Involvement of Y-family DNA polymerases in mutagenesis caused by oxidized nucleotides in Escherichia coli

Escherichia coli DNA polymerase IV incorporated 2-hydroxy-dATP opposite template guanine or thymine and 8-hydroxy-dGTP exclusively opposite adenine in vitro. Mutator phenotypes in sod/fur strains were substantially diminished by deletion of dinB and/or umuDC. DNA polymerases IV and V may be involved in mutagenesis caused by incorporation of the oxidized deoxynucleoside triphosphates.     

Base excision repair enzyme endonuclease III suppresses mutagenesis caused by 8-hydroxy-dGTP

To examine whether base excision repair suppresses mutations induced by oxidized deoxyribonucleotide 5'-triphosphates in the nucleotide pool, 8-hydroxy-dGTP (8-OH-dGTP) and 2-hydroxy-dATP were introduced into Escherichia coli strains deficient in endonucleases III (Nth) and VIII (Nei) and MutY, and mutations in the chromosomal rpoB gene were analyzed. The spontaneous rpoB mutant frequency was also examined in mutT/nth and mutT/nei strains, to assess the influence on the mutations induced by the endogenous 8-OH-dGTP accumulated in the mutT mutant. The mutations induced by exogenous 2-hydroxy-dATP were similar in all of the strains tested. Exogenous 8-OH-dGTP increased the rpoB mutant frequency more efficiently in the nth strain than that in the wild-type strain. The spontaneous mutant frequency in the mutT/nth strain was 2-fold higher than that in the mutT strain. These results suggest that E. coli endonuclease III also acts as a defense against the mutations caused by 8-OH-dGTP in the nucleotide pool.     

Frameshift mutagenesis by eucaryotic DNA polymerases in vitro

The frequency and specificity of frameshift errors produced during a single round of in vitro DNA synthesis by DNA polymerases-alpha, -beta, and -gamma (pol-alpha, -beta, and -gamma, respectively) have been determined. DNA polymerase-beta is the least accurate enzyme, producing frameshift errors at an average frequency of one error for each 1,000-3,000 nucleotides polymerized, a frequency similar to its average base substitution accuracy. DNA polymerase-alpha is approximately 10-fold more accurate, producing frameshifts at an average frequency of one error for every 10,000-30,000 nucleotides polymerized, a frequency which is about 2- to 6-fold lower than the average pol-alpha base substitution accuracy. DNA polymerase-gamma is highly accurate, producing on the average less than one frameshift error for every 200,000-400,000 nucleotides polymerized. This represents a more than 10-fold higher fidelity than for base substitutions. Among the collection of sequenced frameshifts produced by DNA polymerases-alpha and beta, both common features and distinct specificities are apparent. These specificities suggest a major role for eucaryotic DNA polymerases in modulating frameshift fidelity. Possible mechanisms for production of frameshifts are discussed in relation to the observed biases. One of these models has been experimentally supported using site-directed mutagenesis to change the primary DNA sequence of the template. Alteration of a pol-beta frameshift hotspot sequence TTTT to CTCT reduced the frequency of pol-beta-dependent minus-one-base errors at this site by more than 30-fold, suggesting that more than 97% of the errors at the TTTT run involve a slippage mechanism.     

The overexpression of specialized DNA polymerases in cancer

Specialized DNA polymerases are required to bypass DNA damage lesions that would otherwise cause replication arrest and cell death. When operating on non-canonical templates, such as undamaged DNA or on non-cognate lesions, these polymerases exhibit considerably reduced fidelity, resulting in the generation of mutations. Ectopic overexpression of these polymerases can also lead to an increased mutation rate and an enhanced capability of DNA repair, suggesting that they could potentially act as oncogenes if they were overexpressed in cancers. Here, we examine expression patterns of DNA polymerases in matched normal and tumor samples from a diverse range of tissues. As well as investigating the specialized polymerases beta, lambda, iota and kappa, we also investigate the expression of the replicative polymerases alpha, delta and epsilon. The data presented provide evidence for the overexpression of specialized polymerases in tumors, with more than 45% of the 68 tumor samples studied demonstrating greater than two-fold enhanced expression of at least one specialized polymerase. Of particular note, DNA polymerase beta (pol beta) was found to be overexpressed at both the mRNA and protein level in approximately one third of all tumor types studied, with overexpression being particularly frequent in uterus, ovary, prostate and stomach samples. Pols lambda, and iota were also found to be overexpressed to a significant extent in a range of tumor types, albeit less frequently than pol beta. In contrast, pol kappa was rarely found to be overexpressed in tumors but was found to be commonly underexpressed in many samples. Downregulation of pol beta expression by siRNA resulted in an increased sensitivity to the chemotherapeutic agent cisplatin, suggesting a role for this polymerase in providing tolerance to cisplatin-induced damage. These observations suggest that specialised DNA polymerases, and particularly pol beta, could be considered both as caretaker genes altered during tumorigenesis, and as potential drug targets to sensitise tumors to chemotherapy.     

SOS-inducible DNA polymerases and adaptive mutagenesis

Stability of genomes of living organisms is maintained by various mechanisms that ensure high fidelity of DNA replication. However, cells can reversibly enhance the level of replication errors in response to external factors. As mutable states are potentially involved in carcinogenesis, aging, and resistance for pathogenic agents, the existence of these states is of great importance for human health. A well-known system of inducible mutation is SOS response, whose key component is replication of damaged DNA regions. Inducible mutation implies a contribution of SOS response to the adaptation of a bacterial population to adverse environments. There is ample evidence indicating the primary role of SOS response genes in the phenomenon of adaptive mutation. The involvement of the SOS system in adaptive mutagenesis is discussed.     

Mutagenic effects of 8-hydroxy-dGTP in live mammalian cells

The mutagenicity of an oxidized form of dGTP, 8-hydroxy-2'-deoxyguanosine 5'-triphosphate (8-OH-dGTP), was examined using COS-7 cells. 8-OH-dGTP and supF shuttle plasmid DNA were cointroduced by means of cationic liposomes, and the DNAs replicated in the cells were recovered and then transfected into Escherichia coli. 8-OH-dGTP induced A:T-->C:G substitution mutations in the COS-7 cells. This result agrees with previous observations indicating that DNA polymerases misincorporate 8-OH-dGTP opposite A in vitro, and that the oxidized deoxyribonucleotide induces A:T-->C:G transversions in E. coli. These results constitute the first direct evidence to show that 8-OH-dGTP actually induces mutations in living mammalian cells.     

A real-time fluorescence method for enzymatic characterization of specialized human DNA polymerases

Specialized DNA polymerases are involved in DNA synthesis during base-excision repair and translesion synthesis across a wide range of chemically modified DNA templates. Notable features of these enzymes include low catalytic efficiency, low processivity and low fidelity. Traditionally, in vitro studies of these enzymes have utilized radiolabeled substrates and gel electrophoretic separation of products. We have developed a simple homogeneous fluorescence-based method to study the enzymology of specialized DNA polymerases in real time. The method is based on fluorescent reporter strand displacement from a tripartite substrate containing a quencher-labeled template strand, an unlabeled primer and a fluorophore-labeled reporter. With this method, we could follow the activity of human DNA polymerases β, η, ι and κ under different reaction conditions, and we investigated incorporation of the aberrant nucleotide, 8-oxodGTP, as well as bypass of an abasic site or 8-oxoG DNA template lesion in different configurations. Lastly, we demonstrate that the method can be used for small molecule inhibitor discovery and characterization in highly miniaturized settings, and we report the first nanomolar inhibitors of Y-family DNA polymerases ι and η. The fluorogenic method presented here should facilitate mechanistic and inhibitor investigations of these polymerases and is also applicable to the study of highly processive replicative polymerases.     

Roles of replicative and specialized DNA polymerases in frameshift mutagenesis: mutability of Salmonella typhimurium strains lacking one or all of SOS-inducible DNA polymerases to 26 chemicals

Progression of DNA replication is occasionally blocked by endogenous and exogenous DNA damage. To circumvent the stalling of DNA replication, cells possess a variety of specialized DNA polymerases that replicate through DNA damage. Salmonella typhimurium strain TA1538 has six DNA polymerases and four of them are encoded by damage-inducible SOS genes, i.e. polB(ST) (pol II), dinB(ST) (pol IV), umuDC(ST) (pol V) and samAB. The strain has been used for the detection of a variety of chemical mutagens because of the high sensitivity to -2 frameshift occurring in CGCGCGCG sequence. To assign the role of each DNA polymerase in the frameshift mutagenesis, we have constructed the derivatives lacking one or all of SOS-inducible DNA polymerases and examined the mutability to 26 chemical mutagens. Interestingly, the chemicals could be categorized into four classes: class I whose mutagenicity was reduced by the deletion of dinB(ST) (1-aminoanthracene and other four chemicals); class II whose mutagenicity was reduced by the deletion of either dinB(ST) or umuDC(ST) plus samAB (7,12-dimethylbenz[a]anthracene and other three chemicals); class III whose mutagenicity largely depended on the presence of umuDC(ST) plus samAB (1-N-6-azabenzo[a]pyrene and other three chemicals) and class IV whose mutagenicity was not reduced by deletion of any of the genes encoding SOS-inducible DNA polymerases (Glu-P-1 and other 12 chemicals). Deletion of polB(ST) reduced by 30-60% the mutagenicity of six chemicals of classes II and III. These results suggest that multiple DNA polymerases including the replicative DNA polymerase, i.e. DNA polymerase III holoenzyme, play important roles in chemically induced -2 frameshift and also that different sets of DNA polymerases are engaged in the translesion bypass of different DNA lesions.     

Suppression of spontaneous mutagenesis in human cells by DNA base excision-repair

The chemical instability of the covalent structure of DNA, and in vivo exposure of DNA to reactive oxygen species and endogenously produced alkylating agents, has triggered the evolution of several specific DNA repair pathways. A major strategy of repair involves the initial removal of an altered base from DNA by a member of the enzyme family of DNA glycosylases. The currently known enzymes of this type in mammalian cells are reviewed, and the subsequent base excision-repair (BER) steps that achieve restoration of the intact DNA structure are also described. The specific problem of retaining high accuracy in this essentially error-free repair process is discussed.     

DNA polymerases and repair synthesis in NER in human cells

The late steps of nucleotide excision repair, following incisions to remove the damaged section of DNA, comprise repair synthesis and ligation. In vitro and in vivo studies have shown the size of the repaired patch to be about 30 nucleotides. In vitro studies implicated the replicative polymerases in repair synthesis, but recent in vivo data have shown that several DNA polymerases and ligases are involved in these steps in human cells.     

DNA polymerases of anucleated cells. Isolation and characterization of two DNA polymerases from human platelets.

Two different DNA polymerases have been purified and characterized from human platelets. In the mitochondrial fraction a unique activity of the polymerase gamma type has been found. The same enzyme is found in the extramitochondrial supernatant. A second DNA polymerase, called 'cytoplasmic' DNA polymerase has been found in the 10000 x g supernatant of human platelets. The following properties of the latter DNA polymerase from human platelets are identical to those of DNA polymerase alpha from normal cells: DEAE-cellulose and phosphocellulose chromatography, size, thermal stability, phosphonoacetic acid and ethidium bromide inhibition. However, some of its properties, like high resistance to N-ethylmaleimide and the lack of DNA polymerization using synthetic RNA primers, are those of DNA polymerase beta.     

Replication of UV-damaged DNA: new insights into links between DNA polymerases, mutagenesis and human disease

The existence of homologous genes in diverse species is intriguing. A detailed comparison of the structure and function of gene families may provide important insights into gene regulation and evolution. An unproven assumption is that homologous genes have a common ancestor. During evolution, the original function of the ancestral gene might be retained in the different species which evolved along separate courses. In addition, new functions could have developed as the sequence began to diverge. This may also explain partly the presence of multipurpose genes, which have multiple functions at different stages of development and in different tissues. The Drosophila gene snail is a multipurpose gene; it has been demonstrated that snail is critical for mesoderm formation, for CNS development, and for wing cell fate determination. The related vertebrate Snail and Slug genes have also been proposed to participate in mesoderm formation, neural crest cell migration, carcinogenesis, and apoptosis. In this review, we will discuss the Snail/Slug family of regulators in species ranging from insect to human. We will present the protein structures, expression patterns, and functions based on molecular genetic analyses. We will also include the studies that helped to elucidate the molecular mechanisms of repression and the relationship between the conserved and divergent functions of these genes. Moreover, the studies may enable us to trace the evolution of this gene family.     

DNA polymerases and human disease

The human genome encodes at least 14 DNA-dependent DNA polymerases--a surprisingly large number. These include the more abundant, high-fidelity enzymes that replicate the bulk of genomic DNA, together with eight or more specialized DNA polymerases that have been discovered in the past decade. Although the roles of the newly recognized polymerases are still being defined, one of their crucial functions is to allow synthesis past DNA damage that blocks replication-fork progression. We explore the reasons that might justify the need for so many DNA polymerases, describe their function and mode of regulation, and finally consider links between mutations in DNA polymerases and human disease.     

Mutational specificities of brominated DNA adducts catalyzed by human DNA polymerases

Chronic inflammation is known to lead to an increased risk for the development of cancer. Under inflammatory condition, cellular DNA is damaged by hypobromous acid, which is generated by myeloperoxidase and eosinophil peroxidase. The reactive brominating species induced brominated DNA adducts such as 8-bromo-2′-deoxyguanosine (8-Br-dG), 8-bromo-2′-deoxyadenosine (8-Br-dA), and 5-bromo-2′-deoxycytidine (5-Br-dC). These DNA lesions may be implicated in carcinogenesis. In this study, we analyzed the miscoding properties of the brominated DNA adducts generated by human DNA polymerases (pols). Site-specifically modified oligodeoxynucleotides containing a single 8-Br-dG, 8-Br-dA, or 5-Br-dC were used as a template in primer extension reactions catalyzed by human pols α, κ, and η. When 8-Br-dG-modified template was used, pol α primarily incorporated dCMP, the correct base, opposite the lesion, along with a small amount of one-base deletion (4.8%). Pol κ also promoted one-base deletion (14.2%), accompanied by misincorporation of dGMP (9.5%), dAMP (8.0%), and dTMP (6.1%) opposite the lesion. Pol η, on the other hand, readily bypassed the 8-Br-dG lesion in an error-free manner. As for 8-Br-dA and 5-Br-dC, all the pols bypassed the lesions and no miscoding events were observed. These results indicate that only 8-Br-dG, and not 5-Br-dC and 8-Br-dA, is a mutagenic lesion; the miscoding frequency and specificity vary depending on the DNA pol used. Thus, hypobromous acid-induced 8-Br-dG adduct may increase mutagenic potential at the site of inflammation.     

DNA polymerases and human diseases

DNA polymerases function in DNA replication, repair, recombination and translesion synthesis. Currently, 15 DNA polymerase genes have been identified in human cells, belonging to four distinct families. In this review, we briefly describe the biochemical activities and known cellular roles of each DNA polymerase. Our major focus is on the phenotypic consequences of mutation or ablation of individual DNA polymerase genes. We discuss phenotypes of current mouse models and altered polymerase functions and the relationship of DNA polymerase gene mutations to human cell phenotypes. Interestingly, over 120 single nucleotide polymorphisms (SNPs) have been identified in human populations that are predicted to result in nonsynonymous amino acid substitutions of DNA polymerases. We discuss the putative functional consequences of these SNPs in relation to human disease.     

Involvement of the essential yeast DNA polymerases in induced gene conversion

In the yeast Saccharomyces cerevisiae three different DNA polymerases alpha, delta and epsilon are involved in DNA replication. DNA polymerase alpha is responsible for initiation of DNA synthesis and polymerases delta and epsilon are required for elongation of DNA strand during replication. DNA polymerases delta and epsilon are also involved in DNA repair. In this work we studied the role of these three DNA polymerases in the process of recombinational synthesis. Using thermo-sensitive heteroallelic mutants in genes encoding DNA polymerases we studied their role in the process of induced gene conversion. Mutant strains were treated with mutagens, incubated under permissive or restrictive conditions and the numbers of convertants obtained were compared. A very high difference in the number of convertants between restrictive and permissive conditions was observed for polymerases alpha and delta, which suggests that these two polymerases play an important role in DNA synthesis during mitotic gene conversion. Marginal dependence of gene conversion on the activity of polymerase epsilon indicates that this DNA polymerase may be involved in this process but rather as an auxiliary enzyme.     

Alkylation damage, DNA repair and mutagenesis in human cells

17 human cell lines that differ significantly in level of O⁶-alkylguanine-DNA alkyltransferase (AGT) activity were identified by comparing their sensitivity to the cytotoxic effect of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and determining the level of AGT activity in cell extracts from the various lines by measuring the decrease in radiolabeled O⁶-methylguanine from DNA, using high-performance liquid chromatography. 9 lines exhibited high levels of AGT activity, 2 showed an intermediate level (25–50% of the mean of those with the higher levels), and 6 exhibited very low or virtually undetectable levels of AGT. Included were several lines that are very deficient in capacity for nucleotide excision repair. When representatives from the 3 categories of cell lines defined by the level of AGT activity were compared for sensitivity to the cytotoxic and mutagenic effect of MNNG, they showed an inverse correlation between the degree of cell killing and frequency of mutants induced and the level of AGT activity. The cells' capacity for nucleotide excision repair did not affect these results. Exposure of cells with a high level of AGT activity to O⁶-methylguanine in the medium reduced the AGT activity 60–80%. These pre-treated cells exhibited a significantly higher frequency of MNNG-induced mutants than did cells that were not pre-treated, suggesting that the O⁶-methylguanine lesion in DNA is responsible for a significant proportion of the mutations induced. Cell strains containing substrates for assaying intrachromosomal homologous recombination were constructed using parental cell lines from each of the 3 categories of AGT activity. These strains showed an inverse correlation between the level of AGT activity and the frequency of MNNG-induced recombination. When various cell lines representing the 3 categories of AGT activity were compared for sensitivity to ethylnitrosourea, the results were consistent with AGT and nucleotide excision repair playing a role in preventing cell killing and mutation induction by this agent.