Interaction of nucleotide excision repair factors XPC-HR23B, XPA, and RPA with damaged DNA

Abstract

The interaction of nucleotide excision repair factors--xeroderma pigmentosum complementation group C protein in complex with human homolog of yeast Rad23 protein (XPC-HR23B), replication protein A (RPA), and xeroderma pigmentosum complementation group A protein (XPA)--with 48-mer DNA duplexes imitating damaged DNA structures was investigated. All studied proteins demonstrated low specificity in binding to damaged DNA compared with undamaged DNA duplexes. RPA stimulates formation of XPC-HR23B complex with DNA, and when XPA and XPC-HR23B are simultaneously present in the reaction mixture a synergistic effect in binding of these proteins to DNA is observed. RPA crosslinks to DNA bearing photoreactive 5I-dUMP residue on one strand and fluorescein-substituted dUMP analog as a lesion in the opposite strand of DNA duplex and also stimulates cross-linking with XPC-HR23B. Therefore, RPA might be one of the main regulation factors at various stages of nucleotide excision repair. The data are in agreement with the cooperative binding model of nucleotide excision repair factors participating in pre-incision complex formation with DNA duplexes bearing damages.

Comparative Analysis of Interaction of Human and Yeast DNA Damage Recognition Complexes with Damaged DNA in Nucleotide Excision Repair

“…These effects are most likely provided by protein-protein interactions. This assumption is partially confirmed by our earlier observation that an RPA mutant form depleted of all the domains responsible for protein-protein interactions is unable to stimulate XPC-RAD23B interaction with DNA (20). Moreover, no reports so far existed about a physical interaction between RPA and XPC.…”

“…Our data show that XPA (20) and RPA stimulate XPC binding to the damaged DNA duplex. It was demonstrated previously that XPC physically interacts with XPA (35), and when XPA and XPC are simultaneously present in a reaction mixture, these NER factors mutually stimulate their binding to DNA (20). Photocross-linking experiments reveal that the intensity of XPC cross-linking in the presence of XPA was increased, but the localization of XPC on damaged DNA was not changed.…”

“…6C). We have shown previously that RPA influences XPC-RAD23B modification due to protein-protein interactions (15,20). The level of RPA labeling significantly decreases with the simultaneous appearance of the products of XPC-RAD23B modification.…”

Localization of xeroderma pigmentosum group A protein and replication protein A on damaged DNA in nucleotide excision repair

“…Recombinant hXPA bearing N-terminal polyhistidine fragment was expressed in E. coli BL21(DE3)LysS strain, using pETI5b-XPA recombinant plasmid kindly provided by Dr O. Schärer (SUNY Stony Brook, USA). Protein isolation was performed according to (26) with one modification: EDTA was not added during purification.…”

Recognition and Repair Pathways of Damaged DNA in Higher Plants

“…XPC is then needed for the recruitment of the core NER repair factors XPA, TFIIH, and RPA (Evans et al, 1997;Araujo et al, 2001;Thoma and Vasquez, 2003). XPA and the basal transcription factor complex TFIIH bind to the damaged site and unwind the DNA around the lesion (Reardon and Sancar, 2003;Maltseva et al, 2006;Yang et al, 2006;Kesseler et al, 2007;Krasikova et al, 2008). Unwinding is specifically performed by two subunits of TFIIH, the helicases XPB (ERCC3) and XPD (ERCC2).…”