title: The N-terminus of RPA large subunit and its spatial position are important for the 5'->3' resection of DNA double-strand breaks.
abstract: The first step of homology-dependent repair of DNA double-strand breaks (DSBs) is the resection of the 5' strand to generate 3' ss-DNA. Of the two major nucleases responsible for resection, EXO1 has intrinsic 5'->3' directionality, but DNA2 does not. DNA2 acts with RecQ helicases such as the Werner syndrome protein (WRN) and the heterotrimeric eukaryotic ss-DNA binding protein RPA. We have found that the N-terminus of the RPA large subunit (RPA1N) interacts with both WRN and DNA2 and is essential for stimulating WRN's 3'->5' helicase activity and DNA2's 5'->3' ss-DNA exonuclease activity. A mutant RPA complex that lacks RPA1N is unable to support resection in Xenopus egg extracts and human cells. Furthermore, relocating RPA1N to the middle subunit but not to the small subunit causes severe defects in stimulating DNA2 and WRN and in supporting resection. Together, these findings suggest that RPA1N and its spatial position are critical for restricting the directionality of the WRN-DNA2 resection pathway.
authors.affiliation: Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA.
authors.forename: Margaret
authors.initials: M
authors.lastname: Tammaro
authors.affiliation: Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA.
authors.forename: Shuren
authors.initials: S
authors.lastname: Liao
authors.affiliation: Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA.
authors.forename: Jill
authors.initials: J
authors.lastname: McCane
authors.affiliation: Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA Hong.Yan@fccc.edu.
authors.forename: Hong
authors.initials: H
authors.lastname: Yan
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D053903:DNA Breaks, Double-Stranded; D004265:DNA Helicases / Q000378:metabolism*; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004720:Endonucleases / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000378:metabolism; D006801:Humans; D010063:Ovum / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D051716:Replication Protein A / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D000071657:Werner Syndrome Helicase; D014981:Xenopus; D029867:Xenopus Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; D021122:Protein Subunits; C000604691:RPA1 protein, Xenopus; C497167:RPA1 protein, human; D051716:Replication Protein A; D029867:Xenopus Proteins; D004720:Endonucleases; D005090:Exodeoxyribonucleases; D004265:DNA Helicases; C533186:DNA2 protein, Xenopus; D000071657:Werner Syndrome Helicase; C507159:WRN protein, Xenopus