An SOS-regulated operon involved in damage-inducible mutagenesis in Caulobacter crescentus

DNA polymerases of the Y-family, such as Escherichia coli UmuC and DinB, are specialized enzymes induced by the SOS response, which bypass lesions allowing the continuation of DNA replication. umuDC orthologs are absent in Caulobacter crescentus and other bacteria, raising the question about the existence of SOS mutagenesis in these organisms. Here, we report that the C.crescentus dinB ortholog is not involved in damage-induced mutagenesis. However, an operon composed of two hypothetical genes and dnaE2, encoding a second copy of the catalytic subunit of Pol III, is damage inducible in a recA-dependent manner, and is responsible for most ultraviolet (UV) and mitomycin C-induced mutations in C.crescentus. The results demonstrate that the three genes are required for the error-prone processing of DNA lesions. The two hypothetical genes were named imuA and imuB, after inducible mutagenesis. ImuB is similar to proteins of the Y-family of polymerases, and possibly cooperates with DnaE2 in lesion bypass. The mutations arising as a consequence of the activity of the imuAB dnaE2 operon are rather unusual for UV irradiation, including G:C to C:G transversions.     

Involvement of DnaE, the second replicative DNA polymerase from Bacillus subtilis, in DNA mutagenesis

In a large group of organisms including low G + C bacteria and eukaryotic cells, DNA synthesis at the replication fork strictly requires two distinct replicative DNA polymerases. These are designated pol C and DnaE in Bacillus subtilis. We recently proposed that DnaE might be preferentially involved in lagging strand synthesis, whereas pol C would mainly carry out leading strand synthesis. The biochemical analysis of DnaE reported here is consistent with its postulated function, as it is a highly potent enzyme, replicating as fast as 240 nucleotides/s, and stalling for more than 30 s when encountering annealed 5'-DNA end. DnaE is devoid of 3' --> 5'-proofreading exonuclease activity and has a low processivity (1-75 nucleotides), suggesting that it requires additional factors to fulfill its role in replication. Interestingly, we found that (i) DnaE is SOS-inducible; (ii) variation in DnaE or pol C concentration has no effect on spontaneous mutagenesis; (iii) depletion of pol C or DnaE prevents UV-induced mutagenesis; and (iv) purified DnaE has a rather relaxed active site as it can bypass lesions that generally block other replicative polymerases. These results suggest that DnaE and possibly pol C have a function in DNA repair/mutagenesis, in addition to their role in DNA replication.     

Translesion-synthesis DNA polymerases participate in replication of the telomeres in Streptomyces

Linear chromosomes and linear plasmids of Streptomyces are capped by terminal proteins that are covalently bound to the 5'-ends of DNA. Replication is initiated from an internal origin, which leaves single-stranded gaps at the 3'-ends. These gaps are patched by terminal protein-primed DNA synthesis. Streptomyces contain five DNA polymerases: one DNA polymerase I (Pol I), two DNA polymerases III (Pol III) and two DNA polymerases IV (Pol IV). Of these, one Pol III, DnaE1, is essential for replication, and Pol I is not required for end patching. In this study, we found the two Pol IVs (DinB1 and DinB2) to be involved in end patching. dinB1 and dinB2 could not be co-deleted from wild-type strains containing a linear chromosome, but could be co-deleted from mutant strains containing a circular chromosome. The resulting ΔdinB1 ΔdinB2 mutants supported replication of circular but not linear plasmids, and exhibited increased ultraviolet sensitivity and ultraviolet-induced mutagenesis. In contrast, the second Pol III, DnaE2, was not required for replication, end patching, or ultraviolet resistance and mutagenesis. All five polymerase genes are relatively syntenous in the Streptomyces chromosomes, including a 4-bp overlap between dnaE2 and dinB2. Phylogenetic analysis showed that the dinB1-dinB2 duplication occurred in a common actinobacterial ancestor.     

DNA polymerase I acts in translesion synthesis mediated by the Y-polymerases in Bacillus subtilis

Translesion synthesis (TLS) across damaged DNA bases is most often carried out by the ubiquitous error-prone DNA polymerases of the Y-family. Bacillus subtilis encodes two Y-polymerases, Pol Y1 and Pol Y2, that mediate TLS resulting in spontaneous and ultraviolet light (UV)-induced mutagenesis respectively. Here we show that TLS is a bipartite dual polymerase process in B. subtilis, involving not only the Y-polymerases but also the A-family polymerase, DNA polymerase I (Pol I). Both the spontaneous and the UV-induced mutagenesis are abolished in Pol I mutants affected solely in the polymerase catalytic site. Physical interactions between Pol I and either of the Pol Y polymerases, as well as formation of a ternary complex between Pol Y1, Pol I and the beta-clamp, were detected by yeast two- and three-hybrid assays, supporting the model of a functional coupling between the A- and Y-family polymerases in TLS. We suggest that the Pol Y carries the synthesis across the lesion, and Pol I takes over to extend the synthesis until the functional replisome resumes replication. This key role of Pol I in TLS uncovers a new function of the A-family DNA polymerases.     

Stationary-phase mutations in proofreading exonuclease-deficient strains of the yeast Saccharomyces cerevisiae

In order to understand the role of yeast polymerases in spontaneous mutagenesis in non-growing cells we have studied the effects of mutations that impair the 3'--> 5' exonuclease function of polymerases delta (pol3-01) and epsilon (pol2-4) on the spontaneous reversion frequency of the frameshift mutation his7-2 in cells starved for histidine. We showed that for each exonuclease-deficient mutant the rate of reversion per viable cell per day observed in stationary-phase cells remained constant up to the 9th day of starvation (while the number of viable cells dropped), and was very similar to that observed in the same mutants during the growth phase. These data suggest that both DNA polymerases are involved in the control of mutability in non-growing cells.     

Mutations in human DNA polymerase eta motif II alter bypass of DNA lesions.

Human DNA polymerase eta (hPol eta) is one of the newly identified Y-family of DNA polymerases. These polymerases synthesize past template lesions that are postulated to block replication fork progression. hPol eta accurately bypasses UV-associated cis-syn cyclobutane thymine dimers in vitro and contributes to normal resistance to sunlight-induced skin cancer. We describe here mutational analysis of motif II, a highly conserved sequence, recently reported to reside in the fingers domain and to form part of the active site in Y-family DNA polymerases. We used a yeast-based complementation system to isolate biologically active mutants created by random sequence mutagenesis, synthesized the mutant proteins in vitro and assessed their ability to bypass thymine dimers. The mutability of motif II in 210 active mutants has parallels with natural evolution and identifies Tyr52 and Ala54 as prime candidates for involvement in catalytic activity or bypass. We describe the ability of hPol eta S62G, a mutant polymerase with enhanced activity, to bypass five other site-specific lesions. Our results may serve as a prototype for studying other members of the Y-family DNA polymerases.     

Erroneous incorporation of oxidized DNA precursors by Y-family DNA polymerases

Deranged oxidative metabolism is a property of many tumour cells. Oxidation of the deoxynucleotide triphosphate (dNTP) pool, as well as DNA, is a major cause of genome instability. Here, we report that two Y-family DNA polymerases of the archaeon Sulfolobus solfataricus strains P1 and P2 incorporate oxidized dNTPs into nascent DNA in an erroneous manner: the polymerases exclusively incorporate 8-OH-dGTP opposite adenine in the template, and incorporate 2-OH-dATP opposite guanine more efficiently than opposite thymine. The rate of extension of the nascent DNA chain following on from these incorporated analogues is only slightly reduced. These DNA polymerases have been shown to bypass a variety of DNA lesions. Thus, our results suggest that the Y-family DNA polymerases promote mutagenesis through the erroneous incorporation of oxidized dNTPs during DNA synthesis, in addition to facilitating translesion DNA synthesis. We also report that human DNA polymerase η, a human Y-family DNA polymerase, incorporates the oxidized dNTPs in a similar erroneous manner.     

Structure-based interpretation of missense mutations in Y-family DNA polymerases and their implications for polymerase function and lesion bypass

Our understanding of the molecular mechanisms of error-prone lesion bypass has changed dramatically in the past few years. The concept that the key participants in the mutagenic process were accessory proteins that somehow modified the ability of the cell's main replicase to facilitate bypass of normally blocking lesions has been replaced with one in which the replicase is displaced by a polymerase specialized in lesion bypass. The participants in this process remain the same, only their function has been reassigned. What was once known as the UmuC/DinB/Rev1/Rad30 superfamily of mutagenesis proteins, is now known as the Y-family of DNA polymerases. Quite remarkably, within the space of 3 years, the field has advanced from the initial discovery of intrinsic polymerase function, to the determination of the tertiary structures of several Y-family DNA polymerases.A key to determining the biochemical properties of each DNA polymerase is through structure-function studies that result in the site-specific substitution of particular amino acids at critical sites within each DNA polymerase. However, we should not forget the power of genetic selection that allows us to identify residues within each polymerase that are generated by "random mutagenesis" and which are important for both a gain or loss of function in vivo. In this review, we discuss the structural ramifications of several missense mutations previously identified in various Y-family DNA polymerase and speculate on how each amino acid substitution might modify the enzymatic activity of the respective polymerase or possibly perturb protein-protein interactions necessary for efficient translesion replication in vivo.     

Diverse roles of RAD18 and Y-family DNA polymerases in tumorigenesis

Mutagenesis is a hallmark and enabling characteristic of cancer cells. The E3 ubiquitin ligase RAD18 and its downstream effectors, the ‘Y-family’ Trans-Lesion Synthesis (TLS) DNA polymerases, confer DNA damage tolerance at the expense of DNA replication fidelity. Thus, RAD18 and TLS polymerases are attractive candidate mediators of mutagenesis and carcinogenesis. The skin cancer-propensity disorder xeroderma pigmentosum-variant (XPV) is caused by defects in the Y-family DNA polymerase Pol eta (Polη). However it is unknown whether TLS dysfunction contributes more generally to other human cancers. Recent analyses of cancer genomes suggest that TLS polymerases generate many of the mutational signatures present in diverse cancers. Moreover biochemical studies suggest that the TLS pathway is often reprogrammed in cancer cells and that TLS facilitates tolerance of oncogene-induced DNA damage. Here we review recent evidence supporting widespread participation of RAD18 and the Y-family DNA polymerases in the different phases of multi-step carcinogenesis.     

The essential C family DnaE polymerase is error-prone and efficient at lesion bypass

DnaE-type DNA polymerases belong to the C family of DNA polymerases and are responsible for chromosomal replication in prokaryotes. Like most closely related Gram-positive cells, Streptococcus pyogenes has two DnaE homologs Pol C and DnaE; both are essential to cell viability. Pol C is an established replicative polymerase, and DnaE has been proposed to serve a replicative role. In this report, we characterize S. pyogenes DnaE polymerase and find that it is highly error-prone. DnaE can bypass coding and noncoding lesions with high efficiency. Error-prone extension is accomplished by either of two pathways, template-primer misalignment or direct primer extension. The bypass of abasic sites is accomplished mainly through "dNTP-stabilized" misalignment of template, thereby generating (-1) deletions in the newly synthesized strand. This mechanism may be similar to the dNTP-stabilized misalignment mechanism used by the Y family of DNA polymerases and is the first example of lesion bypass and error-prone synthesis catalyzed by a C family polymerase. Thus, DnaE may function in an error-prone capacity that may be essential in Gram-positive cells but not Gram-negative cells, suggesting a fundamental difference in DNA metabolism between these two classes of bacteria.     

Characterization of Escherichia coli UmuC active-site loops identifies variants that confer UV hypersensitivity

DNA is constantly exposed to chemical and environmental mutagens, causing lesions that can stall replication. In order to deal with DNA damage and other stresses, Escherichia coli utilizes the SOS response, which regulates the expression of at least 57 genes, including umuDC. The gene products of umuDC, UmuC and the cleaved form of UmuD, UmuD', form the specialized E. coli Y-family DNA polymerase UmuD'2C, or polymerase V (Pol V). Y-family DNA polymerases are characterized by their specialized ability to copy damaged DNA in a process known as translesion synthesis (TLS) and by their low fidelity on undamaged DNA templates. Y-family polymerases exhibit various specificities for different types of DNA damage. Pol V carries out TLS to bypass abasic sites and thymine-thymine dimers resulting from UV radiation. Using alanine-scanning mutagenesis, we probed the roles of two active-site loops composed of residues 31 to 38 and 50 to 54 in Pol V activity by assaying the function of single-alanine variants in UV-induced mutagenesis and for their ability to confer resistance to UV radiation. We find that mutations of the N-terminal residues of loop 1, N32, N33, and D34, confer hypersensitivity to UV radiation and to 4-nitroquinoline-N-oxide and significantly reduce Pol V-dependent UV-induced mutagenesis. Furthermore, mutating residues 32, 33, or 34 diminishes Pol V-dependent inhibition of recombination, suggesting that these mutations may disrupt an interaction of UmuC with RecA, which could also contribute to the UV hypersensitivity of cells expressing these variants.     

Comprehensive analysis of DNA polymerase III α subunits and their homologs in bacterial genomes

The analysis of ∼2000 bacterial genomes revealed that they all, without a single exception, encode one or more DNA polymerase III α-subunit (PolIIIα) homologs. Classified into C-family of DNA polymerases they come in two major forms, PolC and DnaE, related by ancient duplication. While PolC represents an evolutionary compact group, DnaE can be further subdivided into at least three groups (DnaE1-3). We performed an extensive analysis of various sequence, structure and surface properties of all four polymerase groups. Our analysis suggests a specific evolutionary pathway leading to PolC and DnaE from the last common ancestor and reveals important differences between extant polymerase groups. Among them, DnaE1 and PolC show the highest conservation of the analyzed properties. DnaE3 polymerases apparently represent an ‘impaired’ version of DnaE1. Nonessential DnaE2 polymerases, typical for oxygen-using bacteria with large GC-rich genomes, have a number of features in common with DnaE3 polymerases. The analysis of polymerase distribution in genomes revealed three major combinations: DnaE1 either alone or accompanied by one or more DnaE2s, PolC + DnaE3 and PolC + DnaE1. The first two combinations are present in Escherichia coli and Bacillus subtilis, respectively. The third one (PolC + DnaE1), found in Clostridia, represents a novel, so far experimentally uncharacterized, set.     

Conformational changes during normal and error-prone incorporation of nucleotides by a Y-family DNA polymerase detected by 2-aminopurine fluorescence

Y-family polymerases are specialized to carry out DNA synthesis past sites of DNA damage. Their active sites make fewer contacts to their substrates, consistent with the remarkably low fidelity of these DNA polymerases when copying undamaged DNA. We have used DNA containing the fluorescent reporter 2-aminopurine (2-AP) to study the reaction pathway of the Y-family polymerase Dbh. We detected 3 rapid noncovalent steps between binding of a correctly paired dNTP and the rate-limiting step for dNTP incorporation. These early steps resemble those seen with high-fidelity DNA polymerases, such as Klenow fragment, and include a step that may be related to the unstacking of the 5' neighbor of the templating base that is seen in polymerase ternary complex crystal structures. A significant difference between Dbh and high-fidelity polymerases is that Dbh generates no fluorescence changes subsequent to dNTP binding if the primer lacks a 3'OH, suggesting that the looser active site of Y-family polymerases may enforce reliance on the correct substrate structure in order to assemble the catalytic center. Dbh, like other bypass polymerases of the DinB subgroup, generates single-base deletion errors at an extremely high frequency by skipping over a template base that is part of a repetitive sequence. Using 2-AP as a reporter to study the base-skipping process, we determined that Dbh uses a mechanism in which the templating base slips back to pair with the primer terminus while the base that was originally paired with the primer terminus becomes unpaired.     

Portraits of a Y-family DNA polymerase

Members of the Y-family of DNA polymerases catalyze template-dependent DNA synthesis but share no sequence homology with other known DNA polymerases. Y-family polymerases exhibit high error rates and low processivity when copying normal DNA but are able to synthesize DNA opposite damaged templates. In the past three years, much has been learned about this family of polymerases including determination of more than a dozen crystal structures with various substrates. In this short review, I will summarize the biochemical properties and structural features of Y-family DNA polymerases.     

DnaE2 polymerase contributes to in vivo survival and the emergence of drug resistance in Mycobacterium tuberculosis

The presence of multiple copies of the major replicative DNA polymerase (DnaE) in some organisms, including important pathogens and symbionts, has remained an unresolved enigma. We postulated that one copy might participate in error-prone DNA repair synthesis. We found that UV irradiation of Mycobacterium tuberculosis results in increased mutation frequency in the surviving fraction. We identified dnaE2 as a gene that is upregulated in vitro by several DNA damaging agents, as well as during infection of mice. Loss of this protein reduces both survival of the bacillus after UV irradiation and the virulence of the organism in mice. Our data suggest that DnaE2, and not a member of the Y family of error-prone DNA polymerases, is the primary mediator of survival through inducible mutagenesis and can contribute directly to the emergence of drug resistance in vivo. These results may indicate a potential new target for therapeutic intervention.     

Ubiquitin-family modifications in the replication of DNA damage

The cell uses specialised Y-family DNA polymerases or damage avoidance mechanisms to replicate past damaged sites in DNA. These processes are under complex regulatory systems, which employ different types of post-translational modification. All the Y-family polymerases have ubiquitin binding domains that bind to mono-ubiquitinated PCNA to effect the switching from replicative to Y-family polymerase. Ubiquitination and de-ubiquitination of PCNA are tightly regulated. There is also evidence for another as yet unidentified ubiquitinated protein being involved in recruitment of Y-family polymerases to chromatin. Poly-ubiquitination of PCNA stimulates damage avoidance, and, at least in yeast, PCNA is SUMOylated to prevent unwanted recombination events at the replication fork. The Y-family polymerases themselves can be ubiquitinated and, in the case of DNA polymerase η, this results in the polymerase being excluded from chromatin.     

Y-family DNA polymerases and their role in tolerance of cellular DNA damage

The past 15 years have seen an explosion in our understanding of how cells replicate damaged DNA and how this can lead to mutagenesis. The Y-family DNA polymerases lie at the heart of this process, which is commonly known as translesion synthesis. This family of polymerases has unique features that enable them to synthesize DNA past damaged bases. However, as they exhibit low fidelity when copying undamaged DNA, it is essential that they are only called into play when they are absolutely required. Several layers of regulation ensure that this is achieved.     

Damage repair DNA polymerases Y

The newly found Y-family DNA polymerases are characterized by low fidelity replication using an undamaged template and the ability to carry out translesion DNA synthesis. The crystal structures of three Y-family polymerases, alone or complexed with DNA and nucleotide substrate, reveal a conventional right-hand-like catalytic core consisting of finger, thumb and palm domains. The finger and thumb domains are unusually small resulting in an open and spacious active site, which can accommodate mismatched base pairs as well as various DNA lesions. Although devoid of a 3'-->5' exonuclease activity, the Y-family polymerases possess a unique "little finger" domain that facilitates DNA association, catalytic efficiency and interactions with auxiliary factors. Expression of Y-family polymerases is often induced by DNA damage, and their recruitment to the replication fork is mediated by beta-clamp, clamp loader, single-strand-DNA-binding protein and RecA in Escherichia coli, and by ubiquitin-modified proliferating cell nuclear antigen in yeast.     

Involvement of Escherichia coli DNA polymerase IV in tolerance of cytotoxic alkylating DNA lesions in vivo

Escherichia coli PolIV, a DNA polymerase capable of catalyzing synthesis past replication-blocking DNA lesions, belongs to the most ubiquitous branch of Y-family DNA polymerases. The goal of this study is to identify spontaneous DNA damage that is bypassed specifically and accurately by PolIV in vivo. We increased the amount of spontaneous DNA lesions using mutants deficient for different DNA repair pathways and measured mutation frequency in PolIV-proficient and -deficient backgrounds. We found that PolIV performs an error-free bypass of DNA damage that accumulates in the alkA tag genetic background. This result indicates that PolIV is involved in the error-free bypass of cytotoxic alkylating DNA lesions. When the amount of cytotoxic alkylating DNA lesions is increased by the treatment with chemical alkylating agents, PolIV is required for survival in an alkA tag-proficient genetic background as well. Our study, together with the reported involvement of the mammalian PolIV homolog, Polkappa, in similar activity, indicates that Y-family DNA polymerases from the DinB branch can be added to the list of evolutionarily conserved molecular mechanisms that counteract cytotoxic effects of DNA alkylation. This activity is of major biological relevance because alkylating agents are continuously produced endogenously in all living cells and are also present in the environment.     

Characterization of physical and functional interactions between eukaryote-like Orc1/Cdc6 proteins and Y-family DNA polymerase in the hyperthermophilic archaeon Sulfolobus solfataricus

The roles of Y-family DNA polymerases and the regulation mechanisms are not well defined in Archaea. In this study, we performed in vitro and in vivo characterization of the physical interaction between the archaeon Sulfolobus solfataricus Y-family DNA polymerase (SsoPolY) and three eukaryote-like Orc1/Cdc6 proteins (SsoCdc6-1, SsoCdc6-2, and SsoCdc6-3). The effect of SsoCdc6-2 was the strongest, and the three SsoCdc6 proteins were shown to have very different effects on the function of SsoPolY. SsoCdc6-2 inhibited both the DNA-binding activity and DNA polymerization activity of SsoPolY on the DNA substrates containing mismatched bases, while it formed a large complex with SsoPolY and stimulated DNA-binding activity on paired primer-template DNA substrates. SsoCdc6-2 and S. solfataricus PCNA (SsoPCNA) showed a cooperative effect on polymerization by SsoPolY on paired DNA templates, but SsoCdc6 reduced the stimulating effect of SsoPCNA on this polymerization on mismatched DNA substrates. Therefore, we uncovered a DNA substrate-dependent SsoCdc6/SsoPolY interaction mechanism. This is the first evidence for a physical and functional linkage between archaeal eukaryote-like Orc1/Cdc6 proteins and Y-family DNA polymerase.