relative contribution of each SOS polymerase to copying damaged DNA appears to be determined by the lesion's identity.     

Roles of chromosomal and episomal dinB genes encoding DNA pol IV in targeted and untargeted mutagenesis in Escherichia coli

DNA polymerase IV (pol IV) in Escherichia coli is a member of a novel family of DNA polymerases (the DinB/UmuC/Rad30/Rev1 super-family or the DNA polymerase Y family). Although expression of the dinB gene encoding DNA pol IV is known to result in an enhancement of untargeted mutagenesis, it remains uncertain whether DNA pol IV is involved in a variety of lesion-induced mutagenesis (targeted mutagenesis), and the relationship between expression levels of dinB and the mutagenesis that DNA pol IV promotes has not been investigated thoroughly. Here, we report that DNA pol IV is involved in -1 frameshift mutagenesis induced by 4-nitroquinoline N-oxide (4-NQO) and that the expression level of the chromosomal pol IV gene is 6-12 times higher than those for other SOS-inducible DNA polymerases in E. coli, i.e., DNA pol II (PolB) or DNA pol V (UmuDC), respectively. Interestingly, the dinB gene is present not only on the chromosome but also on the F' plasmid in the E. coli CC108 strain. In this strain, 750 molecules of DNA pol IV are expressed from the F' dinB gene in the uninduced state and 250 molecules are expressed from the chromosomal gene. These cellular expression levels strongly affect -1 frameshifts induced by 4-NQO in runs of six guanine bases: mutagenicity was highest in the strain CC108, followed by strains YG2242 (chromosome deltadinB/F' dinB+), YG2247 (chromosome dinB+/F' deltadinB) and FC1243 (chromosome deltadinB/F' deltadinB). The incidence of untargeted -1 frameshifts was reduced by two-thirds on deletion of dinB from the F' episome. The chromosomal dinB gene appeared to have little or no effect on the untargeted mutagenesis. These results suggest that DNA pol IV efficiently mediates targeted mutagenesis by 4-NQO, and that the cellular levels of expression substantially affect targeted and untargeted mutagenesis.     

Production of "authentic" poliovirus RNA-dependent RNA polymerase (3D(pol)) by ubiquitin-protease-mediated cleavage in Escherichia coli.

The first amino acid of "authentic" poliovirus RNA-dependent RNA polymerase, 3D(pol), is a glycine. As a result, production of 3D(pol) in Escherichia coli requires addition of an initiation codon; thus, a formylmethionine is added to the amino terminus. The formylmethionine should be removed by the combined action of a cellular deformylase and methionine aminopeptidase. However, high-level expression of 3D(pol) in E. coli yields enzyme with a heterogeneous amino terminus. To preclude this problem, we developed a new expression system for 3D(pol). This system exploits the observation that proteins fused to the carboxyl terminus of ubiquitin can be processed in E. coli to produce proteins with any amino acid as the first residue when expressed in the presence of a ubiquitin-specific, carboxy-terminal protease. By using this system, authentic 3D(pol) can be obtained in yields of 30-60 mg per liter of culture. While addition of a single glycine, alanine, serine, or valine to the amino terminus of 3D(pol) produced derivatives with a specific activity reduced by at least 25-fold relative to wild-type enzyme, addition of a methionine to the amino terminus resulted in some processing to yield enzyme with a glycine amino terminus. Addition of a hexahistidine tag to the carboxyl terminus of 3D(pol) had no deleterious effect on the activity of the enzyme. The utility of this expression system for production of other viral polymerases and accessory proteins is discussed.     

Synthetic activity of Sso DNA polymerase Y1, an archaeal DinB-like DNA polymerase, is stimulated by processivity factors proliferating cell nuclear antigen and replication factor C

DNA replication efficiency is dictated by DNA polymerases (pol) and their associated proteins. The recent discovery of DNA polymerase Y family (DinB/UmuC/RAD30/REV1 superfamily) raises a question of whether the DNA polymerase activities are modified by accessory proteins such as proliferating cell nuclear antigen (PCNA). In fact, the activity of DNA pol IV (DinB) of Escherichia coli is enhanced upon interaction with the beta subunit, the processivity factor of DNA pol III. Here, we report the activity of Sso DNA pol Y1 encoded by the dbh gene of the archaeon Sulfolobus solfataricus is greatly enhanced by the presence of PCNA and replication factor C (RFC). Sso pol Y1 per se was a distributive enzyme but a substantial increase in the processivity was observed on poly(dA)-oligo(dT) in the presence of PCNA (039p or 048p) and RFC. The length of the synthesized DNA product reached at least 200 nucleotides. Sso pol Y1 displayed a higher affinity for DNA compared with pol IV of E. coli, suggesting that the two DNA polymerases have distinct reason(s) to require the processivity factors for efficient DNA synthesis. The abilities of pol Y1 and pol IV to bypass DNA lesions and their sensitive sites to protease are also discussed.     

Resolving a fidelity paradox: why Escherichia coli DNA polymerase II makes more base substitution errors in AT- compared with GC-rich DNA.

The activity of DNA polymerase-associated proofreading 3'-exonucleases is generally enhanced in less stable DNA regions leading to a reduction in base substitution error frequencies in AT- versus GC-rich sequences. Unexpectedly, however, the opposite result was found for Escherichia coli DNA polymerase II (pol II). Nucleotide misincorporation frequencies for pol II were found to be 3-5-fold higher in AT- compared with GC-rich DNA, both in the presence and absence of polymerase processivity subunits, beta dimer and gamma complex. In contrast, E. coli pol III holoenzyme, behaving "as expected," exhibited 3-5-fold lower misincorporation frequencies in AT-rich DNA. A reduction in fidelity in AT-rich regions occurred for pol II despite having an associated 3'-exonuclease proofreading activity that preferentially degrades AT-rich compared with GC-rich DNA primer-template in the absence of DNA synthesis. Concomitant with a reduction in fidelity, pol II polymerization efficiencies were 2-6-fold higher in AT-rich DNA, depending on sequence context. Pol II paradoxical fidelity behavior can be accounted for by the enzyme's preference for forward polymerization in AT-rich sequences. The more efficient polymerization suppresses proofreading thereby causing a significant increase in base substitution error rates in AT-rich regions.