Human Nucleotide Excision Repair Research Articles

Similar Nucleotide Excision Repair Capacity in Melanocytes and Melanoma Cells

Sunlight UV exposure produces DNA photoproducts in skin that are repaired solely by nucleotide excision repair in humans. A significant fraction of melanomas are thought to result from UV-induced DNA damage that escapes repair; however, little evidence is available about the functional capacity of normal human melanocytes, malignant melanoma cells, and metastatic melanoma cells to repair UV-induced photoproducts in DNA. In this study, we measured nucleotide excision repair in both normal melanocytes and a panel of melanoma cell lines. Our results show that in 11 of 12 melanoma cell lines tested, UV photoproduct repair occurred as efficiently as in primary melanocytes. Importantly, repair capacity was not affected by mutation in the N-RAS or B-RAF oncogenes, nor was a difference observed between a highly metastatic melanoma cell line (A375SM) or its parental line (A375P). Lastly, we found that although p53 status contributed to photoproduct removal efficiency, its role did not seem to be mediated by enhanced expression or activity of DNA binding protein DDB2. We concluded that melanoma cells retain capacity for nucleotide excision repair, the loss of which probably does not commonly contribute to melanoma progression.

XPA impacts formation but not proteasome-sensitive repair of DNA-protein cross-links induced by chromate

DNA-protein cross-links (DPCs) are caused by a large number of human carcinogens and anti-cancer drugs. However, cellular processes involved in decreasing a burden of these genotoxic lesions remain poorly understood. Here, we examined the impact of nucleotide excision repair (NER), which is a principal repair pathway for bulky DNA adducts, and the main cellular reducers on removal of chromium(VI)-induced DPC. We found that standard and ascorbate-restored cultures of isogenic XPA-null (NER deficient) and XPA-complemented human fibroblasts had very similar repair of Cr-DPC (60-65% average DPC removal after 24 h). However, XPA absence caused depletion of G1 and accumulation of G2 cells at low Cr(VI) doses, suggesting that Cr-DPC were not a significant cause of cell cycle perturbations. Interestingly, although pro-oxidant metabolism of Cr(VI) in glutathione-depleted cells generated significantly fewer DPC, they were repair resistant irrespective of the NER status of cells. Inhibition of proteasome activity by MG132 abolished DPC repair in both XPA-null and XPA-complemented cells. XPA loss caused two to three times higher initial DPC formation, demonstrating the importance of NER in removal of the precursor lesions. Our results indicate that human NER is not involved in removal of Cr-DPC containing non-histone proteins but it acts as a defence mechanism against these large lesions by preventing their formation. Therefore, individual differences in NER activity are expected to alter sensitivity but not persistence of DPC as a biomarker of hexavalent Cr.

Distant Neighbor Base Sequence Context Effects in Human Nucleotide Excision Repair of a Benzo[a]pyrene-derived DNA Lesion

The effects of non-nearest base sequences, beyond the nucleotides flanking a DNA lesion on either side, on nucleotide excision repair (NER) in extracts from human cells were investigated. We constructed two duplexes containing the same minor groove-aligned 10S (+)-trans-anti-B[a]P-N2-dG (G⁎) DNA adduct, derived from the environmental carcinogen benzo[a]pyrene (B[a]P): 5′-C-C-A-T-C-G⁎-C-T-A-C-C-3′ (CG⁎C-I), and 5′-C-A-C3-A4-C5-G⁎-C-A-C-A-C-3′ (CG⁎C-II). We used polyacrylamide gel electrophoresis to compare the extent of DNA bending, and molecular dynamics simulations to analyze the structural characteristics of these two DNA duplexes. The NER efficiencies are 1.6(±0.2)-fold greater in the case of the CG⁎C-II than the CG⁎C-I sequence context in 135-mer duplexes. Gel electrophoresis and self-ligation circularization experiments revealed that the CG⁎C-II duplex is more bent than the CG⁎C-I duplex, while molecular dynamics simulations showed that the unique -C3-A4-C5- segment in the CG⁎C-II duplex plays a key role. The presence of a minor groove-positioned guanine amino group, the Watson–Crick partner to C3, acts as a wedge; facilitated by a highly deformable local -C3-A4- base step, this amino group allows the B[a]P ring system to produce a more enlarged minor groove in CG⁎C-II than in CG⁎C-I, as well as a local untwisting and enlarged and flexible Roll only in the CG⁎C-II sequence. These structural properties fit well with our earlier findings that in the case of the family of minor groove 10S (+)-trans-anti-B[a]P-N2-dG lesions, flexible bends and enlarged minor groove widths constitute NER recognition signals, and extend our understanding of sequence context effects on NER to the neighbors that are distant to the lesion.

Three DNA Polymerases, Recruited by Different Mechanisms, Carry Out NER Repair Synthesis in Human Cells

Nucleotide excision repair (NER) is the most versatile DNA repair system that deals with the major UV photoproducts in DNA, as well as many other DNA adducts. The early steps of NER are well understood, whereas the later steps of repair synthesis and ligation are not. In particular, which polymerases are definitely involved in repair synthesis and how they are recruited to the damaged sites has not yet been established. We report that, in human fibroblasts, approximately half of the repair synthesis requires both pol kappa and pol delta, and both polymerases can be recovered in the same repair complexes. Pol kappa is recruited to repair sites by ubiquitinated PCNA and XRCC1 and pol delta by the classical replication factor complex RFC1-RFC, together with a polymerase accessory factor, p66, and unmodified PCNA. The remaining repair synthesis is dependent on pol epsilon, recruitment of which is dependent on the alternative clamp loader CTF18-RFC.

Binding of the human nucleotide excision repair proteins XPA and XPC/HR23B to the 5 R -thymine glycol lesion and structure of the cis -(5 R ,6 S ) thymine glycol epimer in the 5′-GTgG-3′ sequence: destabilization of two base pairs at the lesion site

The 5R thymine glycol (5R-Tg) DNA lesion exists as a mixture of cis-(5R,6S) and trans-(5R,6R) epimers; these modulate base excision repair. We examine the 7:3 cis-(5R,6S):trans-(5R,6R) mixture of epimers paired opposite adenine in the 5′-GTgG-3′ sequence with regard to nucleotide excision repair. Human XPA recognizes the lesion comparably to the C8-dG acetylaminoflourene (AAF) adduct, whereas XPC/HR23B recognition of Tg is superior. 5R-Tg is processed by the Escherichia coli UvrA and UvrABC proteins less efficiently than the C8-dG AAF adduct. For the cis-(5R, 6S) epimer Tg and A are inserted into the helix, remaining in the Watson–Crick alignment. The Tg N3H imine and A N6 amine protons undergo increased solvent exchange. Stacking between Tg and the 3′-neighbor G•C base pair is disrupted. The solvent accessible surface and T2 relaxation of Tg increases. Molecular dynamics calculations predict that the axial conformation of the Tg CH3 group is favored; propeller twisting of the Tg•A pair and hydrogen bonding between Tg OH6 and the N7 atom of the 3′-neighbor guanine alleviate steric clash with the 5′-neighbor base pair. Tg also destabilizes the 5′-neighbor G•C base pair. This may facilitate flipping both base pairs from the helix, enabling XPC/HR23B recognition prior to recruitment of XPA.

Coordination of dual incision and repair synthesis in human nucleotide excision repair

Nucleotide excision repair (NER) requires the coordinated sequential assembly and actions of the involved proteins at sites of DNA damage. Following damage recognition, dual incision 5' to the lesion by ERCC1-XPF and 3' to the lesion by XPG leads to the removal of a lesion-containing oligonucleotide of about 30 nucleotides. The resulting single-stranded DNA (ssDNA) gap on the undamaged strand is filled in by DNA repair synthesis. Here, we have asked how dual incision and repair synthesis are coordinated in human cells to avoid the exposure of potentially harmful ssDNA intermediates. Using catalytically inactive mutants of ERCC1-XPF and XPG, we show that the 5' incision by ERCC1-XPF precedes the 3' incision by XPG and that the initiation of repair synthesis does not require the catalytic activity of XPG. We propose that a defined order of dual incision and repair synthesis exists in human cells in the form of a 'cut-patch-cut-patch' mechanism. This mechanism may aid the smooth progression through the NER pathway and contribute to genome integrity.

The Sequence Dependence of Human Nucleotide Excision Repair Efficiencies of Benzo[ a]pyrene-derived DNA Lesions: Insights into the Structural Factors that Favor Dual Incisions

Nucleotide excision repair (NER) is a vital cellular defense system against carcinogen–DNA adducts, which, if not repaired, can initiate cancer development. The structural features of bulky DNA lesions that account for differences in NER efficiencies in mammalian cells are not well understood. In vivo, the predominant DNA adduct derived from metabolically activated benzo[ a]pyrene (BP), a prominent environmental carcinogen, is the 10 S (+)- trans- anti-[BP]- N 2-dG adduct ( G*), which resides in the B-DNA minor groove 5′-oriented along the modified strand. We have compared the structural distortions in double-stranded DNA, imposed by this adduct, in the different sequence contexts 5′-…CG G*C…, 5′-…C G*GC…, 5′-…CI G*C… (I is 2′-deoxyinosine), and 5′-…C G*C…. On the basis of electrophoretic mobilities, all duplexes manifest moderate bends, except the 5′-…CG G*C…duplex, which exhibits an anomalous, slow mobility attributed to a pronounced flexible kink at the site of the lesion. This kink, resulting from steric hindrance between the 5′-flanking guanine amino group and the BP aromatic rings, both positioned in the minor groove, is abolished in the 5′-…CI G*C…duplex (the 2′-deoxyinosine group, I, lacks this amino group). In contrast, the sequence-isomeric 5′-…C G*GC…duplex exhibits only a moderate bend, but displays a remarkably increased opening rate at the 5′-flanking base pair of G*, indicating a significant destabilization of Watson–Crick hydrogen bonding. The NER dual incision product yields were compared for these different sequences embedded in otherwise identical 135-mer duplexes in cell-free human HeLa extracts. The yields of excision products varied by a factor of as much as ∼ 4 in the order 5′-...C G*GC…> 5′...CG G*C…≥ 5′...CI G*C…≥ 5′-…C G*C…. Overall, destabilized Watson–Crick hydrogen bonding, manifested in the 5′-...C G*GC...duplex, elicits the most significant NER response, while the flexible kink displayed in the sequence-isomeric 5′-...CG G*C...duplex represents a less significant signal in this series of substrates. These results demonstrate that the identical lesion can be repaired with markedly variable efficiency in different local sequence contexts that differentially alter the structural features of the DNA duplex around the lesion site.

Characterization of CHO XPF mutant UV41: Influence of XPF heterozygosity on double-strand break-induced intrachromosomal recombination

The UV hypersensitive CHO cell mutant UV41 is the archetypal XPF mammalian cell mutant, and was essential for cloning the human nucleotide excision repair (NER) gene XPF by DNA transfection and rescue. The ERCC1 and XPF genes encode proteins that form the heterodimer responsible for making incisions required in NER and the processing of certain types of recombination intermediates. In this study, we cloned and sequenced the CHO cell XPF cDNA, determining that the XPF mutation in UV41 is a +1 insertion in exon 8 generating a premature stop codon at amino acid position 499; however, the second allele of XPF is apparently unaltered in UV41, resulting in XPF heterozygosity. XPF expression was found to be several-fold lower in UV41 compared to its parental cell line, AA8. Using approaches we previously developed to study intrachromosomal recombination in CHO cells, we modified UV41 and its parental cell line AA8 to allow site-specific gene targeting at a Flp recombination target (FRT) in intron 3 of the endogenous adenine phosphoribosyltransferase (APRT) locus. Using FLP/FRT targeting, we integrated a plasmid containing an I-SceI endonuclease sequence into this site in the paired cell lines to generate a heteroallelic APRT duplication. Frequencies of intrachromosomal recombination between APRT heteroalleles and the structures of resulting recombinants were analyzed after I-SceI induction of site-specific double-strand breaks (DSBs) in a non-homologous insertion contained within APRT homology. Our results show that I-SceI induced a small proportion of aberrant recombinants reflecting DSB-induced deletions/rearrangements in parental, repair-proficient AA8 cells. However, in XPF mutant UV41, XPF heterozygosity is responsible for a similar, but much more pronounced genomic instability phenotype, manifested independently of DSB induction. In addition, gene conversions were suppressed in UV41 cells compared to wild-type cells. These observations suggest that UV41 exhibits a genomic instability phenotype of aberrant recombinational repair, confirming a critical role for XPF in mammalian cell recombination.

A Sensitive Flow Cytometry-based Nucleotide Excision Repair Assay Unexpectedly Reveals That Mitogen-activated Protein Kinase Signaling Does Not Regulate the Removal of UV-induced DNA Damage in Human Cells

In response to diverse genotoxic stimuli (e.g. UV and cisplatin), the mitogen-activated protein kinases ERK1/2, JNK1/2, and p38alpha/beta become rapidly phosphorylated and in turn activate multiple downstream effectors that modulate apoptosis and/or growth arrest. Furthermore, previous lines of evidence have strongly suggested that ERK1/2 and JNK1/2 participate in global-genomic nucleotide excision repair, a critical antineoplastic pathway that removes helix-distorting DNA adducts induced by a variety of mutagenic agents, including UV. To rigorously evaluate the potential role of mitogen-activated protein kinases in global-genomic nucleotide excision repair, various human cell strains (primary skin fibroblasts, primary lung fibroblasts, and HCT116 colon carcinoma cells) were treated with highly specific chemical inhibitors, which, following UV exposure, (i) abrogated the capacities of ERK1/2, JNK1/2, or p38alpha/beta to phosphorylate specific downstream effectors and (ii) characteristically modulated cellular proliferation, clonogenic survival, and/or apoptosis. A highly sensitive flow cytometry-based nucleotide excision repair assay recently optimized and validated in our laboratory was then employed to directly demonstrate that the kinetics of UV DNA photoadduct repair are highly similar in mock-treated versus mitogen-activated protein kinase inhibitor-treated cells. These data on primary and tumor cells treated with pharmacological inhibitors were fully corroborated by repair studies using (i) short hairpin RNA-mediated knockdown of ERK1/2 or JNK1/2 in human U2OS osteosarcoma cells and (ii) expression of a dominant negative p38alpha mutant in human primary lung fibroblasts. Our results provide solid evidence for the first time, in disaccord with a burgeoning perception, that mitogen-activated protein kinase signaling does not influence the efficiency of human global-genomic nucleotide excision repair.

Nucleotide excision repair and the degradation of RNA pol II by the Caenorhabditis elegans XPA and Rsp5 orthologues, RAD-3 and WWP-1

The Caenorhabditis elegans rad-3 gene was identified in a genetic screen for radiation sensitive (rad) mutants. Here, we report that the UV sensitivity of rad-3 mutants is caused by a nonsense mutation in the C. elegans orthologue of the human nucleotide excision repair gene XPA. We have used the xpa-1/rad-3 mutant to examine how a defect in nucleotide excision repair (NER) perturbs development. We find that C. elegans carrying a mutation in xpa-1/rad-3 are hypersensitive and hypermutable in response to UV irradiation, but do not display hypersensitivity to oxidative stress or show obvious developmental abnormalities in the absence of UV exposure. Consistent with these observations, non-irradiated xpa-1 mutants have a similar lifespan as wild type. We further show that UV irradiated xpa-1 mutants undergo a stage-dependent decline in growth and survival, which is associated with a loss in transcriptional competence. Surprisingly, transcriptionally quiescent dauer stage larvae are able to survive a dose of UV irradiation, which is otherwise lethal to early stage larvae. We show that the loss of transcriptional competence in UV irradiated xpa-1 mutants is associated with the degradation of the large RNA polymerase II (RNA pol II) subunit, AMA-1, and have identified WWP-1 as the putative E3 ubiquitin ligase mediating this process. The absence of wwp-1 by itself does not cause sensitivity to UV irradiation, but it acts synergistically with a mutation in xpa-1 to enhance UV hypersensitivity.

Ultraviolet light exposure triggers nuclear accumulation of p21WAF1 and accelerated senescence in human normal and nucleotide excision repair‐deficient fibroblast strains

Induction of the p21(WAF1) protein (hereafter called p21) following genotoxic stress is known to inhibit proliferating cell nuclear antigen (PCNA)-dependent DNA repair, downregulate apoptosis, and trigger a sustained growth-arrested phenotype called accelerated senescence. Studies with immortalized human and murine cell lines have revealed that exposure to ultraviolet light (UVC; 254 nm) results in the degradation of p21 to facilitate DNA repair and promote cell survival, or may lead to apoptotic cell death. The objective of the present study was to determine whether exposure of non-transformed human fibroblast strains to relatively low fluences of UVC (i.e., fluences typically used in the clonogenic survival assay) might induce sustained nuclear accumulation of p21, leading to accelerated senescence. We have evaluated the responses of normal human fibroblast (NHF) strains and nucleotide excision repair (NER)-deficient fibroblast strains representing xeroderma pigmentosum (XP) complementation groups A and G and Cockayne syndrome (CS) complementation groups A and B. We report that exposure of NHFs to < or =15 J/m(2) of UVC, and NER-deficient fibroblasts to < or =5 J/m(2) of UVC, results in sustained nuclear accumulation of p21 and growth arrest through accelerated senescence. With each fibroblast strain examined, exposure to UVC fluences that resulted in approximately 90% loss of clonogenic potential triggered significant (>60%) accelerated senescence, but only marginal (<5%) apoptosis. We conclude that nuclear accumulation of p21 accompanied by accelerated senescence may be an integral component of the response of human fibroblasts to UVC-induced DNA damage, irrespective of their DNA repair capabilities.

A mathematical model for human nucleotide excision repair: Damage recognition by random order assembly and kinetic proofreading

A mathematical model of human nucleotide excision repair was constructed and validated. The model incorporates cooperative damage recognition by RPA, XPA, and XPC followed by three kinetic proofreading steps by the TFIIH transcription/repair factor. The model yields results consistent with experimental data regarding excision rates of UV photoproducts by the reconstituted human excision nuclease system as well as the excision of oligonucleotides from undamaged DNA. The model predicts the effect that changes in the initial concentrations of repair factors have on the excision rate of damaged DNA and provides a testable hypothesis on the biochemical mechanism of cooperativity in protein assembly, suggesting experiments to determine if cooperativity in protein assembly results from an increased association rate or a decreased dissociation rate. Finally, a comparison between the random order assembly with kinetic proofreading model and a sequential assembly model is made. This investigation reveals the advantages of the random order assembly/kinetic proofreading model.

Quantitative electrospray ionization mass spectrometry of zinc finger oxidation: The reaction of XPA zinc finger with H 2O 2

Oxidation plays an important role in the functioning of zinc fingers (ZFs). Electrospray ionization mass spectrometry (ESI–MS) is a very useful technique to study products of ZF oxidation, but its application has been limited largely to qualitative analysis of reaction products. On the other hand, ESI–MS has been applied successfully on several occasions to determine binding constants in metalloproteins. We used a synthetic 37-residue peptide acetyl-DYVICEECGKEFMDSYLMNHFDLPTCDNCRDADDKHK-amide (XPAzf), which corresponds to the Cys4 ZF sequence of human nucleotide excision repair protein XPA, to find out whether ESI–MS might be used quantitatively to study ZF reaction kinetics. For this purpose, we studied oxidation of the Zn(II) complex of XPAzf (ZnXPAzf) by H 2O 2 using three techniques in parallel: high-performance liquid chromatography (HPLC) of covalent reaction products, 4-(2-pyridylazo)-resorcinol monosodium salt (PAR)-based spectrophotometric zinc release assay, and ESI–MS. Single and double intrapeptide disulfides were detected by ESI–MS to be the sole reaction products. All three techniques yielded independently the same reaction rate, thereby demonstrating that ESI–MS may indeed be used in quantitative kinetic studies of ZF reactions. The comparison of experimental information demonstrated that the formation of the Cys5–Cys8 single disulfide was responsible for zinc release.

Crosslinking of the NER damage recognition proteins XPC-HR23B, XPA and RPA to photoreactive probes that mimic DNA damages

A new assay to probe the mechanism of mammalian nucleotide excision repair (NER) was developed. Photoreactive arylazido analogues of dNMP in DNA were shown to be substrates for the human NER system. Oligonucleotides carrying photoreactive “damages” were prepared using the multi-stage protocol including one-nucleotide gap filling by DNA polymerase β using photoreactive dCTP or dUTP analogues followed by ligation of the resulting nick. Photoreactive 60-mers were annealed with single-stranded pBluescript II SK (+) and subsequently primer extension reactions were performed. Incubation of HeLa extracts with the plasmids containing photoreactive moieties resulted in an excision pattern typical of NER. DNA duplexes containing photoreactive analogues were used to analyze the interaction of XPC-HR23B, RPA, and XPA with damaged DNA using the photocrosslinking assay. Crosslinking of the XPC-HR23B complex with photoreactive 60-mers resulted in modification of its XPC subunit. RPA crosslinked to ssDNA or mismatched dsDNA more efficiently than to dsDNA, whereas XPA did not show a preference for any of the DNA species. XPC and XPA photocrosslinking to DNA decreased in the presence of Mg 2+ whereas RPA crosslinking to DNA was not sensitive to this cofactor. Our data establish a photocrosslinking assay for the investigation of the damage recognition step in human nucleotide excision repair.

A mathematical model for human nucleotide excision repair: Damage recognition by random order assembly and kinetic proofreading

A mathematical model for human nucleotide excision repair: Damage recognition by random order assembly and kinetic proofreading

The protein shuffle

Xeroderma pigmentosum (XP) is an inherited disease in which cells from patients exhibit defects in nucleotide excision repair (NER). XP proteins A-G are crucial in the processes of DNA damage recognition and incision, and patients with XP can carry mutations in any of the genes that specify these proteins. In mammalian cells, NER is a dynamic process in which a variety of proteins interact with one another, via modular domains, to carry out their functions. XP proteins are key players in several steps of the NER process, including DNA strand discrimination (XPA, in complex with replication protein A), repair complex formation (XPC, in complex with hHR23B; XPF, in complex with ERCC1) and repair factor recruitment (transcription factor IIH, in complex with XPG). Through these protein-protein interactions, various types of bulky DNA adducts can be recognized and repaired. Communication between the NER system and other cellular pathways is also achieved by selected binding of the various structural domains. Here, we summarize recent studies on the domain structures of human NER components and the regulatory networks that utilize these proteins. Data provided by these studies have helped to illuminate the complex molecular interactions among NER factors in the context of DNA repair.

Repair of DNA–polypeptide crosslinks by human excision nuclease

DNA-protein crosslinks are relatively common DNA lesions that form during the physiological processing of DNA by replication and recombination proteins, by side reactions of base excision repair enzymes, and by cellular exposure to bifunctional DNA-damaging agents such as platinum compounds. The mechanism by which pathological DNA-protein crosslinks are repaired in humans is not known. In this study, we investigated the mechanism of recognition and repair of protein-DNA and oligopeptide-DNA crosslinks by the human excision nuclease. Under our assay conditions, the human nucleotide excision repair system did not remove a 16-kDa protein crosslinked to DNA at a detectable level. However, 4- and 12-aa-long oligopeptides crosslinked to the DNA backbone were recognized by some of the damage recognition factors of the human excision nuclease with moderate selectivity and were excised from DNA at relatively efficient rates. Our data suggest that, if coupled with proteolytic degradation of the crosslinked protein, the human excision nuclease may be the major enzyme system for eliminating protein-DNA crosslinks from the genome.

Activity of nucleotide excision repair enzymes for oxanine cross-link lesions

Nitric oxide and nitrous acid induce deamination of DNA bases, resulting in uracil, hypoxanthine, xanthine, and oxanine (Oxa) as major damage. Oxa reacts further with polyamines and DNA binding proteins, generating bulky cross-link adducts. Recently we have shown Oxa and cross-link adducts are potentially genotoxic lesions. In the present study, we have assessed the role of base excision repair (BER) and nucleotide excision repair (NER) systems in the repair of Oxa and Oxa-spermine (Oxa-Sp) cross-link adducts. Oxa was very poorly removed from DNA by both BER glycosylases and NER enzymes, whereas Oxa-Sp was efficiently excised by E. coli and human NER enzymes.

Human XPC-hHR23B interacts with XPA-RPA in the recognition of triplex-directed psoralen DNA interstrand crosslinks

DNA interstrand crosslinks (ICLs) represent a severe form of damage that blocks DNA metabolic processes and can lead to cell death or carcinogenesis. The repair of DNA ICLs in mammals is not well characterized. We have reported previously that a key protein complex of nucleotide excision repair (NER), XPA-RPA, recognizes DNA ICLs. We now report the use of triplex technology to direct a site-specific psoralen ICL to a target DNA substrate to determine whether the human global genome NER damage recognition complex, XPC-hHR23B, recognizes this lesion. Our results demonstrate that XPC-hHR23B recognizes psoralen ICLs, which have a structure fundamentally different from other lesions that XPC-hHR23B is known to bind, with high affinity and specificity. XPC-hHR23B and XPA-RPA protein complexes were also observed to bind psoralen ICLs simultaneously, demonstrating not only that psoralen ICLs are recognized by XPC-hHR23B alone, but also that XPA-RPA may interact cooperatively with XPC-hHR23B on damaged DNA, forming a multimeric complex. Since XPC-hHR23B and XPA-RPA participate in the recognition and verification of DNA damage, these results support the hypothesis that interplay between components of the global genome repair sub-pathway of NER is critical for the recognition of psoralen DNA ICLs in the mammalian genome.

Epigenetic Silencing of the Human Nucleotide Excision Repair Gene, hHR23B, in Interleukin-6-responsive Multiple Myeloma KAS-6/1 Cells

During tumorigenesis, selective proliferative advantage in certain cell subsets is associated with accumulation of multiple genetic alterations. For instance, multiple myeloma is characterized by frequent karyotypic instability at the earliest stage, progressing to extreme genetic abnormalities as the disease progresses. These successive genetic alterations can be attributed, in part, to defects in DNA repair pathways, perhaps based on epigenetic gene silencing of proteins involved in DNA damage repair. Here we report epigenetic hypermethylation of the hHR23B gene, a key component of the nucleotide excision repair in response to DNA damage, in interleukin-6 (IL-6)-responsive myeloma KAS-6/1 cells. This hypermethylation was significantly abated by Zebularine, a potent demethylating agent, with a consequent increase in the hHR23B mRNA level. Subsequent removal of this drug and supplementation with IL-6 in the culture medium re-established DNA hypermethylation of the hHR23B gene and silencing of mRNA expression levels. The inclination of DNA to be remethylated, at least within the hHR23B gene promoter region, reflects an epigenetic driving force by the cytogenetic/tumorigenic status of KAS-6/1 myeloma. The IL-6 response of KAS-6/1 myeloma also raises a question of whether the proneoplastic growth factor, such as IL-6, supports the epigenetic silencing of important DNA repair genes via promoter hypermethylation during the development of multiple myeloma.