Artemis is a negative regulator of p53 in response to oxidative stress.

Artemis is a multifunctional phospho-protein with roles in V(D)J recombination, repair of double-strand breaks by nonhomologous end-joining and regulation of cell-cycle checkpoints after DNA damage. Here, we describe a new function of Artemis as a negative regulator of p53 in response to oxidative stress in both primary cells and cancer cell lines. We show that depletion of Artemis under typical culture conditions (21% oxygen) leads to a spontaneous phosphorylation and stabilization of p53, and resulting cellular G1 arrest and apoptosis. These effects are suppressed by co-depletion of DNA-PKcs, but not ATM, indicating that Artemis is an inhibitor of DNA-PKcs-mediated stabilization of p53. Culturing of cellsat 3% oxygen or treatment with an antioxidant abrogated p53 stabilization, indicating that oxidative stress is the responsible cellular stimulus. Treatment with ionizing radiation or hydrogen peroxide did not cause activation of this signaling pathway, whereas inhibitors of mitochondrial electron transport were effective in reducing its activation. In addition, we show that p53-inducible genes involved in reducing reactive oxygen species are upregulated by Artemis depletion. These findings indicate that Artemis and DNA-PKcs participate in a new, signaling pathway to modulate p53 function in response to oxidative stress produced by mitochondrial respiration.

Snm1B/Apollo mediates replication fork collapse and S Phase checkpoint activation in response to DNA interstrand cross-links.

The removal of DNA interstrand cross-links (ICLs) has proven to be notoriously complicated due to the involvement of multiple pathways of DNA repair, which include the Fanconi anemia/BRCA pathway, homologous recombination and components of the nucleotide excision and mismatch repair pathways. Members of the SNM1 gene family have also been shown to have a role in mediating cellular resistance to ICLs, although their precise function has remained elusive. Here, we show that knockdown of Snm1B/Apollo in human cells results in hypersensitivity to mitomycin C (MMC), but not to IR. We also show that Snm1B-deficient cells exhibit a defective S phase checkpoint in response to MMC, but not to IR, and this finding may account for the specific sensitivity to the cross-linking drug. Interestingly, although previous studies have largely implicated ATR as the major kinase activated in response to ICLs, we show that it is activation of the ATM-mediated checkpoint that is defective in Snm1B-deficient cells. The requirement for Snm1B in ATM checkpoint activation specifically after ICL damage is correlated with its role in promoting double-strand break formation, and thus replication fork collapse. Consistent with this result Snm1B was found to interact directly with Mus81-Eme1, an endonuclease previously implicated in fork collapse. In addition, we also show that Snm1B interacts with the Mre11-Rad50-Nbs1 (MRN) complex and with FancD2 further substantiating its role as a checkpoint/DNA repair protein.

Cellular responses to ionizing radiation damage.

PURPOSE: The purpose of this report is to provide current perspectives on studies of DNA damage and cell cycle response after ionizing radiation, and their applications in radiation oncology. METHODS AND MATERIALS: Presentations at the Seventh Annual Radiation Oncology Workshop, held at the International Festival Institute at Round Top, TX, were summarized. RESULTS: Eighteen speakers presented their current work covering a wide range of studies on cellular responses to ionizing radiation. These presentations and discussions form the framework of our report. CONCLUSION: In response to ionizing radiation, cells immediately activate a series of biochemical pathways that promote cell survival while maintaining genetic integrity. The main cellular defense system against ionizing radiation exposure is composed of two distinct types of biochemical pathways, that is, the DNA damage cell cycle checkpoint pathways and the DNA repair pathways. The DNA damage checkpoint pathways are activated directly by DNA damage, while the repair pathways are constitutively active and are likely modulated by checkpoint signals. Discussions here emphasize that the ATM protein is a central component of the ionizing radiation-responsive pyramid and is essential for activating divergent molecular responses that involve transcriptional regulation, cell cycle arrest, and modulation of DNA repair. The relationship between homologous recombinational repair and nonhomologous end joining of double-strand breaks is also discussed.

Involvement of nucleotide excision repair in a recombination-independent and error-prone pathway of DNA interstrand cross-link repair.

DNA interstrand cross-links (ICLs) block the strand separation necessary for essential DNA functions such as transcription and replication and, hence, represent an important class of DNA lesion. Since both strands of the double helix are affected in cross-linked DNA, it is likely that conservative recombination using undamaged homologous regions as a donor may be required to repair ICLs in an error-free manner. However, in Escherichia coli and yeast, recombination-independent mechanisms of ICL repair have been identified in addition to recombinational repair pathways. To study the repair mechanisms of interstrand cross-links in mammalian cells, we developed an in vivo reactivation assay to examine the removal of interstrand cross-links in cultured cells. A site-specific psoralen cross-link was placed between the promoter and the coding region to inactivate the expression of green fluorescent protein or luciferase genes from reporter plasmids. By monitoring the reactivation of the reporter gene, we showed that a single defined psoralen cross-link was removed in repair-proficient cells in the absence of undamaged homologous sequences, suggesting the existence of an ICL repair pathway that is independent of homologous recombination. Mutant cell lines deficient in the nucleotide excision repair pathway were examined and found to be highly defective in the recombination-independent repair of ICLs, while mutants deficient in homologous recombination were found to be proficient. Mutation analysis of plasmids recovered from transfected cells showed frequent base substitutions at or near positions opposing a cross-linked thymidine residue. Based on these results, we suggest a distinct pathway for DNA interstrand cross-link repair involving nucleotide excision repair and a putative lesion bypass mechanism.

Differential processing of UV mimetic and interstrand crosslink damage by XPF cell extracts.

We have recently developed a mammalian cell free assay in which interstrand crosslinks induce DNA synthesis in both damaged and undamaged plasmids co-incubated in the same extract. We have also shown using hamster mutants that both ERCC1 and XPF are required for the observed incorporation. Here, we show that extracts from an XPF patient cell line differentially process UV mimetic damage and interstrand crosslinks in vitro. XPF extracts are highly defective in the stimulation of repair synthesis by N:-acetoxy-N:- acetylaminofluorene, but are proficient in the stimulation of DNA synthesis by psoralen interstrand crosslinks. In addition, we show that extracts from the hamster UV140 mutant, which has high UV sensitivity, but moderate mitomycin C sensitivity, are similar in both assays to XPF cell extracts. These findings support the hypothesis that the activities of XPF in nucleotide excision repair (NER) and crosslink repair are separable, and that mutations in XPF patients result in the abolition of NER, but not recombinational repair pathways, which are likely to be essential as has been observed in ERCC1 homozygous -/- mice.

Requirement for PCNA and RPA in interstrand crosslink-induced DNA synthesis.

Proliferating nuclear cell antigen (PCNA) and replication protein A (RPA) have proven to be essential elements in many aspects of DNA metabolism including replication, repair and recombination. We have developed an in vitro assay in which the presence of an interstrand crosslink stimulates the incorporation of radiolabeled nucleotides into both damaged and undamaged plasmid DNAs. Using this assay we have investigated the roles of PCNA and RPA in crosslink-induced DNA synthesis. p21, a potent inhibitor of PCNA, was found to strongly inhibit crosslink-induced incorporation. Addition of exogenous PCNA partially restored the resynthesis activity. Likewise, neutralization of RPA by monoclonal antibodies also inhibited incorporation, but the effect was somewhat more pronounced on the undamaged plasmid than the damaged plasmid. Addition of excess RPA also partially reversed antibody inhibition. These results indicate that both PCNA and RPA are required for efficient in vitro DNA resynthesis induced by interstrand crosslinks.

Expression in normal human tissues of five nucleotide excision repair genes measured simultaneously by multiplex reverse transcription-polymerase chain reaction.

DNA repair is central to the integrity of the human genome. Reduced DNA repair capacity has been linked to genetic susceptibility to cancer. An adequate expression level of DNA repair genes is essential for normal DNA repair activities. Although there is tissue specificity in the expression, searching for a surrogate tissue is needed for molecular epidemiological studies. In this study, the relative expression levels of five selected human nucleotide excision repair (NER) genes (ERCC1, XPB/ERCC3, XPG/ERCC5, CSB/ERCC6, and XPC) in 20 different types of human normal tissue were simultaneously measured by a new multiplex reverse transcription (RT)-PCR assay using the expression level of the beta-actin gene as an internal control. Transcripts of each of the five NER genes were detectable, but the levels varied in these normal tissues. Both mitogen (phytohemagglutinin)-stimulated and unstimulated human peripheral lymphocytes showed similar expression patterns for the five NER genes. In general, the expression levels of stimulated lymphocytes were also similar to most of the rapidly proliferating tissues, such as the skin, breast, intestine, liver, testis, ovary, placenta, or prostate, but was relatively higher than that of the slowly proliferating or nonproliferating tissues such as adipose, brain, hippocampus, muscle, spleen, or lung. The data suggested that although the five NER genes were expressed at different levels in the normal tissues examined, PHA-stimulated peripheral lymphocytes may be used as a surrogate tissue for estimating expression levels of these genes in proliferating tissues. This new multiplex RT-PCR assay may help detect aberrant expression of these NER genes in both normal and tumor tissues.

Interstrand cross-links induce DNA synthesis in damaged and undamaged plasmids in mammalian cell extracts.

Mammalian cell extracts have been shown to carry out damage-specific DNA repair synthesis induced by a variety of lesions, including those created by UV and cisplatin. Here, we show that a single psoralen interstrand cross-link induces DNA synthesis in both the damaged plasmid and a second homologous unmodified plasmid coincubated in the extract. The presence of the second plasmid strongly stimulates repair synthesis in the cross-linked plasmid. Heterologous DNAs also stimulate repair synthesis to variable extents. Psoralen monoadducts and double-strand breaks do not induce repair synthesis in the unmodified plasmid, indicating that such incorporation is specific to interstrand cross-links. This induced repair synthesis is consistent with previous evidence indicating a recombinational mode of repair for interstrand cross-links. DNA synthesis is compromised in extracts from mutants (deficient in ERCC1, XPF, XRCC2, and XRCC3) which are all sensitive to DNA cross-linking agents but is normal in extracts from mutants (XP-A, XP-C, and XP-G) which are much less sensitive. Extracts from Fanconi anemia cells exhibit an intermediate to wild-type level of activity dependent upon the complementation group. The DNA synthesis deficit in ERCC1- and XPF-deficient extracts is restored by addition of purified ERCC1-XPF heterodimer. This system provides a biochemical assay for investigating mechanisms of interstrand cross-link repair and should also facilitate the identification and functional characterization of cellular proteins involved in repair of these lesions.

hRAD17, a structural homolog of the Schizosaccharomyces pombe RAD17 cell cycle checkpoint gene, stimulates p53 accumulation.

The RAD17 gene product of S. Pombe is an essential component of the checkpoint control pathway which responds to both DNA damage and disruption of replication. We have identified a human cDNA that encodes a polypeptide which is structurally conserved with the S. Pombe Rad17 protein. The human gene, designated hRAD17, predicts an encoded protein of 590 amino acids and a molecular weight of 69 kD. Amino acid sequence alignment revealed that hRadl7 has 28.3% and 52.5% similarity with the S. Pombe Rad17 protein, and 21.8% identity and 45.8% similarity to the budding yeast cell cycle checkpoint protein, Rad 24. When introduced into the S. Pombe rad17 mutant, hRAD17 was able to partially revert its hydroxyurea and ionizing radiation hypersensitivity, but not its UV hypersensitivity. Permanent overexpression of the hRAD17 gene in human fibrosarcoma cells resulted in p53 activation and a significant reduction of S- and G2/M-phase cells accompanied by an accumulation of the G1-phase population, suggesting that hRAD17 may have a role in cell cycle checkpoint control. Immunostaining of HT-1080 cells transiently transfected with a hRAD17 construct confirmed the nuclear accumulation of p53, which mimics the induction caused by DNA damage. Using FISH analysis, we have mapped the hRAD17 locus to human chromosome 5q11.2.

RAD1, a human structural homolog of the Schizosaccharomyces pombe RAD1 cell cycle checkpoint gene.

Cell cycle checkpoints are gating mechanisms that govern cell cycle progression in the presence of DNA damage and incomplete DNA replication. The Schizosaccharomyces pombe Rad1 protein is an essential component of cell cycle checkpoints activated by both types of genomic stress. In this study, we report the isolation of a human homolog of the S. pombe RAD1 gene. The hRAD1 protein is also similar to the Saccharomyces cerevisiae cell cycle checkpoint protein Rad17 and the Ustilago maydis 3' --> 5' exonuclease, Rec1. We show that human RAD1 partially complements the hydroxyurea and ionizing radiation hypersensitivities of a S. pombe rad1 mutant, suggesting phylogenetic conservation of the DNA damage and replication checkpoints. The human RAD1 locus was mapped to human chromosome 5p13.2, a locus frequently altered in non-small-cell lung cancer and bladder cancer.

Xeroderma pigmentosum group C splice mutation associated with autism and hypoglycinemia.

A 4 y old boy of Korean ancestry had xeroderma pigmentosum (XP) with sun sensitivity, multiple cutaneous neoplasms, and inability to speak. Neurologic examination revealed hyperactivity and autistic features without typical XP neurologic abnormalities. Cultured skin fibroblasts (XP22BE) showed decreased post-UV survival, reduced post-UV plasmid host cell reactivation and defective DNA repair (16% of normal unscheduled DNA synthesis in intact cells and undetectable excision repair in a cell free extract). In vitro and in vivo complementation assigned XP22BE to XP group C (XPC) and a markedly reduced level of XPC mRNA was found. Two XPC cDNA bands were identified. One band had a deletion of 161 bases comprising the entire exon 9, which resulted in premature termination of the mutant XPC mRNA. The larger band also had the same deletion of exon 9 but, in addition, had an insertion of 155 bases in its place (exon 9a), resulting in an in-frame XPC mRNA. Genomic DNA analysis revealed a T-->G mutation at the splice donor site of XPC exon 9, which markedly reduced its information content. The 155 base pair XPC exon 9a insertion was located in intron 9 and was flanked by strong splice donor and acceptor sequences. Analysis of the patient's blood showed persistently low levels of glycine (68 microM; NL, 125-318 microM). Normal glycine levels were maintained with oral glycine supplements and his hyperactivity diminished. These data provide evidence of an association of an XPC splice site mutation with autistic neurologic features and hypoglycinemia.

XPC interacts with both HHR23B and HHR23A in vivo.

XP group C protein (XPC) and a human homologue of RAD23, HHR23B, have previously been shown to copurify in a tightly associated complex. Here, we show that XPC interacts in vivo, by means of the yeast two-hybrid system, with both HHR23B and a second homologue of RAD23, HHR23A. Domain mapping studies have revealed that both RAD23 homologues interact with XPC at the same highly conserved region in the C-terminal half of the protein. XPC mutants deleted within this domain and that are highly deficient in binding both RAD23 homologues are also highly defective in complementing XPC cells in vivo. Domain mapping studies have also identified a region in the N-terminal half of HHR23B that contains the XPC interactive site. This domain is highly conserved among HHR23B, HHR23A, and RAD23.

Confirmation of homozygosity for a single nucleotide substitution mutation in a Cockayne syndrome patient using monoallelic mutation analysis in somatic cell hybrids.

The identification of individuals homozygous for a specific mutation offers advantages for the elucidation of molecular mechanisms of hereditary disease states. Cockayne syndrome is a rare autosomal recessive disorder, the molecular basis of which is complicated by significant genetic and clinical heterogeneity. The genes associated with both genetic complementation groups, CSA and CS-B, have been identified. We have previously identified a number of CSA mutations, including a single base substitution that introduces a stop codon (322Tyr-->Stop) mutation in the C-terminal region for at least one allele of the CSA gene in a severely affected patient. We now present data confirming the existence of homozygosity in this patient using a strategy with general applicability. Somatic cell hybrids were established by fusing patient cells with mouse A9 cells. Screening with chromosome 5 specific polymorphic markers facilitated identification of hybrid clones bearing only one of the distinct CSA alleles. Sequencing of a portion of the human CSA gene in a subset of these hybrids permitted monoallelic mutation analysis and confirmed the presence of the 322Tyr-->Stop mutation in both alleles.

Expression of five selected human mismatch repair genes simultaneously detected in normal and cancer cell lines by a nonradioactive multiplex reverse transcription-polymerase chain reaction.

Abnormalities in at least 1 of 5 mismatch repair (MMR) genes (hMSH2, hMLH1, hPMS1, hPMS2 and GTBP/hMSH6) are found in hereditary nonpolyposis colon cancer and sporadic colon cancers. We used a single-reaction multiplex reverse transcription (RT)-polymerase chain reaction (PCR), with the beta-actin gene as an internal control, to simultaneously evaluate expression of these 5 known human MMR genes in normal and tumor cell lines with known or uncharacterized mutations in MMR genes. The relative quantitation of the transcripts is demonstrated by controlling the number of PCR cycles and titrating cDNA with a dose-curve. The 13 normal cell lines tested were derived from normal lymphocytes, skin, thymus, breast, lung, colon, liver and kidney. The 26 cancer cell lines were derived from melanoma and cancers of the brain, breast, lung, colon, pancreas and prostate. All 5 MMR genes were ubiquitously expressed in all normal cell lines tested, suggesting their housekeeping roles. Aberrant MMR gene expression was only observed in the colon cancer cell lines. Two previously uncharacterized colon cancer cell lines did not express hMLH1. These data suggest that this nonradioactive multiplex RT-PCR assay for MMR gene expression may be useful for fast screening for genetic alterations that may affect gene expression and so may aid molecular analysis of MMR-related colon cancer.

Sequence of the mouse XPC cDNA and genomic structure of the human XPC gene.

The full length-mouse XPC cDNA contains a 2703 bp orf which encodes a polypeptide containing 900 amino acids. Overall, there is 75% identity in nucleotide sequence and 73% identity in amino acid sequence between mouse and human genes. The C-terminal half is more conserved (80%) than the N-terminal half (65%). Northern analysis has revealed a constitutive expression pattern for both human and mouse transcripts in various tissues examined. However, high level expression was observed in liver, testis and kidney in both species. The human XPC gene was cloned from a cosmid library and the full-length gene was found to span -24 kb. Analysis of the genomic structure indicated that the transcribed sequence is divided into 15 exons.

Genetic complementation of radiation response by 3' untranslated regions (UTR) of RNA.

The molecular basis of radiosensitivity was studied using a cDNA complementation approach to correct radiosensitivity in cells. Four cDNAs of sizes 1.6, 2.0, 2.2 and 2.5 kb were isolated that corrected several aspects of the phenotype of cells from patients with the human genetic disorder ataxia-telangiectasia, characterized by hypersensitivity to ionizing radiation. The criteria used to assess correction included cell viability, induced chromosome aberrations, G2 phase delay and induction of p53 after exposure to radiation. One cDNA (2.5 kb) was identified as the complete sequence of the RNA helicase p68, which was capable of correcting radiosensitivity based on two of the above four criteria, with p53 induction post irradiation being partially corrected. The 2.2 kb cDNA was shown to correspond to the complete sequence of arginyl tRNA synthetase and the other two cDNAs were identical to the 3' untranslated regions (UTR) of the transcription factor TFIIS (1.6 kb) and phospholipase A2 (2.0 kb) respectively. Additional transfections with the 3'UTR (198 nucleotides) of p68 RNA helicase and its inverse sequence revealed that the 3'UTR had the same complementation capacity as the full-length cDNA, whereas the inverse construct failed to complement radiosensitivity. These data provide additional support for a novel role for 3'UTRs in the regulation of gene expression.

Nucleotide excision repair genes as determinants of cellular sensitivity to cyclophosphamide analogs.

UNLABELLED: The objective of this study was to determine the relative importance of the first six complementation groups of the nucleotide excision repair cross-complementing genes (ERCC1-ERCC6) and the first complementation group of the X-ray repair cross-complementing genes (XRCC1), in the repair of DNA damage induced by the in vitro active cyclophosphamide (CP) derivatives 4-hydroperoxycyclophosphamide (4HC) and phosphorodiamidic mustard (PM). We compared the sensitivity of the wild-type CHO cell line, AA8, with that of the CHO mutant cell lines UV4 and UV20 (ERCC1-), UV5 (ERCC2-), UV24 (ERCC3-), UV41 (ERCC4-), UV135 (ERCC5-), UV61 (ERCC6-), and EM9 (XRCC1-). Cell survival was determined using both growth inhibition and conventional clonogenic assays. The yield of DNA crosslinks in selected cell lines was determined using an ethidium bromide fluorescence assay. RESULTS: The rank ordering of sensitivity to both 4HC and PM, based on the combined survival data, was UV41/UV4/UV20 > > UV61/UV24/UV135/EM9 > or = UV5 approximately AA8. Thus mutations in the ERCC1 and ERCC4 genes impart a hypersensitivity to CP analogs. To confirm the importance of the ERCC1 gene for cellular resistance to 4HC and PM, UV20 cells were transfected with the human ERCC1 gene and subsequently exposed to 4HC and PM. The transfected cells displayed essentially wild-type resistance to both drugs. Furthermore, two interspecific hybrids derived from UV41, both of which retained the region of human chromosome 16 that harbors the ERCC4 gene, displayed essentially wild-type resistance to 4HC and PM, confirming the importance of ERCC4 for the repair of 4HC-induced DNA damage. When crosslinks were assayed after a 60-min treatment with 4HC or a 15-min treatment with PM, their yield paralleled the sensitivity of the cell lines to both drugs: UV41 cells showed markedly elevated levels of crosslinks, whereas AA8 and UV5 cells showed similar (low) levels of crosslinks. CONCLUSIONS: Our findings confirm the general pattern indicating that the ERCC1 and ERCC4 gene products are crucial for the repair of 4HC-induced DNA damage, while the other nucleotide excision repair genes examined are relatively unimportant. These data suggest that the hypersensitivity of ERCC1- and ERCC4- mutants to DNA crosslinking agents may reflect a defect in recombinational repair rather than nucleotide excision repair.

Simultaneous amplification of four DNA repair genes and beta-actin in human lymphocytes by multiplex reverse transcriptase-PCR.

We describe here the development, optimization, and use of a non-radioactive, quantitative, multiplex reverse transcriptase-PCR technique to measure, in a single reaction, the relative levels of the transcripts of four DNA repair genes (XPCC, hMSH2, XRCC1, and ERCC1) and the beta-actin gene in lymphoblastoid cell lines and frozen peripheral blood lymphocytes. Expression of defective DNA repair genes was not detected in DNA repair-deficient human cell lines, whereas the intact genes were detected in repair-proficient cell lines and in lymphocytes from a normal donor. The assay was reproducible, and repeated determinations of the same samples generated highly consistent results for each target gene. This approach should facilitate molecular epidemiological studies that incorporate screening for germline alterations that may affect gene expression and for changes in the levels of gene expression.

An interaction between the DNA repair factor XPA and replication protein A appears essential for nucleotide excision repair.

Replication protein A (RPA) is required for simian virus 40-directed DNA replication in vitro and for nucleotide excision repair (NER). Here we report that RPA and the human repair protein XPA specifically interact both in vitro and in vivo. Mapping of the RPA-interactive domains in XPA revealed that both of the largest subunits of RPA, RPA-70 and RPA-34, interact with XPA at distinct sites. A domain involved in mediating the interaction with RPA-70 was located between XPA residues 153 and 176. Deletion of highly conserved motifs within this region identified two mutants that were deficient in binding RPA in vitro and highly defective in NER both in vitro and in vivo. A second domain mediating the interaction with RPA-34 was identified within the first 58 residues in XPA. Deletion of this region, however, only moderately affects the complementing activity of XPA in vivo. Finally, the XPA-RPA complex is shown to have a greater affinity for damaged DNA than XPA alone. Taken together, these results indicate that the interaction between XPA and RPA is required for NER but that only the interaction with RPA-70 is essential.

Inhibition of nucleotide excision repair by the cyclin-dependent kinase inhibitor p21.

p21, a p53-induced gene product that blocks cell cycle progression at the G1 phase, interacts with both cyclin-dependent kinases and proliferating cell nuclear antigen (PCNA). PCNA functions as a processivity factor for DNA polymerases delta and epsilon and is required for both DNA replication and nucleotide excision repair. Previous studies have shown that p21 inhibits simian virus 40 (SV40) DNA replication in HeLa cell extracts by interacting with PCNA. In this report we show that p21 blocks nucleotide excision repair of DNA that has been damaged by either ultraviolet radiation or alkylating agents, and that this inhibition can be reversed following addition of PCNA. We have determined that p21 is more effective in blocking DNA resynthesis than in inhibiting the excision step. We further show that a peptide derived from the carboxyl terminus of p21, which specifically interacts with PCNA, inhibits polymerase delta-catalyzed elongation of DNA chains almost stoichiometrically relative to the concentration of PCNA. When added at higher levels, this peptide also blocks both SV40 DNA replication and nucleotide excision repair in HeLa cell extracts. These results indicate that p21 interferes with the function of PCNA in both in vitro DNA replication and nucleotide excision repair.