Replication Protein A Confers Structure-specific Endonuclease Activities to the XPF-ERCC1 and XPG Subunits of Human DNA Repair Excision Nuclease

Abstract

XPF-ERCC1 and XPG proteins are nucleases that are involved in human nucleotide excision repair. In this study, we characterized the structure-specific junctioncutting activities of both nucleases using DNA substrates containing a bubble or loop structure. We found that the junction-cutting activities of XPF-ERCC1 and XPG were greatly stimulated by human replication protein A (RPA), while heterologous single-stranded DNAbinding proteins could not substitute for human RPA. To test for specific interaction between RPA and XPF-ERCC1 as is known to occur between RPA and XPG, we employed a pull-down assay with immobilized "bubble" substrate. We found that the binding of XPF-ERCC1 complex to the bubble substrate was enhanced by RPA, suggesting a possible mechanism for RPA in the excision nuclease system, that is the targeting of the nuclease subunits to their specific sites of action. Furthermore, the RPA-promoted junction cutting by XPF-ERCC1 and XPG nucleases was observed with "loop" substrates as well, raising the possibility that XPF-ERCC1, XPG, and RPA may function in removing loop structures from DNA, independent of the other subunits of the human excinuclease.Nucleotide excision repair is a general repair system that plays an important role in maintaining genetic integrity (1, 2). This repair system removes damaged nucleotides from DNA by dual incisions on both sides of the lesion in the damaged strand (3). Recently, the human and the highly homologous yeast nucleotide excision repair systems have been reconstituted from extensively purified proteins, which demonstrated that 14 -15 polypeptides comprising replication protein A (RPA, 1 also known as HSSB or RF-A) and the general transcription factor, TFIIH, in addition to XP and ERCC proteins were essential for the dual incision step (4 -6).It was found that the two subunits of the excision nuclease had intrinsic endonuclease activities in the absence of the other components of the repair system. XPG was reported to possess a single-stranded DNA endonuclease activity (7,8) and an exonuclease activity with 5Ј to 3Ј directionality (9). Similarly, it was found that XPF-ERCC1 had a single strand-specific endonuclease activity and a weak activity on double-stranded UVirradiated DNA, which was stimulated by RPA (10). Furthermore, it has been reported that the RAD1-RAD10 complex (11), the yeast counterpart of the human XPF-ERCC1 nuclease, and XPG nuclease (12) have structure-specific junction-cutting activities with unique polarities. These results obtained with the model "undamaged" substrates suggested that XPF-ERCC1 and XPG were the 5Ј-and 3Ј-endonucleases of the dual incisions, respectively. A recent study using damaged DNA and specific antibodies supported this model (13).Recently, it has been reported that RPA, which is absolutely required for dual incisions (4), specifically binds to XPA protein and XPG nuclease (14). These findings raised the possibility that RPA may play an important role in recognizing DNA lesions and then targeting the nuclease subunits of hu...

RPA activates the XPF ‐ ERCC 1 endonuclease to initiate processing of DNA interstrand crosslinks

“…RPA is a well‐established XPF‐interacting factor, required to position and activate XPF‐ERCC1 for incisions made 5′ to the lesion during nucleotide excision repair (Matsunaga et al , 1996; Bessho et al , 1997; de Laat et al , 1998b). To explore the effect of RPA on XPF‐ERCC1 activity, a simple fork substrate was pre‐incubated with increasing concentrations of recombinant human RPA on ice for 10 min prior to reaction with 40 nM XPF‐ERCC1 for 60 min at 30°C.…”

“…Consequently, we determined whether any known XPF‐ERCC1‐interacting factors relevant to replication‐coupled ICL repair could modulate this reaction. Consequently, we investigated a very well‐established XPF‐ERCC1‐interacting partner relevant to replication‐coupled repair, RPA (Matsunaga et al , 1996; Bessho et al , 1997; de Laat et al , 1998b). Strikingly, for simple fork structures RPA was able to dramatically stimulate XPF‐ERCC1 cleavage.…”

Preferential binding of ATR protein to UV-damaged DNA

“…The T [6 -4]T photoproduct containing substrate was constructed by ligating the damage-containing oligomer (8-mer), which was 5Ј-terminally labeled, with other partially overlapping oligonucleotides to obtain the internally labeled duplex as described (19,20). The resulting 46-bp duplex contains the (6-4) photoproduct at positions 23 and 24.…”

An Alternative Form of Replication Protein A Expressed in Normal Human Tissues Supports DNA Repair

“…Formation of these complexes may be promoted by the strand separation activity of RPA (22). In addition, RPA participates in the dual incision by stimulating the XPF-ERCC1 endonuclease (23)(24)(25). Lastly, RPA has been implicated in the coordination of DNA synthesis after removal of DNA lesions (26).…”

“…We previously found that RPA highly stimulates the structure-specific endonuclease activity of XPF-ERCC1 using a 32 P-labeled substrate containing a stretch of 30 unpaired nucleotides (23,24). This bubble struc- ture mimics the unwound state of DNA generated around the lesion by the helicase activity of the excision repair factor TFIIH (12,49).…”