title: HARP preferentially co-purifies with RPA bound to DNA-PK and blocks RPA phosphorylation.
abstract: The HepA-related protein (HARP/SMARCAL1) is an ATP-dependent annealing helicase that is capable of rewinding DNA structures that are stably unwound due to binding of the single-stranded DNA (ssDNA)-binding protein Replication Protein A (RPA). HARP has been implicated in maintaining genome integrity through its role in DNA replication and repair, two processes that generate RPA-coated ssDNA. In addition, mutations in HARP cause a rare disease known as Schimke immuno-osseous dysplasia. In this study, we purified HARP containing complexes with the goal of identifying the predominant factors that stably associate with HARP. We found that HARP preferentially interacts with RPA molecules that are bound to the DNA-dependent protein kinase (DNA-PK). We also found that RPA is phosphorylated by DNA-PK in vitro, while the RPA-HARP complexes are not. Our results suggest that, in addition to its annealing helicase activity, which eliminates the natural binding substrate for RPA, HARP blocks the phosphorylation of RPA by DNA-PK.
authors.affiliation: Radiation Oncology; Dana-Farber Cancer Institute; Biological Chemistry and Molecular Pharmacology; Harvard Medical School; Boston, MA USA.
authors.forename: Jinhua
authors.initials: J
authors.lastname: Quan
authors.affiliation: Radiation Oncology; Dana-Farber Cancer Institute; Biological Chemistry and Molecular Pharmacology; Harvard Medical School; Boston, MA USA.
authors.forename: Timur
authors.initials: T
authors.lastname: Yusufzai
mesh_terms: D004265:DNA Helicases / Q000302:isolation & purification* / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D051747:DNA-Activated Protein Kinase / Q000302:isolation & purification* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins / Q000302:isolation & purification* / Q000378:metabolism; D010766:Phosphorylation; D051716:Replication Protein A / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D004277:DNA, Single-Stranded; D009687:Nuclear Proteins; D051716:Replication Protein A; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; C412482:SMARCAL1 protein, human; D004265:DNA Helicases
keywords: DNA-PK; HARP; RPA; SMARCAL1; annealing helicase