Oxygen free radical damage to DNA. Translesion synthesis by human DNA polymerase eta and resistance to exonuclease action at cyclopurine deoxynucleoside residues.

Cyclopurine deoxynucleosides are common DNA lesions generated by exposure to reactive oxygen species under hypoxic conditions. The S and R diastereoisomers of cyclodeoxyadenosine on DNA were investigated separately for their ability to block 3' to 5' exonucleases. The mammalian DNA-editing enzyme DNase III (TREX1) was blocked by both diastereoisomers, whereas only the S diastereoisomer was highly efficient in preventing digestion by the exonuclease function of T4 DNA polymerase. Digestion in both cases was frequently blocked one residue before the modified base. Oligodeoxyribonucleotides containing a cyclodeoxyadenosine residue were further employed as templates for synthesis by human DNA polymerase eta (pol eta). pol eta could catalyze translesion synthesis on the R diastereoisomer of cyclodeoxyadenosine. On the S diastereoisomer, pol eta could catalyze the incorporation of one nucleotide opposite the lesion but could not continue elongation. Although pol eta preferentially incorporated dAMP opposite the R diastereoisomer, elongation continued only when dTMP was incorporated, suggesting bypass of this lesion by pol eta with reasonable fidelity. With the S diastereoisomer, pol eta mainly incorporated dAMP or dTMP opposite the lesion but could not elongate even after incorporating a correct nucleotide. These data suggest that the S diastereoisomer may be a more cytotoxic DNA lesion than the R diastereoisomer.

Error rate and specificity of human and murine DNA polymerase eta.

We describe here the error specificity of mammalian DNA polymerase eta (pol eta), an enzyme that performs translesion DNA synthesis and may participate in somatic hypermutation of immunoglobulin genes. Both mouse and human pol eta lack intrinsic proofreading exonuclease activity and both copy undamaged DNA inaccurately. Analysis of more than 1500 single-base substitutions by human pol eta indicates that error rates for all 12 mismatches are high and variable depending on the composition and symmetry of the mismatch and its location. pol eta also generates tandem base substitutions at an unprecedented rate, and kinetic analysis indicates that it extends a tandem double mismatch about as efficiently as other replicative enzymes extend single-base mismatches. This ability to use an aberrant primer terminus and the high rate of single and double-base substitutions support the idea that pol eta may forego strict shape complementarity in order to facilitate highly efficient lesion bypass. Relaxed discrimination is further indicated by pol eta infidelity for a wide variety of nucleotide deletion and addition errors. The nature and location of these errors suggest that some may be initiated by strand slippage, while others result from additional mechanisms.

Translesion DNA synthesis catalyzed by human pol eta and pol kappa across 1,N6-ethenodeoxyadenosine.

1,N(6)-Ethenodeoxyadenosine, a DNA adduct generated by exogenous and endogenous sources, severely blocks DNA synthesis and induces miscoding events in human cells. To probe the mechanism for in vivo translesion DNA synthesis across this adduct, in vitro primer extension studies were conducted using newly identified human DNA polymerases (pol) eta and kappa, which have been shown to catalyze translesion DNA synthesis past several DNA lesions. Steady-state kinetic analyses and analysis of translesion products have revealed that the synthesis is >100-fold more efficient with pol eta than with pol kappa and that both error-free and error-prone syntheses are observed with these enzymes. The miscoding events include both base substitution and frameshift mutations. These results suggest that both polymerases, particularly pol eta, may contribute to the translesion DNA synthesis events observed for 1,N(6)-ethenodeoxyadenosine in human cells.

ATP-dependent chromatin remodeling facilitates nucleotide excision repair of UV-induced DNA lesions in synthetic dinucleosomes.

To investigate the relationship between chromatin dynamics and nucleotide excision repair (NER), we have examined the effect of chromatin structure on the formation of two major classes of UV-induced DNA lesions in reconstituted dinucleosomes. Furthermore, we have developed a model chromatin-NER system consisting of purified human NER factors and dinucleosome substrates that contain pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) either at the center of the nucleosome or in the linker DNA. We have found that the two classes of UV-induced DNA lesions are formed efficiently at every location on dinucleosomes in a manner similar to that of naked DNA, even in the presence of histone H1. On the other hand, excision of 6-4PPs is strongly inhibited by dinucleosome assembly, even within the linker DNA region. These results provide direct evidence that the human NER machinery requires a space greater than the size of the linker DNA to excise UV lesions efficiently. Interestingly, NER dual incision in dinucleosomes is facilitated by recombinant ACF, an ATP-dependent chromatin remodeling factor. Our results indicate that there is a functional connection between chromatin remodeling and the initiation step of NER.

Mutagenic and nonmutagenic bypass of DNA lesions by Drosophila DNA polymerases dpoleta and dpoliota.

cDNA sequences were identified and isolated that encode Drosophila homologues of human Rad30A and Rad30B called drad30A and drad30B. Here we show that the C-terminal-truncated forms of the drad30A and drad30B gene products, designated dpoletaDeltaC and dpoliotaDeltaC, respectively, exhibit DNA polymerase activity. dpoletaDeltaC and dpoliotaDeltaC efficiently bypass a cis-syn-cyclobutane thymine-thymine (TT) dimer in a mostly error-free manner. dpoletaDeltaC shows limited ability to bypass a 6-4-photoproduct ((6-4)PP) at thymine-thymine (TT-(6-4)PP) or at thymine-cytosine (TC-(6-4)PP) in an error-prone manner. dpoliotaDeltaC scarcely bypasses these lesions. Thus, the fidelity of translesion synthesis depends on the identity of the lesion and on the polymerase. The human XPV gene product, hpoleta, bypasses cis-syn-cyclobutane thymine-thymine dimer efficiently in a mostly error-free manner but does not bypass TT-(6-4)PP, whereas Escherichia coli DNA polymerase V (UmuD'(2)C complex) bypasses both lesions, especially TT-(6-4)PP, in an error-prone manner (Tang, M., Pham, P., Shen, X., Taylor, J. S., O'Donnell, M., Woodgate, R., and Goodman, M. F. (2000) Nature 404, 1014-1018). Both dpoletaDeltaC and DNA polymerase V preferentially incorporate GA opposite TT-(6-4)PP. The chemical structure of the lesions and the similarity in the nucleotides incorporated suggest that structural information in the altered bases contribute to nucleotide selection during incorporation opposite these lesions by these polymerases.

Diversity of the damage recognition step in the global genomic nucleotide excision repair in vitro.

The XPC-HR23B complex, a mammalian factor specifically involved in global genomic nucleotide excision repair (NER) has been shown to bind various forms of damaged DNA and initiate DNA repair in cell-free reactions. To characterize the binding specificity of this factor in more detail, a method based on immunoprecipitation was developed to assess the relative affinity of XPC-HR23B for defined lesions on DNA. Here we show that XPC-HR23B preferentially binds to UV-induced (6-4) photoproducts (6-4PPs) as well as to cholesterol, but not to the cyclobutane pyrimidine dimer (CPD), 8-oxoguanine (8-oxo-G), O6-methylguanine (O6-Me-G), or a single mismatch. Human whole cell extracts could efficiently excise 6-4PPs and cholesterol in an XPC-HR23B-dependent manner, but not 8-oxo-G, O6-Me-G or mismatches. Thus, there was good correlation between the binding specificity of XPC-HR23B for certain types of lesion and the ability of human cell extracts to excise these lesions, supporting the model that XPC-HR23B initiates global genomic NER. Although, XPC-HR23B does not preferentially bind to CPDs, the excision of CPDs in human whole cell extracts was found to be absolutely dependent on XPC-HR23B, in agreement with the in vivo observation that CPDs are not removed from the global genome in XP-C mutant cells. These results suggest that, in addition to the excision repair pathway initiated by XPC-HR23B, there exists another sub-pathway for the global genomic NER that still requires XPC-HR23B but is not initiated by XPC-HR23B. Possible mechanisms will be discussed.

A multistep damage recognition mechanism for global genomic nucleotide excision repair.

A mammalian nucleotide excision repair (NER) factor, the XPC-HR23B complex, can specifically bind to certain DNA lesions and initiate the cell-free repair reaction. Here we describe a detailed analysis of its binding specificity using various DNA substrates, each containing a single defined lesion. A highly sensitive gel mobility shift assay revealed that XPC-HR23B specifically binds a small bubble structure with or without damaged bases, whereas dual incision takes place only when damage is present in the bubble. This is evidence that damage recognition for NER is accomplished through at least two steps; XPC-HR23B first binds to a site that has a DNA helix distortion, and then the presence of injured bases is verified prior to dual incision. Cyclobutane pyrimidine dimers (CPDs) were hardly recognized by XPC-HR23B, suggesting that additional factors may be required for CPD recognition. Although the presence of mismatched bases opposite a CPD potentiated XPC-HR23B binding, probably due to enhancement of the helix distortion, cell-free excision of such compound lesions was much more efficient than expected from the observed affinity for XPC-HR23B. This also suggests that additional factors and steps are required for the recognition of some types of lesions. A multistep mechanism of this sort may provide a molecular basis for ensuring the high level of damage discrimination that is required for global genomic NER.

Centrosome protein centrin 2/caltractin 1 is part of the xeroderma pigmentosum group C complex that initiates global genome nucleotide excision repair.

Nucleotide excision repair (NER) is carried out by xeroderma pigmentosum (XP) factors. Before the excision reaction, DNA damage is recognized by a complex originally thought to contain the XP group C responsible gene product (XPC) and the human homologue of Rad23 B (HR23B). Here, we show that centrin 2/caltractin 1 (CEN2) is also a component of the XPC repair complex. We demonstrate that nearly all XPC complexes contain CEN2, that CEN2 interacts directly with XPC, and that CEN2, in cooperation with HR23B, stabilizes XPC, which stimulates XPC NER activity in vitro. CEN2 has been shown to play an important role in centrosome duplication. Thus, those findings suggest that the XPC-CEN2 interaction may reflect coupling of cell division and NER.

Proofreading of DNA polymerase eta-dependent replication errors.

Human DNA polymerase eta, the product of the skin cancer susceptibility gene XPV, bypasses UV photoproducts in template DNA that block synthesis by other DNA polymerases. Pol eta lacks an intrinsic proofreading exonuclease and copies DNA with low fidelity, such that pol eta errors could contribute to mutagenesis unless they are corrected. Here we provide evidence that pol eta can compete with other human polymerases during replication of duplex DNA, and in so doing it lowers replication fidelity. However, we show that pol eta has low processivity and extends mismatched primer termini less efficiently than matched termini. These properties could provide an opportunity for extrinsic exonuclease(s) to proofread pol eta-induced replication errors. When we tested this hypothesis during replication in human cell extracts, pol eta-induced replication infidelity was found to be modulated by changing the dNTP concentration and to be enhanced by adding dGMP to a replication reaction. Both effects are classical hallmarks of exonucleolytic proofreading. Thus, pol eta is ideally suited for its role in reducing UV-induced mutagenesis and skin cancer risk, in that its relaxed base selectivity may facilitate efficient bypass of UV photoproducts, while subsequent proofreading by extrinsic exonuclease(s) may reduce its mutagenic potential.

Genomic structure, chromosomal localization and identification of mutations in the xeroderma pigmentosum variant (XPV) gene.

The xeroderma pigmentosum variant (XP-V) is one of the most common forms of this cancer-prone syndrome. XP groups A through G are characterized by defective nucleotide excision repair, whereas the XP-V phenotype is proficient in this pathway. The XPV gene encodes DNA polymerase eta, which catalyzes an accurate translesion synthesis, indicating that the XPV gene contributes tumor suppression in normal individuals. Here we describe the genomic structure and chromosomal localization of the XPV gene, which includes 11 exons covering the entire coding sequence, lacks a TATA sequence in the upstream region of the transcription-initiation, and is located at the chromosome band 6p21.1-6p12. Analyses of patient-derived XP-V cell lines strongly suggested that three of four cell lines carried homozygous mutations in the XPV gene. The fourth cell line, XP1RO, carried heterozygous point mutations in the XPV gene, one of which was located at the splice acceptor site of exon 2, resulting in the omission of exon 2 from the mature mRNA. These findings provide a basis for diagnosis and therapy of XP-V patients.

Modulation of TFIIH-associated kinase activity by complex formation and its relationship with CTD phosphorylation of RNA polymerase II.

BACKGROUND: The general transcription factor TFIIH plays important roles in initiation and the transition to elongation steps of transcription by RNA polymerase II (PolII). Both roles are dependent on the protein kinase, DNA-dependent ATPase and DNA helicase activities of TFIIH. However, how these enzyme activities of TFIIH contribute to transcription has remained elusive. TFIIH consists of nine subunits, and one of them, Cdk7, possesses kinase activity. Here the substrate specificities of TFIIH and two forms of the Cdk7-containing kinase complex are compared, and the relationship between transcription activity and the TFIIH-dependent phosphorylation of the carboxy terminal domain of the largest subunit of PolII (CTD) is studied. RESULTS: We prepared TFIIH and two Cdk7-containing kinase complexes, Cdk7/Cyclin H and CAK (Cdk7/Cyclin H/MAT1). Consistent with previous reports, CAK strongly phosphorylated Cdk2, Cdk4, CTD and intact PolII. In contrast, Cdk7/Cyclin H, which lacks MAT1, did not phosphorylate these substrates, except for weak phosphorylation of Cdk2. The kinase activity of TFIIH displayed stronger substrate preference for Cdk4 than did CAK. In addition, TFIIH phosphorylation of PolII was stimulated by TFIIE both in solution and during preinitiation complex formation, whereas Cdk7/Cyclin H and CAK phosphorylation of PolII was not. In combination with other general transcription factors, TFIIH, but not Cdk7/CycH or CAK, promoted transcription on a linear DNA template. This transcription was well correlated with TFIIE stimulated TFIIH phosphorylation of serine at position 5 (Ser-5) within the heptapeptide repeat of the PolII CTD. CONCLUSION: These results provide clues about the roles of CTD phosphorylation at Ser-5 in transcription.

Error-prone bypass of certain DNA lesions by the human DNA polymerase kappa.

The Escherichia coli protein DinB is a newly identified error-prone DNA polymerase. Recently, a human homolog of DinB was identified and named DINB1. We report that the DINB1 gene encodes a DNA polymerase (designated polkappa), which incorporates mismatched bases on a nondamaged template with a high frequency. Moreover, polkappa bypasses an abasic site and N-2-acetylaminofluorene (AAF)-adduct in an error-prone manner but does not bypass a cis-syn or (6-4) thymine-thymine dimer or a cisplatin-adduct. Therefore, our results implicate an important role for polkappa in the mutagenic bypass of certain types of DNA lesions.

3-Methyladenine-DNA glycosylase (MPG protein) interacts with human RAD23 proteins.

Human 3-methyladenine-DNA glycosylase (MPG protein) initiates base excision repair by severing the glycosylic bond of numerous damaged bases. In comparison, homologues of the Rad23 proteins (hHR23) and the hXPC protein are involved in the recognition of damaged bases in global genome repair, a subset of nucleotide excision repair. In this report, we show that the hHR23A and -B also interact with the MPG protein and can serve as accessory proteins for DNA damage recognition in base excision repair. Furthermore, the MPG.hHR23 protein complex elevates the rate of MPG protein-catalyzed excision from hypoxanthine-containing substrates. This increased excision rate is correlated with a greater binding affinity of the MPG protein-hHR23 protein complex for damaged DNA. These data suggest that the hHR23 proteins function as universal DNA damage recognition accessory proteins in both of these major excision repair pathways.

Complementation of defective translesion synthesis and UV light sensitivity in xeroderma pigmentosum variant cells by human and mouse DNA polymerase eta.

Defects in the human gene XPV result in the variant form of the genetic disease xeroderma pigmentosum (XP-V). XPV encodes DNA polymerase eta, a novel DNA polymerase that belongs to the UmuC/DinB/Rad30 superfamily. This polymerase catalyzes the efficient and accurate translesion synthesis of DNA past cis-syn cyclobutane di-thymine lesions. In this report we present the cDNA sequence and expression profiles of the mouse XPV gene and demonstrate its ability to complement defective DNA synthesis in XP-V cells. The mouse XPV protein shares 80.3% amino acid identity and 86.9% similarity with the human XPV protein. The recombinant mouse XPV protein corrected the inability of XP-V cell extracts to carry out DNA replication, by bypassing thymine dimers on template DNA. Transfection of the mouse or human XPV cDNA into human XP-V cells corrected UV sensitivity. Northern blot analysis revealed that the mouse XPV gene is expressed ubiquitously, but at a higher level in testis, liver, skin and thymus compared to other tissues. Although the mouse XPV gene was not induced by UV irradiation, its expression was elevated approximately 4-fold during cell proliferation. These results suggest that DNA polymerase eta plays a role in DNA replication, though the enzyme is not essential for viability.

Mechanisms of accurate translesion synthesis by human DNA polymerase eta.

The XPV (xeroderma pigmentosum variant) gene encodes human DNA polymerase eta (pol eta), which is involved in the replication of damaged DNA. Pol eta catalyzes efficient and accurate translesion synthesis past cis-syn cyclobutane di-thymine lesions. Here we show that human pol eta can catalyze translesion synthesis past an abasic (AP) site analog, N-2-acetylaminofluorene (AAF)-modified guanine, and a cisplatin-induced intrastrand cross-link between two guanines. Pol eta preferentially incorporated dAMP and dGMP opposite AP, and dCMP opposite AAF-G and cisplatin-GG, but other nucleotides were also incorporated opposite these lesions. However, after incorporating an incorrect nucleotide opposite a lesion, pol eta could not continue chain elongation. In contrast, after incorporating the correct nucleotide opposite a lesion, pol eta could continue chain elongation, whereas pol alpha could not. Thus, the fidelity of translesion synthesis by human pol eta relies not only on the ability of this enzyme to incorporate the correct nucleotide opposite a lesion, but also on its ability to elongate only DNA chains that have a correctly incorporated nucleotide opposite a lesion.

Low fidelity DNA synthesis by human DNA polymerase-eta.

A superfamily of DNA polymerases that bypass lesions in DNA has been described. Some family members are described as error-prone because mutations that inactivate the polymerase reduce damage-induced mutagenesis. In contrast, mutations in the skin cancer susceptibility gene XPV, which encodes DNA polymerase (pol)-eta, lead to increased ultraviolet-induced mutagenesis. This, and the fact that pol-eta primarily inserts adenines during efficient bypass of thymine-thymine dimers in vitro, has led to the description of pol-eta as error-free. However, here we show that human pol-eta copies undamaged DNA with much lower fidelity than any other template-dependent DNA polymerase studied. Pol-eta lacks an intrinsic proofreading exonuclease activity and, depending on the mismatch, makes one base substitution error for every 18 to 380 nucleotides synthesized. This very low fidelity indicates a relaxed requirement for correct base pairing geometry and indicates that the function of pol-eta may be tightly controlled to prevent potentially mutagenic DNA synthesis.

Efficient translesion replication past oxaliplatin and cisplatin GpG adducts by human DNA polymerase eta.

Platinum anticancer agents form bulky DNA adducts which are thought to exert their cytotoxic effect by blocking DNA replication. Translesion synthesis, one of the pathways of postreplication repair, is thought to account for some resistance to DNA damage and much of the mutagenicity of bulky DNA adducts in dividing cells. Oxaliplatin has been shown to be effective in cisplatin-resistant cell lines and less mutagenic than cisplatin in the Ames assay. We have shown that the eukaryotic DNA polymerases yeast pol zeta, human pol beta, and human pol gamma bypass oxaliplatin-GG adducts more efficiently than cisplatin-GG adducts. Human pol eta, a product of the XPV gene, has been shown to catalyze efficient translesion synthesis past cis, syn-cyclobutane pyrimidine dimers. In the present study we compared translesion synthesis past different Pt-GG adducts by human pol eta. Our data show that, similar to other eukaryotic DNA polymerases, pol eta bypasses oxaliplatin-GG adducts more efficiently than cisplatin-GG adducts. However, pol eta-catalyzed translesion replication past Pt-DNA adducts was more efficient and less accurate than that seen for previously tested polymerases. We show that the efficiency and fidelity of translesion replication past Pt-DNA adducts appear to be determined by both the structure of the adduct and the DNA polymerase active site.

The xeroderma pigmentosum group C protein complex XPC-HR23B plays an important role in the recruitment of transcription factor IIH to damaged DNA.

The xeroderma pigmentosum group C protein complex XPC-HR23B was first isolated as a factor that complemented nucleotide excision repair defects of XP-C cell extracts in vitro. Recent studies have revealed that this protein complex plays an important role in the early steps of global genome nucleotide excision repair, especially in damage recognition, open complex formation, and repair protein complex formation. However, the precise function of XPC-HR23B in global genome repair is still unclear. Here we demonstrate that XPC-HR23B interacts with general transcription factor IIH (TFIIH) both in vivo and in vitro. This interaction is thought to be mediated through the specific affinity of XPC for the TFIIH subunits XPB and/or p62, which are essential for both basal transcription and nucleotide excision repair. Interestingly, association of TFIIH with DNA was observed in both wild-type and XP-A cell extracts but not in XP-C cell extracts, and XPC-HR23B could restore the association of TFIIH with DNA in XP-C cell extracts. Moreover, we found that XPC-HR23B was necessary for efficient association of TFIIH with damaged DNA in cell-free extracts. We conclude that the XPC-HR23B protein complex plays a crucial role in the recruitment of TFIIH to damaged DNA in global genome repair.