Molecular Anatomy of the Human Excision Nuclease Assembled at Sites of DNA Damage

Abstract

Human nucleotide excision repair is initiated by six repair factors (XPA, RPA, XPC-HR23B, TFIIH, XPF-ERCC1, and XPG) which sequentially assemble at sites of DNA damage and effect excision of damagecontaining oligonucleotides. We here describe the molecular anatomy of the human excision nuclease assembled at the site of a psoralen-adducted thymine. Three polypeptides, primarily positioned 5 to the damage, are in close physical proximity to the psoralen lesion and thus are cross-linked to the damaged DNA: these proteins are RPA70, RPA32, and the XPD subunit of TFIIH. While both XPA and XPC bind damaged DNA and are required for XPD cross-linking to the psoralen-adducted base, neither XPA nor XPC is cross-linked to the psoralen adduct. The presence of other repair factors, in particular TFIIH, alters the mode of RPA binding and the position of its subunits relative to the psoralen lesion. Based on these results, we propose that RPA70 makes the initial contact with psoralen-damaged DNA but that within preincision complexes, it is RPA32 and XPD that are in close contact with the lesion.The basic mechanism of nucleotide excision repair includes (i) damage recognition, (ii) assembly of repair factors at the site of damage, (iii) dual incisions that result in excision of damage-containing oligomers, (iv) resynthesis to fill in the gap, and (v) ligation. In humans excision repair is initiated by the combined action of six repair factors, XPA, RPA, XPC, TFIIH, XPG, and XPF-ERCC1, which excise the damaged nucleotide(s) in the form of 24-to 32-nucleotide (nt)-long oligomers (7,30,33,47). In yeast the same six factors are both essential and sufficient for excision of 24-to 27-nt-long oligomers (10). XPA, RPA, and XPC are involved in damage recognition, TFIIH unwinds the duplex around the lesion and stabilizes the damage recognition complex, and XPG and XPF-ERCC1 make the 3Ј and 5Ј incisions, respectively. There is general agreement about the above-stated roles of these factors, but there is limited information about the location of these proteins relative to one another and relative to the lesion within preincision and incision complexes.Previously we identified three stable DNA-protein complexes (25) which we named preincision complex 1 (PIC1) (RPA-XPA-XPC-TFIIH-DNA), PIC2 (RPA-XPA-TFIIH-XPG-DNA), and PIC3 (RPA-XPA-TFIIH-XPG-XPF-ERCC1-DNA). In this study we attempted to locate the various repair factors with a protein-DNA cross-linking assay using a furan-side psoralen-thymine adduct both as a substrate and as a cross-linker (5,16,27). Our results show that RPA is in close contact with the DNA lesion prior to unwinding by TFIIH and that following unwinding, the mode of RPA-DNA interaction changes drastically such as to affect RPA-DNA cross-linking both qualitatively and quantitatively. Surprisingly, neither XPA nor XPC are cross-linked to DNA either when bound in isolation or in the preincision complexes. In contrast, the XPD subunit of TFIIH is cross-linked to psoralen in PIC1, PIC2, and PIC3. Furthermore, we find that plac...

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

“…In the sequential model, it has been proposed that XPA [12], RPA [18], the RPA-XPA complex [25], or XPC [22] bind to the damage site first and then the other factors assemble in a rigid order. In the random order assembly/kinetic proofreading model, RPA, XPA, or XPC-TFIIH may bind to the damage first and, regardless of which of the three is bound first, protein-protein interactions among the three facilitate the assembly of the RPA-XPA-XPC-TFIIH complex and improves specificity ( [19], [20]).…”

“…In one model the damage is recognized by RPA, XPA, or XPC first and the other factors assemble in a rigid sequential order ( [22], [24], [25], [18]). In the second model the three damage recognition factors, RPA, XPA, and XPC, assemble at the damage site in a random order but cooperatively, and the specificity conferred by these factors is enhanced by the kinetic proofreading activity of TFIIH to achieve a physiologically relevant specificity [19].…”

Nucleotide Excision Repair by Mutant Xeroderma Pigmentosum Group A (XPA) Proteins with Deficiency in Interaction with RPA

“…At least in mammalian cells, NER factors are considered to assemble sequentially at DNA damage sites (35)(36)(37)(38). XPA is a critical factor in NER because a deficiency of XPA results in a high sensitivity to killing by UV light.…”

Ordered Conformational Changes in Damaged DNA Induced by Nucleotide Excision Repair Factors

“…Whether or not the NER reaction occurs by sequential arrival of the various factors or by a pre-assembled complex referred to as the repairosome or the holoenzyme is still under debate (17)(18)(19). Although the hypothesis of the sequential assembly, which has gained a lot of support from recent biological studies, seems to be more accepted, the order of assembly of the NER factors on the damaged DNA and their contribution to the DNA remodeling to allow the repair are not fully understood (10,20,21). As an example, to further learn about the role of TFIIH and its XPB and XPD helicases in NER, it is necessary to determine how it associates with the damaged DNA and recruits the additional factors such as XPA and RPA to promote the formation of an open intermediate essential for the dual incision by XPG and XPF endonucleases.…”