Identification of a novel streptococcal gene cassette mediating SOS mutagenesis in Streptococcus uberis

Streptococci have been considered to lack the classical SOS response, defined by increased mutation after UV exposure and regulation by LexA. Here we report the identification of a potential self-regulated SOS mutagenesis gene cassette in the Streptococcaceae family. Exposure to UV light was found to increase mutations to antibiotic resistance in Streptococcus uberis cultures. The mutational spectra revealed mainly G:C-->A:T transitions, and Northern analyses demonstrated increased expression of a Y-family DNA polymerase resembling UmuC under DNA-damaging conditions. In the absence of the Y-family polymerase, S. uberis cells were sensitive to UV light and to mitomycin C. Furthermore, the UV-induced mutagenesis was almost completely abolished in cells deficient in the Y-family polymerase. The gene encoding the Y-family polymerase was localized in a four-gene operon including two hypothetical genes and a gene encoding a HdiR homolog. Electrophoretic mobility shift assays demonstrated that S. uberis HdiR binds specifically to an inverted repeat sequence in the promoter region of the four-gene operon. Database searches revealed conservation of the gene cassette in several Streptococcus species, including at least one genome each of Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus mitis, Streptococcus sanguinis, and Streptococcus thermophilus strains. In addition, the umuC operon was localized in several mobile DNA elements of Streptococcus and Lactococcus species. We conclude that the hdiR-umuC-ORF3-ORF4 operon represents a novel gene cassette capable of mediating SOS mutagenesis among members of the Streptococcaceae.     

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.     

Effect of SOS-induced levels of imuABC on spontaneous and damage-induced mutagenesis in Caulobacter crescentus

imuABC (imuAB dnaE2) genes are responsible for SOS-mutagenesis in Caulobacter crescentus and other bacterial species devoid of umuDC. In this work, we have constructed operator-constitutive mutants of the imuABC operon. We used this genetic tool to investigate the effect of SOS-induced levels of these genes upon both spontaneous and damage-induced mutagenesis. We showed that constitutive expression of imuABC does not increase spontaneous or damage-induced mutagenesis, nor increases cellular resistance to DNA-damaging agents. Nevertheless, the presence of the operator-constitutive mutation rescues mutagenesis in a recA background, indicating that imuABC are the only genes required at SOS-induced levels for translesion synthesis (TLS) in C. crescentus. Furthermore, these data also show that TLS mediated by ImuABC does not require RecA, unlike umuDC-dependent mutagenesis in E. coli.     

In vivo bypass efficiencies and mutational signatures of the guanine oxidation products 2-aminoimidazolone and 5-guanidino-4-nitroimidazole

The in vivo mutagenic properties of 2-aminoimidazolone and 5-guanidino-4-nitroimidazole, two products of peroxynitrite oxidation of guanine, are reported. Two oligodeoxynucleotides of identical sequence, but containing either 2-aminoimidazolone or 5-guanidino-4-nitroimidazole at a specific site, were ligated into single-stranded M13mp7L2 bacteriophage genomes. Wild-type AB1157 Escherichia coli cells were transformed with the site-specific 2-aminoimidazolone- and 5-guanidino-4-nitroimidazole-containing genomes, and analysis of the resulting progeny phage allowed determination of the in vivo bypass efficiencies and mutational signatures of the DNA lesions. 2-Aminoimidazolone was efficiently bypassed and 91% mutagenic, producing almost exclusively G to C transversion mutations. In contrast, 5-guanidino-4-nitroimidazole was a strong block to replication and 50% mutagenic, generating G to A, G to T, and to a lesser extent, G to C mutations. The G to A mutation elicited by 5-guanidino-4-nitroimidazole implicates this lesion as a novel source of peroxynitrite-induced transition mutations in vivo. For comparison, the error-prone bypass DNA polymerases were overexpressed in the cells by irradiation with UV light (SOS induction) prior to transformation. SOS induction caused little change in the efficiency of DNA polymerase bypass of 2-aminoimidazolone; however, bypass of 5-guanidino-4-nitroimidazole increased nearly 10-fold. Importantly, the mutation frequencies of both lesions decreased during replication in SOS-induced cells. These data suggest that 2-aminoimidazolone and 5-guanidino-4-nitroimidazole in DNA are substrates for one or more of the SOS-induced Y-family DNA polymerases and demonstrate that 2-aminoimidazolone and 5-guanidino-4-nitroimidazole are potent sources of mutations in vivo.     

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.     

Characterization of the SOS regulon of Caulobacter crescentus

The SOS regulon is a paradigm of bacterial responses to DNA damage. A wide variety of bacterial species possess homologs of lexA and recA, the central players in the regulation of the SOS circuit. Nevertheless, the genes actually regulated by the SOS have been determined only experimentally in a few bacterial species. In this work, we describe 37 genes regulated in a LexA-dependent manner in the alphaproteobacterium Caulobacter crescentus. In agreement with previous results, we have found that the direct repeat GTTCN₇GTTC is the SOS operator of C. crescentus, which was confirmed by site-directed mutagenesis studies of the imuA promoter. Several potential promoter regions containing the SOS operator were identified in the genome, and the expression of the corresponding genes was analyzed for both the wild type and the lexA strain, demonstrating that the vast majority of these genes are indeed SOS regulated. Interestingly, many of these genes encode proteins with unknown functions, revealing the potential of this approach for the discovery of novel genes involved in cellular responses to DNA damage in prokaryotes, and illustrating the diversity of SOS-regulated genes among different bacterial species.     

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.     

Quantitative analysis of the efficiency and mutagenic spectra of abasic lesion bypass catalyzed by human Y-family DNA polymerases

Higher eukaryotes encode various Y-family DNA polymerases to perform global DNA lesion bypass. To provide complete mutation spectra for abasic lesion bypass, we employed short oligonucleotide sequencing assays to determine the sequences of abasic lesion bypass products synthesized by human Y-family DNA polymerases eta (hPolη), iota (hPolι) and kappa (hPolκ). The fourth human Y-family DNA polymerase, Rev1, failed to generate full-length lesion bypass products after 3 h. The results indicate that hPolι generates mutations with a frequency from 10 to 80% during each nucleotide incorporation event. In contrast, hPolη is the least error prone, generating the fewest mutations in the vicinity of the abasic lesion and inserting dAMP with a frequency of 67% opposite the abasic site. While the error frequency of hPolκ is intermediate to those of hPolη and hPolι, hPolκ has the highest potential to create frameshift mutations opposite the abasic site. Moreover, the time (t₅₀^bypass) required to bypass 50% of the abasic lesions encountered by hPolη, hPolι and hPolκ was 4.6, 112 and 1 823 s, respectively. These t₅₀^bypass values indicate that, among the enzymes, hPolη has the highest abasic lesion bypass efficiency. Together, our data suggest that hPolη is best suited to perform abasic lesion bypass in vivo.     

Characterization of polVR391: a Y-family polymerase encoded by rumA'B from the IncJ conjugative transposon, R391

Although best characterized for their ability to traverse a variety of DNA lesions, Y-family DNA polymerases can also give rise to elevated spontaneous mutation rates if they are allowed to replicate undamaged DNA. One such enzyme that promotes high levels of spontaneous mutagenesis in Escherichia coli is polV(R391), a polV-like Y-family polymerase encoded by rumA'B from the IncJ conjugative transposon R391. When expressed in a DeltaumuDC lexA(Def) recA730 strain, polV(R391) promotes higher levels of spontaneous mutagenesis than the related MucA'B (polR1) or UmuD'C (polV) polymerases respectively. Analysis of the spectrum of polV(R391)-dependent mutations in rpoB revealed a unique genetic fingerprint that is typified by an increase in C:G-->A:T and A:T-->T:A transversions at certain mutagenic hot spots. Biochemical characterization of polV(R391) highlights the exceptional ability of the enzyme to misincorporate T opposite C and T in sequence contexts corresponding to mutagenic hot spots. Purified polV(R391) can also bypass a T-T pyrimidine dimer efficiently and displays greater accuracy opposite the 3'T of the dimer than opposite an undamaged T. Our study therefore provides evidence for the molecular basis for the enhanced spontaneous mutator activity of RumA'B, as well as explains its ability to promote efficient and accurate bypass of T-T pyrimidine dimers in vivo.     

Y-family DNA polymerases in Escherichia coli

The observation that mutations in the Escherichia coli genes umuC+ and umuD+ abolish mutagenesis induced by UV light strongly supported the counterintuitive notion that such mutagenesis is an active rather than passive process. Genetic and biochemical studies have revealed that umuC+ and its homolog dinB+ encode novel DNA polymerases with the ability to catalyze synthesis past DNA lesions that otherwise stall replication--a process termed translesion synthesis (TLS). Similar polymerases have been identified in nearly all organisms, constituting a new enzyme superfamily. Although typically viewed as unfaithful copiers of DNA, recent studies suggest that certain TLS polymerases can perform proficient and moderately accurate bypass of particular types of DNA damage. Moreover, various cellular factors can modulate their activity and mutagenic potential.     

Replication of Damaged DNA and the Molecular Mechanism of Ultraviolet Light Mutagenesis

Abstract

On UV irradiation of Escherichia coli cells, DNA replication is transiently arrested to allow removal of DNA damage by DNA repair mechanisms. This is followed by a resumption of DNA replication, a major recovery function whose mechanism is poorly understood. During the post-UV irradiation period the SOS stress response is induced, giving rise to a multiplicity of phenomena, including UV mutagenesis. The prevailing model is that UV mutagenesis occurs by the filling in of single-stranded DNA gaps present opposite UV lesions in the irradiated chromosome. These gaps can be formed by the activity of DNA replication or repair on the damaged DNA. The gap filling involves polymerization through UV lesions (also termed bypass synthesis or error-prone repair) by DNA polymerase III. The primary source of mutations is the incorporation of incorrect nucleotides opposite lesions. UV mutagenesis is a genetically regulated process, and it requires the SOS-inducible proteins RecA, UmuD, and UmuC. It may represent a minor repair pathway or a genetic program to accelerate evolution of cells under environmental stress conditions.     

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.     

Role of damage-specific DNA polymerases in M13 phage mutagenesis induced by a major lipid peroxidation product trans-4-hydroxy-2-nonenal

One of the major lipid peroxidation products trans-4-hydroxy-2-nonenal (HNE), forms cyclic propano- or ethenoadducts bearing six- or seven-carbon atom side chains to G > C ? A > T. To specify the role of SOS DNA polymerases in HNE-induced mutations, we tested survival and mutation spectra in the lacZα gene of M13mp18 phage, whose DNA was treated in vitro with HNE, and which was grown in uvrA^? Escherichia coli strains, carrying one, two or all three SOS DNA polymerases. When Pol IV was the only DNA SOS polymerase in the bacterial host, survival of HNE-treated M13 DNA was similar to, but mutation frequency was lower than in the strain containing all SOS DNA polymerases. When only Pol II or Pol V were present in host bacteria, phage survival decreased dramatically. Simultaneously, mutation frequency was substantially increased, but exclusively in the strain carrying only Pol V, suggesting that induction of mutations by HNE is mainly dependent on Pol V. To determine the role of Pol II and Pol IV in HNE induced mutagenesis, Pol II or Pol IV were expressed together with Pol V. This resulted in decrease of mutation frequency, suggesting that both enzymes can compete with Pol V, and bypass HNE-DNA adducts in an error-free manner. However, HNE-DNA adducts were easily bypassed by Pol IV and only infrequently by Pol II.Mutation spectrum established for strains expressing only Pol V, showed that in uvrA^? bacteria the frequency of base substitutions and recombination increased in relation to NER proficient strains, particularly mutations at adenine sites. Among base substitutions A:T → C:G, A:T → G:C, G:C → A:T and G:C → T:A prevailed.The results suggest that Pol V can infrequently bypass HNE-DNA adducts inducing mutations at G, C and A sites, while bypass by Pol IV and Pol II is error-free, but for Pol II infrequent.     

Quantitative analysis of the mutagenic potential of 1-aminopyrene-DNA adduct bypass catalyzed by Y-family DNA polymerases

N-(Deoxyguanosin-8-yl)-1-aminopyrene (dG(AP)) is the predominant nitro polyaromatic hydrocarbon product generated from the air pollutant 1-nitropyrene reacting with DNA. Previous studies have shown that dG(AP) induces genetic mutations in bacterial and mammalian cells. One potential source of these mutations is the error-prone bypass of dG(AP) lesions catalyzed by the low-fidelity Y-family DNA polymerases. To provide a comparative analysis of the mutagenic potential of the translesion DNA synthesis (TLS) of dG(AP), we employed short oligonucleotide sequencing assays (SOSAs) with the model Y-family DNA polymerase from Sulfolobus solfataricus, DNA Polymerase IV (Dpo4), and the human Y-family DNA polymerases eta (hPolη), kappa (hPolκ), and iota (hPolι). Relative to undamaged DNA, all four enzymes generated far more mutations (base deletions, insertions, and substitutions) with a DNA template containing a site-specifically placed dG(AP). Opposite dG(AP) and at an immediate downstream template position, the most frequent mutations made by the three human enzymes were base deletions and the most frequent base substitutions were dAs for all enzymes. Based on the SOSA data, Dpo4 was the least error-prone Y-family DNA polymerase among the four enzymes during the TLS of dG(AP). Among the three human Y-family enzymes, hPolκ made the fewest mutations at all template positions except opposite the lesion site. hPolκ was significantly less error-prone than hPolι and hPolη during the extension of dG(AP) bypass products. Interestingly, the most frequent mutations created by hPolι at all template positions were base deletions. Although hRev1, the fourth human Y-family enzyme, could not extend dG(AP) bypass products in our standing start assays, it preferentially incorporated dCTP opposite the bulky lesion. Collectively, these mutagenic profiles suggest that hPolk and hRev1 are the most suitable human Y-family DNA polymerases to perform TLS of dG(AP) in humans.     

Global Conformational Dynamics of a Y-Family DNA Polymerase during Catalysis

Replicative DNA polymerases are stalled by damaged DNA while the newly discovered Y-family DNA polymerases are recruited to rescue these stalled replication forks, thereby enhancing cell survival. The Y-family DNA polymerases, characterized by low fidelity and processivity, are able to bypass different classes of DNA lesions. A variety of kinetic and structural studies have established a minimal reaction pathway common to all DNA polymerases, although the conformational intermediates are not well defined. Furthermore, the identification of the rate-limiting step of nucleotide incorporation catalyzed by any DNA polymerase has been a matter of long debate. By monitoring time-dependent fluorescence resonance energy transfer (FRET) signal changes at multiple sites in each domain and DNA during catalysis, we present here a real-time picture of the global conformational transitions of a model Y-family enzyme: DNA polymerase IV (Dpo4) from Sulfolobus solfataricus. Our results provide evidence for a hypothetical DNA translocation event followed by a rapid protein conformational change prior to catalysis and a subsequent slow, post-chemistry protein conformational change. Surprisingly, the DNA translocation step was induced by the binding of a correct nucleotide. Moreover, we have determined the directions, rates, and activation energy barriers of the protein conformational transitions, which indicated that the four domains of Dpo4 moved in a synchronized manner. These results showed conclusively that a pre-chemistry conformational change associated with domain movements was too fast to be the rate-limiting step. Rather, the rearrangement of active site residues limited the rate of correct nucleotide incorporation. Collectively, the conformational dynamics of Dpo4 offer insights into how the inter-domain movements are related to enzymatic function and their concerted interactions with other proteins at the replication fork.     

Analysis of UV-induced mutation spectra in Escherichia coli by DNA polymerase eta from Arabidopsis thaliana

DNA polymerase eta belongs to the Y-family of DNA polymerases, enzymes that are able to synthesize past template lesions that block replication fork progression. This polymerase accurately bypasses UV-associated cis-syn cyclobutane thymine dimers in vitro and therefore may contributes to resistance against sunlight in vivo, both ameliorating survival and decreasing the level of mutagenesis. We cloned and sequenced a cDNA from Arabidopsis thaliana which encodes a protein containing several sequence motifs characteristics of Pol eta homologues, including a highly conserved sequence reported to be present in the active site of the Y-family DNA polymerases. The gene, named AtPOLH, contains 14 exons and 13 introns and is expressed in different plant tissues. A strain from Saccharomyces cerevisiae, deficient in Pol eta activity, was transformed with a yeast expression plasmid containing the AtPOLH cDNA. The rate of survival to UV irradiation in the transformed mutant increased to similar values of the wild type yeast strain, showing that AtPOLH encodes a functional protein. In addition, when AtPOLH is expressed in Escherichia coli, a change in the mutational spectra is detected when bacteria are irradiated with UV light. This observation might indicate that AtPOLH could compete with DNA polymerase V and then bypass cyclobutane pyrimidine dimers incorporating two adenylates.     

The choice of nucleotide inserted opposite abasic sites formed within chromosomal DNA reveals the polymerase activities participating in translesion DNA synthesis

Abasic sites in genomic DNA can be a significant source of mutagenesis in biological systems, including human cancers. Such mutagenesis requires translesion DNA synthesis (TLS) bypass of the abasic site by specialized DNA polymerases. The abasic site bypass specificity of TLS proteins had been studied by multiple means in vivo and in vitro, although the generality of the conclusions reached have been uncertain. Here, we introduce a set of yeast reporter strains for investigating the in vivo specificity of abasic site bypass at numerous random positions within chromosomal DNA. When shifted to 37 °C, these strains underwent telomere uncapping and resection that exposed reporter genes within a long 3′ ssDNA overhang. Human APOBEC3G cytosine deaminase was expressed to create uracils in ssDNA, which were excised by uracil-DNA N-glycosylase. During repair synthesis, error-prone TLS bypassed the resulting abasic sites. Because of APOBEC3G's strict motif specificity and the restriction of abasic site formation to only one DNA strand, this system provides complete information about the location of abasic sites that led to mutations. We recapitulated previous findings on the roles of REV1 and REV3. Further, we found that sequence context can strongly influence the relative frequency of A or C insertion. We also found that deletion of Pol32, a non-essential common subunit of Pols δ and ζ, resulted in residual low-frequency C insertion dependent on Rev1 catalysis. We summarize our results in a detailed model of the interplay between TLS components leading to error-prone bypass of abasic sites. Our results underscore the utility of this system for studying TLS bypass of many types of lesions within genomic DNA.     

Discrimination against major groove adducts by Y-family polymerases of the DinB subfamily

Y-family DNA polymerases bypass DNA adducts in a process known as translesion synthesis (TLS). Y-family polymerases make contacts with the minor groove side of the DNA substrate at the nascent base pair. The Y-family polymerases also contact the DNA major groove via the unique little finger domain, but they generally lack contacts with the major groove at the nascent base pair. Escherichia coli DinB efficiently and accurately copies certain minor groove guanosine adducts. In contrast, we previously showed that the presence in the DNA template of the major groove-modified base 1,3-diaza-2-oxophenothiazine (tC) inhibits the activity of E. coli DinB. Even when the DNA primer is extended up to three nucleotides beyond the site of the tC analog, DinB activity is strongly inhibited. These findings prompted us to investigate discrimination against other major groove modifications by DinB and its orthologs. We chose a set of pyrimidines and purines with modifications in the major groove and determined the activity of DinB and several orthologs with these substrates. DinB, human pol kappa, and Sulfolobus solfataricus Dpo4 show differing specificities for the major groove adducts pyrrolo-dC, dP, N⁶-furfuryl-dA, and etheno-dA. In general, DinB was least efficient for bypass of all of these major groove adducts, whereas Dpo4 was most efficient. DinB activity was essentially completely inhibited by the presence of etheno-dA, while pol kappa activity was strongly inhibited. All three of these DNA polymerases were able to bypass N⁶-furfuryl-dA with modest efficiency, with DinB being the least efficient. We also determined that the R35A variant of DinB enhances bypass of N⁶-furfuryl-dA but not etheno-dA. In sum, we find that whereas DinB is specific for bypass of minor groove adducts, it is specifically inhibited by major groove DNA modifications.     

Translesion DNA Synthesis and Mutagenesis in Prokaryotes

The presence of unrepaired lesions in DNA represents a challenge for replication. Most, but not all, DNA lesions block the replicative DNA polymerases. The conceptually simplest procedure to bypass lesions during DNA replication is translesion synthesis (TLS), whereby the replicative polymerase is transiently replaced by a specialized DNA polymerase that synthesizes a short patch of DNA across the site of damage. This process is inherently error prone and is the main source of point mutations. The diversity of existing DNA lesions and the biochemical properties of Escherichia coli DNA polymerases will be presented. Our main goal is to deliver an integrated view of TLS pathways involving the multiple switches between replicative and specialized DNA polymerases and their interaction with key accessory factors. Finally, a brief glance at how other bacteria deal with TLS and mutagenesis is presented.Within the context of this review, we will limit the notion of DNA lesions to chemically altered bases, although the sugar-phosphodiester backbone is also subject to various types of chemical attack leading, for example, to single-strand breaks. Lesions may be spontaneous (e.g., depurinations), induced endogenously (e.g., by reactive oxygen species), induced by radiations (UV light, X rays) or by chemicals. Treatments that induce DNA lesions cause mutations and cancer and are therefore referred to as mutagens or carcinogens. Carcinogens fall into large chemical families of compounds such as aromatic amides, polycyclic hydrocarbons, and nitrosamines. Carcinogens are not necessarily synthetic; for example, some are natural plant metabolites (e.g., Aflatoxin B1, aristolochic acid, etc.). In addition, some drugs used in cancer chemotherapy such as platinum derivatives form covalent DNA adducts and as such are also carcinogens. Drugs from the thiopurine family, such as azathioprine widely used as immunosuppressants in organ transplant patients, form DNA adducts upon interaction with sunlight and promote skin cancer (Zhang et al. 2007).     

Incarnation of classical pro- and eukaryotic mechanisms of mutagenesis in hypermutagenesis and immunity of vertebrates

M.E. Lobashev has brilliantly postulated in 1947 that error-prone repair contribute to mutations in cells. This was shown to be true once the mechanisms of UV mutagenesis in Escherichia coli were deciphered. Induced mutations are generated during error-prone SOS DNA repair with the involvement of inaccurate DNA polymerases belonging to the Y family. Currently, several distinct mutator enzymes participating in spontaneous and induced mutagenesis have been identified. Upon induction of these proteins, mutation rates increase by several orders of magnitude. These proteins regulate the mutation rates in evolution and in ontogeny during immune response. In jawed vertebrates, somatic hypermutagenesis occurs in the variable regions of immunoglobulin genes, leading to affinity maturation of antibodies. The process is initiated by cytidine deamination in DNA to uracil by AID (Activation-Induced Deaminase). Further repair of uracil-containing DNA through proteins that include the Y family DNA polymerases causes mutations, induce gene conversion, and class switch recombination. In jawless vertebrates, the variable lymphocyte receptors (VLR) serve as the primary molecules for adaptive immunity. Generation of mature VLRs most likely depends on agnathan AID-like deaminases. AID and its orthologs in lamprey (PmCDA1 and PMCDA2) belong to the AID/APOBEC family of RNA/DNA editing cytidine deaminases. This family includes enzymes with different functions: APOBEC1 edits RNA, APOBEC3 restricts retroviruses. The functions of APOBEC2 and APOBEC4 have not been yet determined. Here, we report a new member of the AID/APOBEC family, APOBEC5, in the bacterium Xanthomonas oryzae. The widespread presence of RNA/DNA editing deaminases suggests that they are an ancient means of generating genetic diversity.