Human Xeroderma Pigmentosum Group Research Articles

Mutagenicity Profile Induced by UVB Light in Human Xeroderma Pigmentosum Group C Cells.

Nucleotide excision repair (NER) is one of the main pathways for genome protection against structural DNA damage caused by sunlight, which in turn is extensively related to skin cancer development. The mutation spectra induced by UVB were investigated by whole-exome sequencing of randomly selected clones of NER-proficient and XP-C-deficient human skin fibroblasts. As a model, a cell line unable to recognize and remove lesions (XP-C) was used and compared to the complemented isogenic control (COMP). As expected, a significant increase of mutagenesis was observed in irradiated XP-C cells, mainly C>T transitions, but also CC>TT and C>A base substitutions. Remarkably, the C>T mutations occur mainly at the second base of dipyrimidine sites in pyrimidine-rich sequence contexts, with 5'TC sequence the most mutated. Although T>N mutations were also significantly increased, they were not directly related to pyrimidine dimers. Moreover, the large-scale study of a single UVB irradiation on XP-C cells allowed recovering the typical mutation spectrum found in human skin cancer tumors. Eventually, the data may be used for comparison with the mutational profiles of skin tumors obtained from XP-C patients and may help to understand the mutational process in nonaffected individuals.

Association of human XPA rs1800975 polymorphism and cancer susceptibility: an integrative analysis of 71 case\u2013control studies

BackgroundThe objective of the present study is to comprehensively evaluate the impact of the rs1800975 A/G polymorphism within the human xeroderma pigmentosum group A (XPA) gene on susceptibility to overall cancer by performing an integrative analysis of the current evidence.MethodsWe retrieved possible relevant publications from a total of six electronic databases (updated to April 2020) and selected eligible case–control studies for pooled assessment. P-values of association and odds ratio (OR) were calculated for the assessment of association effect. We also performed Begg’s test and Egger’s test, sensitivity analysis, false-positive report probability (FPRP) analysis, trial sequential analysis (TSA), and expression/splicing quantitative trait loci (eQTL/sQTL) analyses.ResultsIn total, 71 case–control studies with 19,257 cases and 30,208 controls from 52 publications were included for pooling analysis. We observed an enhanced overall cancer susceptibility in cancer cases compared with negative controls in the Caucasian subgroup analysis for the genetic models of allelic G vs. A, carrier G vs. A, homozygotic GG vs AA, heterozygotic AG vs. AA, dominant AG + GG vs. AA and recessive GG vs. AA + AG (P < 0.05, OR > 1). A similar positive conclusion was also detected in the “skin cancer” or “skin basal cell carcinoma (BCC)” subgroup analysis of the Caucasian population. Our FPRP analysis and TSA results further confirmed the robustness of the conclusion. However, our eQTL/sQTL data did not support the strong links of rs1800975 with the gene expression or splicing changes of XPA in the skin tissue. In addition, even though we observed a decreased risk of lung cancer under the homozygotic, heterozygotic and dominant models (P < 0.05, OR < 1) and an enhanced risk of colorectal cancer under the allelic, homozygotic, heterozygotic, dominant (P < 0.05, OR > 1), our data from FPRP analysis and another pooling analysis with only the population-based controls in the Caucasian population did not support the strong links between the XPA rs1800975 A/G polymorphism and the risk of lung or colorectal cancer.ConclusionsOur findings provide evidence of the close relationship between the XPA rs1800975 A/G polymorphism and susceptibility to skin cancer in the Caucasian population. The potential effect of XPA rs1800975 on the risk of developing lung or colorectal cancer still merits the enrollment of larger well-scaled studies.

Investigating the Enhancement of XPD Helicase Processivity by Single-Stranded Binding Protein RPA2

Ferroplasma acidarmanus helicase FacXPD is a Superfamily 2B helicase involved in transcription initiation and nucleotide excision repair. This archaeal protein serves as a model for understanding the molecular mechanisms of yeast Rad3 and human xeroderma pigmentosum group D protein (XPD). Previous work has shown that the unwinding of double-stranded DNA by FacXPD is enhanced by the single-stranded DNA binding protein FacRPA2. However, the mechanism by which unwinding enhancement occurs remains unknown. Here, we monitored the unwinding of a DNA hairpin by XPD in the presence of RPA2 using a single-molecule optical trap assay. By loading XPD and RPA2 onto DNA in a controlled sequence and by analyzing helicase unwinding dynamics, we distinguish between different potential mechanisms of regulation. Our data point toward a model of processivity enhancement in which RPA2 transiently interacts with the XPD-DNA complex and stabilizes a faster, more processive state of the helicase.

In silico Structural and Functional Analysis of Arabidopsis thaliana’s XPB Homologs

Arabidopsis thaliana has two homologs of the corresponding human xeroderma pigmentosum group B (XPB) helicase protein. The proteins namely AtXPB1 and AtXPB2, which are 95% similar, participate in transcription and Nucleotide Excision Repair. We used bioinformatic tools to investigate the phylogeny, structure, domains, docking site, and interactome of the two paralogs in order to reveal their functional differences. This study suggests that AtXPB1 interacts with Farnesyl Pyrophosphate Synthase 2 (FPS2), a rate-limiting enzyme in the synthesis of brassinosteroids, and Heat Shock Protein 90 (AtHSP90), such interactions were absent from the AtXPB2 interactome. AtXPB1 may be required to regulate the activity of Farnesyl Pyrophosphate Synthase 2, which controls the levels of brassinosteroids, phytohormones that control multiple physiological processes required for normal plant growth and development. In addition, AtXPB1 may have better oxidative stress tolerance than AtXPB2 as AtHSP90 seems to stabilize and protect the former from the proteasomal degradation induced by oxidative stress. These results were applied to the previously observed phenotypic characteristics of atxpb1-/- mutants which show great sensitivity to the oxidative stress-inducing agent hypochlorous acid, and experience absence of germination synchrony, lower seed germination rate, and delay in organ differentiation Keywords: AtHSP90, brassinosteroids, FPS2, oxidative stress, XPB.

A Distinct Genotype of XP Complementation Group A: Surprisingly Mild Phenotype Highly Prevalent in Northern India/Pakistan/Afghanistan

A Distinct Genotype of XP Complementation Group A: Surprisingly Mild Phenotype Highly Prevalent in Northern India/Pakistan/Afghanistan

The Effect of Single-Stranded DNA Binding Protein RPA2 on XPD Helicase Processivity

FacXPD helicase is the archaeal homolog of yeast Rad3 and human xeroderma pigmentosum group D protein (XPD) from the organism Ferroplasma acidarmanus. This enzyme serves as a model for understanding the molecular mechanism of human superfamily 2B helicase XPD involved in transcription initiation and nucleotide excision repair. Previous work has shown that the unwinding of double-stranded DNA by FacXPD is regulated by the single-stranded DNA binding protein FacRPA2. However, the mechanism by which this occurs is unknown. Here, we present a single molecule study of this regulation using optical traps. We show that XPD is a weak helicase as a monomer, only able to unwind short distances (∼12 bp) under tension applied by the optical traps, with a strong dependence on DNA sequence. In the presence of RPA2, however, XPD monomers are able to unwind more processively (>90 bp). Using a combination of optical traps and a laminar flow cell, we performed experiments in which we loaded XPD, RPA2, and provide ATP in a controlled sequence. We used this approach to distinguish between different potential mechanisms of regulation. Our data disfavor mechanisms by which RPA2 regulates XPD activity by sequestering ssDNA. Instead our results suggest that RPA2 forms a complex with XPD, activating it for processive unwinding. RPA2 can also bind ahead of the traveling helicase and destabilize the duplex junction.

Regulation of XPD Helicase by the Single-Stranded DNA Binding Protein RPA2

Although it is known that single-stranded DNA binding proteins can stimulate helicase activity, the mechanism by which this occurs may be more complex than sequestering ssDNA products of duplex separation. Here, we present a single-molecule helicase assay with base-pair sensitivity, which utilizes high-resolution optical tweezers combined with microfluidics and fluorescence microscopy to decipher how FacXPD helicase is modulated by FacRPA2. FacXPD is the archaeal homolog of yeast Rad3 and human xeroderma pigmentosum group D protein (XPD) helicase from the organism Ferroplasma acidarmanus. This enzyme serves as a model for understanding the molecular mechanism of human Superfamily 2B helicase XPD involved in transcription initiation and nucleotide excision repair and related helicases FANCJ, RTEL and CHLR1 involved in maintenance of the genomic integrity. First, we demonstrated that monomeric XPD unwinds duplex DNA in single base-pair steps, yet is non-processive, unwinding for short distances (∼12 bp) and displaying a strong dependence on DNA sequence. Second, we investigated how RPA2 by itself interacts with DNA. We showed that RPA2 can unwind duplex DNA in steps of ∼5-8 bp in the presence of an assisting force of 12 pN. Finally, we examined the effect of RPA2 on XPD activity. Using our microfluidic platform, we performed experiments in which XPD and RPA2 were sequentially assembled on a DNA substrate in a controlled order. RPA2 increased XPD processivity. Duplex unwinding occurred in 1-bp steps, indicating that RPA2 is not directly involved in strand separation. Importantly, our data suggest that only 1-3 RPA2 molecules are sufficient to turn XPD into a processive helicase. We propose two scenarios. Either RPA2 forms a complex with XPD, or it alters its interaction with DNA upon binding, activating it for processive unwinding. We discuss the biological implications of our findings.

Displaced Strand Regulation of Facxpd Helicase Activity

FacXPD is the archaeal homolog of yeast Rad3 and human xeroderma pigmentosum group D protein (XPD) helicase from Ferroplasma acidarmanus. This enzyme serves as a model for understanding the molecular mechanism of human Superfamily 2 helicase XPD involved in both transcription initiation and nucleotide excision repair, and for the related 5’-3’ helicases FancJ, Rtel and ChlR1 important for maintaining genomic integrity and DNA repair. We developed a single-molecule, high-resolution optical tweezers assay to decipher the mechanism by which a single XPD helicase unwinds dsDNA while translocating in the 5’-3’ direction. This assay monitors the unwinding of an 89-bp DNA hairpin substrate with single base pair resolution. Our substrate design allows us to control the length of a poly-dT ssDNA “translocation” strand (the strand to which XPD binds and along which it translocates), and a “displaced” strand (the strand displaced upon unwinding the duplex), located at the 5’ and 3’ tails of the hairpin, respectively. We found that the displaced strand interacts with XPD and that this interaction controls the helicase activity. When the 3’ tail of the substrate hairpin is substituted for dsDNA, a single XPD molecule displays repetitive “non-processive” bursts of substrate unwinding in which only ∼10-bp of the hairpin is unwound at a time. However, in the presence of a ssDNA displaced strand (of length ranging from 3 to 10 nt), we observe two types of activity: the same 10-bp non-processive mode as above, and also a “processive” mode in which the entire 89-bp hairpin is unwound. These data suggest two different binding modes for XPD resulting in non-processive or processive unwinding which are regulated by its interaction with the displaced strand. We propose a model for how the domains of XPD bind to its DNA substrate in these two modes.

SIRT1 Regulates UV-Induced DNA Repair through Deacetylating XPA

SIRT1, a NAD(+)-dependent histone deacetylase, plays crucial roles in multiple biological processes including gene transcription, cellular metabolism, stress response, and tumorigenesis. Xeroderma pigmentosum group A (XPA) is a core nucleotide excision repair (NER) factor essential for NER process. Here we show that SIRT1 plays an important role in the regulation of NER pathway. Downregulation of SIRT1 significantly sensitizes cells to UV irradiation. SIRT1 interacts with XPA, and the interaction is enhanced after UV irradiation. XPA can be acetylated at lysines 63 and 67. SIRT1 deacetylates XPA both in vitro and in cells. Importantly, SIRT1-mediated deacetylation of XPA is required for optimal NER pathway since XPA-deficient cells complemented with XPA-K6367Q, which mimics hyperacetylated XPA, display significantly higher UV sensitivity compared with the XPA cells complemented with wild-type XPA. Furthermore, SIRT1-mediated XPA deacetylation enhances its interaction with RPA32. Our results demonstrate that SIRT1 regulates NER pathway through modulation of XPA acetylation status.

Conserved XPB Core Structure and Motifs for DNA Unwinding: Implications for Pathway Selection of Transcription or Excision Repair

The human xeroderma pigmentosum group B (XPB) helicase is essential for transcription, nucleotide excision repair, and TFIIH functional assembly. Here, we determined crystal structures of an Archaeoglobus fulgidus XPB homolog (AfXPB) that characterize two RecA-like XPB helicase domains and discover a DNA damage recognition domain (DRD), a unique RED motif, a flexible thumb motif (ThM), and implied conformational changes within a conserved functional core. RED motif mutations dramatically reduce helicase activity, and the DRD and ThM, which flank the RED motif, appear structurally as well as functionally analogous to the MutS mismatch recognition and DNA polymerase thumb domains. Substrate specificity is altered by DNA damage, such that AfXPB unwinds dsDNA with 3' extensions, but not blunt-ended dsDNA, unless it contains a lesion, as shown for CPD or (6-4) photoproducts. Together, these results provide an unexpected mechanism of DNA unwinding with implications for XPB damage verification in nucleotide excision repair.

Cooperative Interaction of Human XPA Stabilizes and Enhances Specific Binding of XPA to DNA Damage

Human xeroderma pigmentosum group A (XPA) is an essential protein for nucleotide excision repair (NER). We have previously reported that XPA forms a homodimer in the absence of DNA. However, what oligomeric forms of XPA are involved in DNA damage recognition and how the interaction occurs in terms of biochemical understanding remain unclear. Using the homogeneous XPA protein purified from baculovirus-infected Sf21 insect cells and the methods of gel mobility shift assays, gel filtration chromatography, and UV-cross-linking, we demonstrated that both monomeric and dimeric XPA bound to the DNA adduct of N-acetyl-2-aminofluorene (AAF), while showing little affinity for nondamaged DNA. The binding occurred in a sequential and protein concentration-dependent manner. At relatively low-protein concentrations, XPA formed a complex with DNA adduct as a monomer, while at the higher concentrations, an XPA dimer was involved in the specific binding. Results from fluorescence spectroscopic and competitive binding analyses indicated that the specific binding of XPA to the adduct was significantly facilitated and stabilized by the presence of the second XPA in a positive cooperative manner. This cooperative binding exhibited a Hill coefficient of 1.9 and the step binding constants of K(1) = 1.4 x 10(6) M(-)(1) and K(2) = 1.8 x 10(7) M(-)(1). When interaction of XPA and RPA with DNA was studied, even though binding of RPA-XPA complex to adducted DNA was observed, the presence of RPA had little effect on the overall binding efficiency. Our results suggest that the dominant form for XPA to efficiently bind to DNA damage is the XPA dimer. We hypothesized that the concentration-dependent formation of different types of XPA-damaged DNA complex may play a role in cellular regulation of XPA activity.

Polymorphisms in the human xeroderma pigmentosum group A gene and their impact on cell survival and nucleotide excision repair

Polymorphisms in DNA repair genes may contribute to defects in DNA repair and increased susceptibility to cancer. The xeroderma pigmentosum group A ( XPA) gene is required for nucleotide excision repair (NER) and mutations in XPA highly predispose humans to skin cancer. We examined DNA samples from 189 individuals for polymorphisms in the XPA gene. First, SSCP analysis was used to examine each of the six exons and their intron boundaries. One frequent single nucleotide polymorphism (SNP) in the untranslated region of exon 1 and two rare SNPs which produce the changes Arg228Gln and Val234Leu in the coding region of exon 6 were identified. Quite surprisingly, no sequence variants were found within the coding regions or the adjacent intron boundaries of exons 1–5. Ecdysone-inducible expression vectors containing wild type XPA cDNA or cDNAs representing the two polymorphisms that we identified in exon 6 were created and independently introduced into the XPA deficient cell line XP12RO-SV. Transcription-coupled repair (TCR), global genome repair (GGR) and cell survival following UV irradiation were studied in each cell line in the absence or presence of the ecdysone hormone analog, ponasterone A. No substantial difference in repair or cell survival was found in cells complemented with wild type or polymorphic alleles of XPA. A 10-fold increase in the expression of XPA by addition of ponasterone A resulted in faster removal of 6–4 photoproducts from the total genomes of cells complemented with wild type or polymorphic alleles of XPA but had no significant impact on TCR or global genome repair of cyclobutane pyrimidine dimers (CPDs). Since our SSCP analysis failed to detect significant numbers of polymorphisms we directly sequenced exons 4–6 in a subset of our samples. One additional rare SNP, which produces the change Leu252Val was found in exon 6 and four rare SNPs and one rare single nucleotide deletion were found in intron 4. Hence, the XPA gene appears to be a cold spot for genetic variation and rare polymorphisms in the coding region of the gene do not reduce NER or cell survival after UV irradiation.

Rapid changes of nucleotide excision repair gene expression following UV-irradiation and cisplatin treatment of Dictyostelium discoideum.

Organisms use different mechanisms to detect and repair different types of DNA damage, and different species vary in their sensitivity to DNA damaging agents. The cellular slime mold Dictyostelium discoideum has long been recognized for its unusual resistance to UV and ionizing radiation. We have recently cloned three nucleotide excision repair (NER) genes from Dictyostelium , the rep B, D and E genes (the homologs of the human xeroderma pigmentosum group B, D and E genes, respectively). Each of these genes has a unique pattern of expression during the multicellular development of this organism. We have now examined the response of these genes to DNA damage. The rep B and D DNA helicase genes are rapidly and transiently induced in a dose dependent manner following exposure to both UV-light and the widely used chemotherapeutic agent cisplatin. Interestingly, the rep E mRNA level is repressed by UV but not by cisplatin, implying unique signal transduction pathways for recognizing and repairing different types of damage. Cells from all stages of growth and development display the same pattern of NER gene expression following exposure to UV-light. These results suggest that the response to UV is independent of DNA replication, and that all the factors necessary for rapid transcription of these NER genes are either stable throughout development, or are continuously synthesized. It is significant that the up-regulation of the rep B and D genes in response to UV and chemical damage has not been observed to occur in cells from other species. We suggest that this rapid expression of NER genes is at least in part responsible for the unusual resistance of Dictyostelium to DNA damage.

Distribution of mutations in the human xeroderma pigmentosum group A gene and their relationships to the functional regions of the DNA damage recognition protein.

A series of xeroderma pigmentosum group A cell lines from 19 patients and cell lines from 13 other family members were examined for XPA mutations to find previously unidentified mutations from American and European patients, to establish pedigrees in represented families, and to develop a database for XPA diagnosis. Most mutations were deletions and splice site mutations observed previously in other XPA patients, in exon III, intron III, or exon IV, that resulted in frameshifts within the DNA binding region-including an Afl III RFLP (G to C) in four unrelated families. One new mutation was a point mutation within intron III (A to G) creating a new splice acceptor site that may compete with the original splice acceptor site. Missplicing at this new site inserts 11 nucleotides in the mRNA creating a frameshift. A small amount of normal splicing to give wild-type XPA protein is the likely molecular mechanism for the relatively mild clinical features of this patient. In another patient, a new 2 bp deletion in the RPA70 binding region was identified in the same region as a 20 bp deletion previously characterized in an unrelated patient. Mutations in the DNA binding region of XPA were from patients with the more severe disease often associated with neurological complications, whereas mutations in the C-terminal end of the protein, which interacts with the TFIIH transcription factor, were from patients with milder skin disease only. The rarity of naturally occurring missense mutations in the DNA binding region of XPA suggests that amino acid changes might be sufficiently tolerated that patients would have mild symptoms and escape detection.

Distribution of mutations in the human xeroderma pigmentosum group A gene and their relationships to the functional regions of the DNA damage recognition protein

Distribution of mutations in the human xeroderma pigmentosum group A gene and their relationships to the functional regions of the DNA damage recognition protein

RepE--the Dictyostelium homolog of the human xeroderma pigmentosum group E gene is developmentally regulated and contains a leucine zipper motif.

We have cloned and characterized the Dictyostelium discoideum repE gene, a homolog of the human xeroderma pigmentosum (XP) group E gene which encodes a UV-damaged DNA binding protein. The repE gene maps to chromosome 4 and it is the first gene identified in Dictyostelium that is homologous to those involved in nucleotide excision repair and their related XP diseases in humans. The predicted protein encodes a leucine zipper motif. The repE gene is not expressed by mitotically dividing cells, and repE mRNA is first detected during the aggregation phase of development when the cells have ceased dividing and replicating genomic DNA. The mRNA level plateaus by the time the developing cells have entered multicellular aggregates and remains at the same steady-state level for the remainder of development. In addition, we have demonstrated that the level of mRNA is very low in developing cells. These observations suggest that repE may play a regulatory role in development. The data indicate that potential developmental roles for XP-related genes can be profitably studied in this system.

Characterization of the human XPA promoter

We cloned the human xeroderma pigmentosum group A gene ( XPA) and characterized the XPA promoter ( pXPA) by transient cat expression. The pXPA is extraordinarily weak in human fibroblasts (1% of RSV-LTR) and appears to function without any of the usual promoter elements. Regions containing positive and negative control elements were localized.

Human Xeroderma Pigmentosum Group A Protein Interacts with Human Replication Protein A and Inhibits DNA Replication

Human replication protein A (RPA; also known as human single-stranded DNA binding protein, or HSSB) is a multisubunit complex involved in both DNA replication and repair. While the role of RPA in replication has been well studied, its function in repair is less clear, although it is known to be involved in the early stages of the repair process. We found that RPA interacts with xeroderma pigmentosum group A complementing protein (XPAC), a protein that specifically recognizes UV-damaged DNA. We examined the effect of this XPAC-RPA interaction on in vitro simian virus 40 (SV40) DNA replication catalyzed by the monopolymerase system. XPAC inhibited SV40 DNA replication in vitro, and this inhibition was reversed by the addition of RPA but not by the addition of DNA polymerase alpha-primase complex, SV40 large tumor antigen, or topoisomerase I. This inhibition did not result from an interaction between XPAC and single-stranded DNA (ssDNA), or from competition between RPA and XPAC for DNA binding, because XPAC does not show any ssDNA binding activity and, in fact, stimulates RPA's ssDNA binding activity. Furthermore, XPAC inhibited DNA polymerase alpha activity in the presence of RPA but not in RPA's absence. These results suggest that the inhibitory effect of XPAC on DNA replication probably occurs through its interaction with RPA.

Caffeine inhibits gene-specific repair of UV-induced DNA damage in hamster cells and in human xeroderma pigmentosum group C cells.

We have studied the effect of caffeine on gene- and strand-specific DNA repair after exposure of Chinese hamster ovary cells and human xeroderma pigmentosum complementation group C (XPC) cells to ultraviolet irradiation (UV). In hamster cells, caffeine inhibited the repair of cyclobutane dimers (CPDs) in the dihydrofolate reductase (DHFR) gene by up to 66% after 8 h of repair incubation. This effect was dose-dependent, with more inhibition at 10 than at 1.5 mM caffeine. The inhibition was due to decreased repair in the transcribed strand of the hamster DHFR gene. This decrease in repair of CPDs in the DHFR gene correlated with an enhancement of UV-induced cell killing by caffeine. DNA repair was also measured in the overall genome by repair-replication analysis. In hamster cells, caffeine caused a modest enhancement of repair. Caffeine did not produce a significant effect on cell cycle progression up to 8 h after UV irradiation, but it caused a distinct block in early S phase during the 24 h post-irradiation period. In XPC cells, 10 mM caffeine inhibited the removal of CPDs from the transcribed strand of the DHFR gene by 92%. The removal of all photoproducts from the overall genome was inhibited by 26% in these cells. Since the residual repair in XPC cells is thought to occur in active genomic regions, we propose that caffeine preferentially inhibits gene-specific repair.

Replacing tryptophan-128 of T4 endonuclease V with a serine residue results in decreased enzymatic activity in vitro and in vivo.

Endonuclease V of bacteriophage T4 possesses two enzymatic activities, a DNA N-glycosylase specific for cyclobutane pyrimidine dimers (CBPD) and an associated apurinic/apyrimidinic (AP) lyase. Extensive structural and functional studies of endonuclease V have revealed that specific amino acids are associated with these two activities. Controversy still exists regarding the role of the aromatic amino acid stretch close to the carboxyl terminus, in particular the tryptophan at position 128. We have expressed wild-type and mutant W128S endonuclease V in Escherichia coli from an inducible tac promoter. Purified W128S endonuclease V demonstrated substantially decreased N-glycosylase (approximately 5-fold) and AP lyase (10- to 20-fold) activities in vitro compared to the wild-type enzyme when a UV-irradiated poly(dA)-poly(dT) substrate was used. However, a much smaller difference in AP lyase activity between the two forms was observed with a site-specific abasic oligonucleotide. The difference in enzymatic activity was qualitatively, but not quantitatively, reflected in the survival of UV-irradiated bacteria, that is the W128S cells were slightly less UV resistant than wild-type cells. No difference was observed in the complementation of UV repair using UV-damaged denV- T4 phage. A more pronounced difference between the wild-type and W128S proteins was observed in human xeroderma pigmentosum group A cells by host-cell reactivation of a UV-irradiated reporter gene. The relatively large discrepancy between the in vitro and in vivo results observed with bacteria may be because saturated levels of DNA repair are obtained in vivo with relatively low levels of endonuclease V. However, under limiting in vitro conditions and in human cells in vivo a considerable difference between the W128S mutant and wild-type endonuclease V activities can be detected. Our results demonstrate that tryptophan-128 is important for endonuclease V activity.