Complex Formation with Damage Recognition Protein Rad14 Is Essential for Saccharomyces cerevisiae Rad1-Rad10 Nuclease To Perform Its Function in Nucleotide Excision Repair In Vivo

Abstract

Nucleotide excision repair (NER) in eukaryotes requires the assembly of a large number of protein factors at the lesion site which then coordinate the dual incision of the damaged DNA strand. However, the manner by which the different protein factors are assembled at the lesion site has remained unclear. Previously, we have shown that in the yeast Saccharomyces cerevisiae, NER proteins exist as components of different protein subassemblies: the Rad1-Rad10 nuclease, for example, forms a tight complex with the damage recognition protein Rad14, and the complex of Rad1-Rad10-Rad14 can be purified intact from yeast cells. As the Rad1-Rad10 nuclease shows no specificity for binding UV lesions in DNA, association with Rad14 could provide an effective means for the targeting of Rad1-Rad10 nuclease to damage sites in vivo. To test the validity of this idea, here we identify two rad1 mutations that render yeast cells as UV sensitive as the rad1⌬ mutation but which have no effect on the recombination function of Rad1. From our genetic and biochemical studies with these rad1 mutations, we conclude that the ability of Rad1-Rad10 nuclease to associate in a complex with Rad14 is paramount for the targeting of this nuclease to lesion sites in vivo. We discuss the implications of these observations for the means by which the different NER proteins are assembled at the lesion site.Nucleotide excision repair (NER) is a highly conserved repair system among eukaryotes. In the yeast Saccharomyces cerevisiae, a combination of Rad14, Rad4-Rad23, RPA, TFIIH, Rad1-Rad10, and Rad2 mediates the dual incision of the damaged DNA strand, releasing an ϳ30-nucleotide (nt) lesioncontaining DNA fragment (6). Rad14, Rad4-Rad23, and RPA function at the damage recognition step. The Rad3 and Rad25 DNA helicases, which are the components of TFIIH, unwind the duplex DNA around the lesion, and the Rad1-Rad10 and Rad2 nucleases incise the damaged DNA strand on the 5Ј and the 3Ј side of the lesion, respectively (24). In humans, a combination of their respective counterparts, XPA, XPC-HR23B, RPA, TFIIH, XPF-ERCC1, and XPG, mediates the dual incision of the damaged DNA strand (19,20). Similar to that in yeast, in humans, XPA, XPC-HR23B, and RPA act in damage recognition, the XPD and XPB helicases in TFIIH unwind the damaged duplex, and the XPF-ERCC1 and XPG nucleases incise the damaged strand on the 5Ј and the 3Ј side of the lesion, respectively (26, 27).Previously, we have shown that the yeast NER proteins exist in vivo as components of separate protein subassemblies, named nucleotide excision repair factors (NEFs) 1 to 4. NEF1 is comprised of the damage recognition protein Rad14 and the Rad1-Rad10 nuclease, which incises the damaged strand on the 5Ј side of the lesion (7). NEF2, the Rad4-Rad23 complex (6), is involved in damage recognition, and NEF3, comprised of TFIIH and the Rad2 endonuclease (12), promotes DNA unwinding and 3Ј incision (24). In yeast, the repair of the nontranscribed DNA strand and of transcriptionally inactive regions additionally req...

Homologous recombination rescues ssDNA gaps generated by nucleotide excision repair and reduced translesion DNA synthesis in yeast G2 cells

“…We created rad14Δ rad30Δ or rad2Δ rad30Δ double mutants that were deficient in NER. Rad14 is a homolog of human XPA protein, which forms a complex with ssDNA endonucleases Rad1 and Rad10 and nicks the damaged DNA strand on the 5′ side of a lesion (29,30), whereas Rad2, a homolog of human XPG, leads to incision at the 3′ side (31). Comparing the results in Fig.…”

Specific and Efficient Binding of Xeroderma Pigmentosum Complementation Group A to Double-Strand/Single-Strand DNA Junctions with 3‘- and/or 5‘-ssDNA Branches

“…Besides its role in DNA damage recognition, XPA also has been suggested to be important for recruitment of other NER factors for nuclease assembly, which repairs the lesion (4,(18)(19)(20)(21). The recruitment of Rad1-Rad10, the yeast homolog of XPF-ERCC1, by Rad14, the yeast homolog of XPA (22), has recently been demonstrated in vivo, even though the mechanism for this process remains unclear.…”

Other Proteins Interacting with XP Proteins

“…4,5 Rad14, the yeast counterpart of XPA, also forms a tight complex with Rad1-Rad10 nuclease, the yeast counterpart of XPF-ERCC1. 30 A recent in vitro study has suggested that the high affinity of XPA for the damage site may not be dependent on the damaged base but rather on the preincision DNA structures generated by local unwinding of the DNA surrounding the damage. 31 Thus, XPA may play a structural role in maintaining the pre-incision DNA bubble and positions the remaining NER factors, particularly XPF-ERCC1, 30 for final incisions while RPA may protect the undamaged strand (which will be used as the template for re-synthesis following incisions) from nuclease attack.…”

“…30 A recent in vitro study has suggested that the high affinity of XPA for the damage site may not be dependent on the damaged base but rather on the preincision DNA structures generated by local unwinding of the DNA surrounding the damage. 31 Thus, XPA may play a structural role in maintaining the pre-incision DNA bubble and positions the remaining NER factors, particularly XPF-ERCC1, 30 for final incisions while RPA may protect the undamaged strand (which will be used as the template for re-synthesis following incisions) from nuclease attack. 32 Other XPA interacting proteins include ATR (ATM and RAD3-Related), 33 a DNA damage checkpoint kinase of the phosphoinositide 3-kinase-like kinase (PIKK) family, XAB1 (XPA-binding protein 1) 34,35 and XAB2 (XPA-binding protein 2).…”