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.Nucleotide excision repair (NER) is a ubiquitously expressed DNA repair pathway that removes a wide range of structurally unrelated DNA damage from the genome. Three human genetic diseases, xeroderma pigmentosum (XP), Cockayne's syndrome, and trichothiodystrophy, have been identified as being caused by defects in this pathway, and much progress has been made recently in identifying and cloning the genes involved in this pathway by using cell lines from these patients or mutant rodent cell lines (for recent reviews, see references 19 and 40). The gene that complements XP group A (XPA) cells encodes a zinc finger protein (45) that preferentially binds to DNA damaged by irradiation with UV light or by treatment with chemical agents such as cis-diamminedichloroplatinum(II) (21, 39). The damage recognition activity of XPA is an essential function in NER, since XPA cells in which the protein has been inactivated by mutation are completely deficient in the incision step of NER. Furthermore, XP group A patients are afflicted by the most severe forms of the disease, which include highly elevated levels of skin cancer and progressive mental retardation. In addition to its function in the early damage recognition step of NER, XPA may also play a further role in subsequent steps of damage processing. In this regard, we and others have reported that XPA interacts with the repair protein ERCC1 (26,29,36). ERCC1 forms a complex with ERCC4 and possibly other factors (2, 38, 46) and contains a putative excision nuclease (excinuclease) activity that makes an incision 5Ј to the site of damage (1). Thus, an additional function of XPA is to load and orient the ERCC1-ERCC4 excinuclease complex at the site of damage.Replication protein A (RPA; also called human singlestranded DNA-binding protein [HSSB]) is a three-subunit complex consisting of polypeptides p70 (RPA-70), p34 (RPA-34), and p14 (RPA-14) (12,49,50) and is re...

Processing ofDNA damage by the nucleotideexcision repair pathway in eukaryotic cells is most likely accomplished by multiprotein complexes. However, the nature of these complexes and the details of the molecular interactions between DNA repair factors are for the most part unknown. Here, we demonstrate both in vivo, using the two-hybrid system, and in vitro, using recombinant proteins, that the human repair factors XPA and ERCC1 specificafly interact. In addition, we report an initial determination of the domains in ERCC1 and XPA that medlate this interaction. These results suggest that XPA may play a role in the iocalation or ld ofan incision complex, composed ofERCC1 and possibly other repair factors, onto a damaged site.Two groups of mutants defective in the early steps of nucleotide-excision repair have been identified in mammalian cells. These are the naturally occurring cell lines obtained from patients with the rare disorders xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy and the laboratory-derived rodent cell lines designated ERCC (excision repair cross-complementing). Eleven genetic complementation groups have been identified in the former series (1, 2) and 11 in the latter (3, 4). Several of the mutants in these two classes fall into the same complementation group; however, the full extent of overlap remains to be determined. Seven of these genes have now been cloned (5-11), setting the stage for investigations into how DNA damage processing is mediated by this system. One aspect of these studies is to determine the nature ofprotein-protein interactions between DNA-repair factors and to define the composition of multifactorial complexes with the eventual goal ofelucidating their function.The yeast two-hybrid system developed by Fields and colleagues (12, 13) is a novel genetic approach that is used for the detection of interacting proteins. In this system, association in yeast between two proteins, one fused to the DNAbinding domain and the second to the activation domain II of GALA, activates promoters containing GALA-binding sites (14). We have used a modification (15, 16) of the original system involving a dual selection scheme for HIS3 and LacZ to screen a human cDNA library for genes whose expression products interact with the human repair factor ERCC1 (5,17). In addition to the isolation of a number of novel genes, this screening also resulted in the identification of the repair protein XPA as a factor that associates with ERCC1.

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 recombinationindependent 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.Alkylating agents were among the first compounds found to be efficacious in cancer therapy and remain important components of many modern chemotherapeutic regimens (38). Many members of this class of drugs have bifunctional groups that can react with both strands of the DNA helix and thus form interstrand and intrastrand cross-links. As profound blocks for both transcription and DNA replication, interstrand cross-links (ICLs) appear to represent the primary cytotoxic lesion induced by most bifunctional alkylating agents.Repair of DNA ICLs has been studied extensively in Escherichia coli (11,12). Both genetic and biochemical evidence has established a combined nucleotide excision repair (NER)-recombination mechanism for the error-free repair of ICLs, in which the gap, created by the Uvr(A)BC excinuclease, is repaired through recA-mediated recombination with a lesionfree homologous chromosome as the donor (10, 37, 41). Although the NER-recombination pathway appears to be the primary mechanism of cross-link repair in E. coli, recent evidence has suggested a recombination-independent pathway for cross-link repair in which the gap created by the uvr(A)BC excinuclease is repaired by translesion bypass in order to circumvent a d...

The removal of interstrand cross-links (ICLs) from DNA in higher eucaryotes is not well understood. Here, we show that processing of psoralen ICLs in mammalian cell extracts is dependent upon the mismatch repair complex hMutS␤ but is not dependent upon the hMutS␣ complex or hMlh1. The processing of psoralen ICLs is also dependent upon the nucleotide excision repair proteins Ercc1 and Xpf but not upon other components of the excision stage of this pathway or upon Fanconi anemia proteins. Products formed during the in vitro reaction indicated that the ICL has been removed or uncoupled from the cross-linked substrate in the mammalian cell extracts. Finally, the hMutS␤ complex is shown to specifically bind to psoralen ICLs, and this binding is stimulated by the addition of PCNA. Thus, a novel pathway for processing ICLs has been identified in mammalian cells which involves components of the mismatch repair and nucleotide excision repair pathways.DNA interstrand cross-linking agents have been widely used for more than 50 years as potent anticancer agents; nevertheless, the repair of the lesions created by these drugs in mammalian cells is not well understood. In Escherichia coli, two mechanisms for the removal of interstrand cross-links (ICLs) have been described as either recombination dependent or independent. The recombination-dependent pathway was first described by Cole and his colleagues and requires the products of the uvrA, uvrB, uvrC, uvrD, recA, and polA genes (17, 18), indicating that elements of both the nucleotide excision repair (NER) and homologous recombination pathways of E. coli were required for ICL repair. Cole (17) was unable to find evidence for a double-strand break (DSB) intermediate and therefore proposed a model for the removal of ICLs in E. coli in which repair is initiated by dual incisions in one strand on either side of the ICL. The resulting gap is then repaired by a recA-mediated recombination process using a homologous template as a donor. The remaining monoadduct is repaired by a second round of incision by the NER apparatus, and the resulting gap is filled in by the product of the polA gene (DNA polymerase I). Current findings suggest that this pathway is also largely conserved in Saccharomyces cerevisiae (36, 51), with the notable exception that DSBs have been observed during the repair of ICLs in yeast (31,48,50). Thus, components of both the RAD3 and RAD52 epistasis groups have been shown to be required for ICL repair in S. cerevisiae. A recombination-independent pathway of ICL repair has also been described for E. coli which requires the products of the uvr and polB genes (6, 7), suggesting that the gap created by NER is repaired by translesion synthesis. Recently a similar recombination-independent pathway requiring NER elements has been described for mammalian cells (61).While NER plays a central role in repair of ICLs in E. coli and apparently yeast, the mammalian NER pathway does not appear to be required for recombination-dependent repair of ICLs. With the exception of muta...

Xeroderma pigmentosum (XP) is a rare human autosomal recessive disease characterized by solar sensitivity, high predisposition for developing cancers on areas exposed to sunlight, and, in some cases, neurological abnormalities. XP cells are defective in DNA repair, and complementation of this defect has been used to identify eight genetic groups (A-G and variant). We have developed a simple, highly efficient complementary DNA expression system for use in human cells. Here we use this system to isolate a cDNA clone that restores the ultraviolet sensitivity and unscheduled DNA synthesis of XP-C cells to normal levels. The XP-C complementing clone XPCC encodes a highly hydrophilic protein which is composed of a predicted 823 amino acids and shares limited homology with the product of the yeast DNA repair gene RAD4. The XPCC transcript is undetectable by northern blotting in most XP-C cell lines examined.

The human repair proteins XPA and ERCC1 have been shown to be absolutely required for the incision step of nucleotide excision repair, and recently we identified an interaction between these two proteins both in vivo and in vitro (L. Li, S. J. Elledge, C. A. Peterson, E. S. Bales, and R. J. Legerski, Proc. Natl. Acad. Sci. USA 91:5012-5016, 1994). In this report, we demonstrate the functional relevance of this interaction. The ERCC1-binding domain on XPA was previously mapped to a region containing two highly conserved XPA sequences, Gly-72 to Phe-75 and Glu-78 to Glu-84, which are termed the G and E motifs, respectively. Site-specific mutagenesis was used to independently delete these motifs and create two XPA mutants referred to as ⌬G and ⌬E. In vitro, the binding of ERCC1 to ⌬E was reduced by approximately 70%, and binding to ⌬G was undetectable; furthermore, both mutants failed to complement XPA cell extracts in an in vitro DNA repair synthesis assay. In vivo, the ⌬E mutant exhibited an intermediate level of complementation of XPA cells and the ⌬G mutant exhibited little or no complementation. In addition, the ⌬G mutant inhibited repair synthesis in wild-type cell extracts, indicating that it is a dominant negative mutant. The ⌬E and ⌬G mutations, however, did not affect preferential binding of XPA to damaged DNA. These results suggest that the association between XPA and ERCC1 is a required step in the nucleotide excision repair pathway and that the probable role of the interaction is to recruit the ERCC1 incision complex to the damaged site. Finally, the affinity of the XPA-ERCC1 complex was found to increase as a function of salt concentration, indicating a hydrophobic interaction; the half-life of the complex was determined to be approximately 90 min.

Xeroderma pigmentosum (XP) is a rare autosomal recessive disease of humans characterized by an accelerated chronic degeneration of sun-exposed areas of the body, including an elevated risk of developing cancers of the skin. We recently reported the isolation of a gene XPCC that complements the repair deficiency of cultured XP-C cells. Here we report the results of a characterization of XPCC at the nucleotide level in five XP-C cell lines. Each cell line exhibited a unique mutation that correlated well with the cellular DNA repair deficiency and the clinical severity of the disease. These results extend our previous observations and indicate that defects in XPCC cause Xeroderma pigmentosum group C.

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.DNA interstrand cross-linking agents are among the oldest and yet still most effective anticancer drugs available in the clinic, and the chemotherapeutic use of the early forms of these chemicals, such as mustard gas and nitrogen mustard, extends back to before the Second World War. The alkylation chemistry of these drugs was also elucidated shortly after that war, and their cellular pharmacology was extensively studied during the 1970s and 1980s (27). In contemporary chemotherapy, interstrand cross-linking agents such as cyclophosamide, melphalan, and cisplatin are among the most potent antitumor agents. Despite this lengthy history of clinical use and pharmacologic investigation, the mechanisms of repair of the lesions produced in DNA by interstrand cross-linking agents have not been extensively studied. This situation of relative neglect of biochemical pathways of cross-link repair contrasts with the striking advances that have been accomplished in the past decade in other DNA damage processing pathways, such as nucleotide excision repair (NER), base excision repair, and mismatch repair (18).Current evidence (44) indicates that the error-free repair of both interstrand cross-links and double-strand breaks involves a recombinational mechanism in which an undamaged donor chromosome provides a homologous copy for the repair of the damaged template. Both of these lesions are highly deleterious, and it has been shown in certain yeast genetic backgrounds in which particul...