The role of DNA polymerase eta in UV mutational spectra

UV irradiation generates predominantly cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts in DNA. CPDs are thought to be responsible for most of the UV-induced mutations. Thymine-thymine CPDs, and probably also CPDs containing cytosine, are replicated in vivo in a largely accurate manner by a DNA polymerase eta (Pol eta) dependent process. Pol eta is encoded by the POLH (XPV) gene in humans. In order to clarify the specific role of Pol eta in UV mutagenesis, we have used an siRNA knockdown approach in combination with a supF shuttle vector which replicates in mammalian cells. This strategy provides an advantage over studying mutagenesis in cell lines derived from normal individuals and XP-V patients, since the genetic background of the cells is identical. Synthetic RNA duplexes were used to inhibit Pol eta expression in 293T cells. The reduction of Pol eta mRNA and protein was greater than 90%. The supF shuttle vector was irradiated with UVC and replicated in 293T cells in presence of anti-Pol eta siRNA. The supF mutant frequency was increased by up to 3.6-fold in the siRNA knockdown cells relative to control cells confirming that Pol eta plays an important role in mutation avoidance and that the pol eta knockdown was efficient. UV-induced supF mutants were sequenced from siRNA-treated cells and controls. Surprisingly, neither the type of mutations nor their distribution along the supF gene were substantially different between controls and siRNA knockdown cells and were predominantly C to T and CC to TT transitions at dipyrimidine sites. The data are compatible with two models. (i) Incorrect replication of cytosine-containing photoproducts by a polymerase other than Pol eta produces similar mutations as when Pol eta is present but at a higher frequency. (ii) Due to lack of Pol eta or low levels of remaining Pol eta, lesion replication is delayed allowing more time for cytosine deamination within CPDs to occur. We provide proof of principle that siRNA technology can be used to dissect the in vivo roles of lesion bypass DNA polymerases in DNA damage-induced mutagenesis.     

Requirement for functional DNA polymerase eta in genome-wide repair of UV-induced DNA damage during S phase

The autosomal recessive disorder Xeroderma pigmentosum-variant (XPV) is characterized (i) at the cellular level by dramatic hypermutability and defective recovery of DNA synthesis following UV exposure, and (ii) clinically by abnormal sunlight sensitivity and remarkable predisposition to skin cancer. These phenotypes are clearly attributable to germline mutations in POLH, encoding DNA polymerase eta (polη) normally required for accurate translesion DNA synthesis (TLS) past UV-induced cyclobutane pyrimidine dimers. Here we demonstrate that patient-derived XPV-skin fibroblasts exposed to 15 J/m² of UV also exhibit (in addition to abnormal TLS) a significant defect in global-genomic nucleotide excision repair (GG-NER) exclusively during S phase. This cell cycle-specific GG-NER defect can be complemented by ectopic expression of wild-type polη, but not of polη variants deficient in either nuclear relocalization or PCNA interaction. We highlight a previous study from our laboratory demonstrating that UV-exposed, ATR-deficient Seckel syndrome fibroblasts, like XPV fibroblasts, manifest strong attenuation of GG-NER uniquely in S phase populations. We now present further evidence suggesting that deficient S phase repair can be rescued in both XPV- and Seckel syndrome-cells if the formation of blocked replication forks post-UV is either prevented or substantially reduced, i.e., following, respectively, pharmacological inhibition of DNA synthesis prior to UV irradiation, or exposure to a relatively low UV dose (5 J/m²). Our findings in cultured cells permit speculation that abrogation of GG-NER during S phase might partially contribute (in a synergistic manner with defective, atypically error-prone TLS) to the extreme state of UV-hypermutability leading to accelerated skin cancer development in XPV patients. Moreover, based on the overall data, we postulate that loss of either functional polη or -ATR engenders abnormal persistence of stalled replication forks at UV-adducted sites in DNA which, in turn, can actively and/or passively trigger GG-NER inhibition.     

DNA sequence context affects UV-induced mutagenesis in Escherichia coli

We investigated the effect of altering the DNA sequence surrounding a mutable target site on the production of ultraviolet light (UV) induced mutations. Site-directed base substitutions were incorporated on both sides of a TAA sequence encoding a UAA nonsense defect in the tyrA14 allele of Escherichia coli. This allele is readily revertable by UV and a total of eight different base substitution mutations can be recovered. Five different strains harboring DNA sequences allowing the formation of 5'-TT, 5'-CT and 5'-TA* photoproducts were constructed and exposed to UV. DNA sequence analysis was used to determine the spectrum of the revertants that were recovered. The results showed that changes at the 3'-base of a TT site were predominantly T to C transitions and T to A transversions. However, unlike the TT site, a 5'-CT site produced a relatively high frequency of T to G transversions. In addition, T to A transversions that could not have been targeted by a cyclobutane-type or [6-4]-type pyrimidine dimer were produced; this result suggested that these mutations may be targeted by a TA* photoproduct. Also, a distinct strand bias was noted for two mechanistically identical base substitutions in a strain having a palindromic target sequence; this result may reflect an unequal damage distribution or processing of photoproducts as a consequence of asymmetric DNA replication. Finally, our results show that DNA sequences expected to allow the greatest density of UV-induced DNA damage produce the highest mutation frequencies. Overall, these findings provide new insights regarding the role of DNA photoproducts in UV mutagenesis.     

Requirement of DNA polymerase eta for error-free bypass of UV-induced CC and TC photoproducts

The yeast RAD30-encoded DNA polymerase eta (Poleta) bypasses a cis-syn thymine-thymine dimer efficiently and accurately. Human DNA polymerase eta functions similarly in the bypass of this lesion, and mutations in human Poleta result in the cancer prone syndrome, the variant form of xeroderma pigmentosum. UV light, however, also elicits the formation of cis-syn cyclobutane dimers and (6-4) photoproducts at 5'-CC-3' and 5'-TC-3' sites, and in both yeast and human DNA, UV-induced mutations occur primarily by 3' C to T transitions. Genetic studies presented here reveal a role for yeast Poleta in the error-free bypass of cyclobutane dimers and (6-4) photoproducts formed at CC and TC sites. Thus, by preventing UV mutagenesis at a wide spectrum of dipyrimidine sites, Poleta plays a pivotal role in minimizing the incidence of sunlight-induced skin cancers in humans.     

UV-induced RPA phosphorylation is increased in the absence of DNA polymerase eta and requires DNA-PK

Signaling from arrested replication forks plays a role in maintaining genome stability. We have investigated this process in xeroderma pigmentosum variant cells that carry a mutation in the POLH gene and lack functional DNA polymerase eta (poleta). Poleta is required for error-free bypass of UV-induced cyclobutane pyrimidine dimers; in the absence of poleta in XPV cells, DNA replication is arrested at sites of UV-induced DNA damage, and mutagenic bypass of lesions is ultimately carried out by other, error-prone, DNA polymerases. The present study investigates whether poleta expression influences the activation of a number of UV-induced DNA damage responses. In a stably transfected XPV cell line (TR30-9) in which active poleta can be induced by addition of tetracycline, expression of poleta determines the extent of DNA double-strand break formation following UV-irradiation. UV-induced phosphorylation of replication protein A (RPA), a key DNA-binding protein involved in DNA replication, repair and recombination, is increased in cells lacking poleta compared to when poleta is expressed in the same cell line. To identify the protein kinase responsible for increased UV-induced hyperphosphorylation of the p34 subunit of RPA, we have used NU7441, a specific small molecule inhibitor of DNA-PK. DNA-PK is necessary for RPA p34 hyperphosphorylation, but DNA-PK-mediated phosphorylation is not required for recruitment of RPA p34 into nuclear foci in response to UV-irradiation. The results demonstrate that activation of a UV-induced DNA damage response pathway, involving phosphorylation of RPA p34 by DNA-PK, is enhanced in cells lacking poleta.     

Role of DNA polymerase eta in the UV mutation spectrum in human cells

In humans, inactivation of the DNA polymerase eta gene (pol eta) results in sunlight sensitivity and causes the cancer-prone xeroderma pigmentosum variant syndrome (XP-V). Cells from XP-V individuals have a reduced capacity to replicate UV-damaged DNA and show hypermutability after UV exposure. Biochemical assays have demonstrated the ability of pol eta to bypass cis-syn-cyclobutane thymine dimers, the most common lesion generated in DNA by UV. In most cases, this bypass is error-free. To determine the actual requirement of pol eta in vivo, XP-V cells (XP30RO) were complemented by the wild type pol eta gene. We have used two pol eta-corrected clones to study the in vivo characteristics of mutations produced by DNA polymerases during DNA synthesis of UV-irradiated shuttle vectors transfected into human host cells, which had or had not been exposed previously to UV radiation. The functional complementation of XP-V cells by pol eta reduced the mutation frequencies both at CG and TA base pairs and restored UV mutagenesis to a normal level. UV irradiation of host cells prior to transfection strongly increased the mutation frequency in undamaged vectors and, in addition, especially in the pol eta-deficient XP30RO cells at 5'-TT sites in UV-irradiated plasmids. These results clearly show the protective role of pol eta against UV-induced lesions and the activation by UV of pol eta-independent mutagenic processes.     

Genetic characterization of late-flowering traits induced by DNA hypomethylation mutation in Arabidopsis thaliana

Arabidopsis DNA hypomethylation mutation, ddm1 , results in a variety of developmental abnormalities by slowly inducing heritable lesions at unlinked loci. Here, late-flowering traits observed at high frequencies in independently-established ddm1 lines were genetically characterized. In all of the four late-flowering lines examined the traits were dominant and mapped to the same chromosomal region, which is close or possibly identical to the FWA locus. The ddm1 -induced phenotypic onsets are apparently not random mutation events, but specific to a group of genes, suggesting the underlying epigenetic mechanism. The DNA methylation mutant provide useful system for identifying epigenetically-regulated genes important for plant development.     

The SMC-like protein complex SbcCD enhances DNA polymerase IV-dependent spontaneous mutation in Escherichia coli

In Escherichia coli, RpoS, the general stress response sigma factor, regulates the activity of the specialized DNA polymerase DNA polymerase IV (Pol IV) both in stationary-phase and in exponential-phase cells. Because during exponential phase dinB, the gene encoding Pol IV, is transcribed independently of RpoS, RpoS must regulate Pol IV activity in growing cells indirectly via one or more intermediate factors. The results presented here show that one of these intermediate factors is SbcCD, an SMC-like protein and an ATP-dependent nuclease. By initiating or participating in double-strand break repair, SbcCD may provide DNA substrates for Pol IV polymerase activity.     

Escherichia coli DNA polymerase II is stimulated by DNA polymerase III holoenzyme auxiliary subunits

DNA polymerase III of Escherichia coli requires multiple auxiliary factors to enable it to serve as a replicative complex. We demonstrate that auxiliary components of the DNA polymerase III holoenzyme, the gamma delta complex and beta subunit, markedly stimulate DNA polymerase II on long single-stranded templates. DNA polymerase II activity is enhanced by single-stranded DNA binding protein, but the stimulation by gamma delta and beta can be observed either in the absence or presence of single-stranded DNA binding protein. In contrast with DNA polymerase III, the requirement of DNA polymerase II for gamma delta cannot be bypassed by large excesses of the beta subunit at low ionic strength in the absence of the single-stranded DNA binding protein. The product of the DNA polymerase II-gamma delta-beta reaction on a uniquely primed single-stranded circle is of full template length; the reconstituted enzyme apparently is incapable of strand displacement synthesis. The possible biological implications of these observations are discussed.     

Methylation-dependent DNA synthesis in Escherichia coli mediated by DNA polymerase I.

An in vitro system was used to study DNA synthesis in lysates of Escherichia coli cells which had been grown in the presence of ethionine. Such lysates showed a reduced capacity to incorporate [3H]TTP into high-molecular-weight material. Activity could be restored by incubation with S-adenosyl methionine and ATP. S-adenosyl methionine-reactivated TTP incorporation required the presence of DNA polymerase I, ATP, and all four deoxyribonucleotide triphosphates. DNA polymerase III was not required.     

Spontaneous and UV-induced mutations in Escherichia coli K-12 strains with altered or absent DNA polymerase I.

The induction of mutations to valine resistance and to rifampin resistance occurs after UV irradiation in bacteria carrying a deletion through the polA gene (delta polA), showing that DNA polymerase I (PolI) is not an essential enzyme for this process. The PolI deletion strain showed a 7- to 10-fold-higher spontaneous mutation frequency than the wild type. The presence in the deletion strain of the 5'----3' exonuclease fragment on an F' episome caused an additional 10-fold increase in spontaneous mutation frequency, resulting in mutation frequencies on the order of 50- to 100-fold greater than wild type. The mutator effect associated with the 5'----3' exonuclease gene fragment together with much of the effect attributable to the polA deletion was blocked in bacteria carrying a umuC mutation. The mutator activity therefore appears to reflect constitutive SOS induction. Excision-proficient polA deletion strains exhibited increased sensitivity to the lethal effect of UV light which was only partially ameliorated by the presence of polA+ on an F' episome. The UV-induced mutation rate to rifampin resistance was marginally lower in delta polA bacteria than in bacteria carrying the polA+ allele. This effect is unlikely to be caused by the existence of a PolI-dependent mutagenic pathway and is probably an indirect effect caused by an alteration in the pattern of excision repair, since it did not occur in excision-deficient (uvrA) bacteria. An excision-deficient polA deletion strain possessed UV sensitivity similar to that of an isogenic strain carrying polA+ on an F' episome, showing that none of the functions of PolI are needed for postreplication repair in the absence of excision repair. Our data provide no evidence for a pathway of UV mutagenesis dependent on PolI, although it remains an open question whether PolI is able to participate when it is present.     

Mechanisms of error discrimination by Escherichia coli DNA polymerase I

The mechanism of base selection by DNA polymerase I of Escherichia coli has been investigated by kinetic analysis. The apparent KM for the insertion of the complementary nucleotide dATP into the hook polymer poly(dT)-oligo(dA) was found to be 6-fold lower than that for the noncomplementary nucleotide dGTP, whereas the Vmax for insertion of dATP was 1600-fold higher than that for dGTP. The ratio of Kcat/KM values for complementary and mismatched nucleotides of 10(4) demonstrates the extremely high specificity of base selection by DNA polymerase I and is in agreement with results obtained with a different template-primer, poly(dC)-oligo(dG) [El-Deiry, W. S., Downey, K. M., & So, A. G. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 7378]. Studies on the effects of phosphate ion on the polymerase and 3'- to 5'-exonuclease activities of DNA polymerase I showed that, whereas the polymerase activity was somewhat stimulated by phosphate, the exonuclease activity was markedly inhibited, being 50% inhibited at 25 mM phosphate and greater than 90% inhibited at 80 mM phosphate. Selective inhibition of the exonuclease activity by phosphate also resulted in inhibition of template-dependent conversion of a noncomplementary dNTP to dNMP and, consequently, markedly affected the kinetic constants for insertion of noncomplementary nucleotides. The mutagenic metal ion Mn2+ was found to affect error discrimination by both the polymerase and 3'- and 5'-exonuclease activities of DNA polymerase I.(ABSTRACT TRUNCATED AT 250 WORDS)     

Error-prone lesion bypass by human DNA polymerase eta

DNA lesion bypass is an important cellular response to genomic damage during replication. Human DNA polymerase η (Polη), encoded by the Xeroderma pigmentosum variant (XPV) gene, is known for its activity of error-free translesion synthesis opposite a TT cis-syn cyclobutane dimer. Using purified human Polη, we have examined bypass activities of this polymerase opposite several other DNA lesions. Human Polη efficiently bypassed a template 8-oxoguanine, incorporating an A or a C opposite the lesion with similar efficiencies. Human Polη effectively bypassed a template abasic site, incorporating an A and less frequently a G opposite the lesion. Significant –1 deletion was also observed when the template base 5′ to the abasic site is a T. Human Polη partially bypassed a template (+)-trans-anti-benzo[a]pyrene-N²-dG and predominantly incorporated an A, less frequently a T, and least frequently a G or a C opposite the lesion. This specificity of nucleotide incorporation correlates well with the known mutation spectrum of (+)-trans-anti-benzo[a]pyrene-N²-dG lesion in mammalian cells. These results show that human Polη is capable of error-prone translesion DNA syntheses in vitro and suggest that Polη may bypass certain lesions with a mutagenic consequence in humans.