title: Interaction between DNA Polymerase β and BRCA1.
abstract: The breast cancer 1 (BRCA1) protein is a tumor suppressor playing roles in DNA repair and cell cycle regulation. Studies of DNA repair functions of BRCA1 have focused on double-strand break (DSB) repair pathways and have recently included base excision repair (BER). However, the function of BRCA1 in BER is not well defined. Here, we examined a BRCA1 role in BER, first in relation to alkylating agent (MMS) treatment of cells and the BER enzyme DNA polymerase β (pol β). MMS treatment of BRCA1 negative human ovarian and chicken DT40 cells revealed hypersensitivity, and the combined gene deletion of BRCA1 and pol β in DT40 cells was consistent with these factors acting in the same repair pathway, possibly BER. Using cell extracts and purified proteins, BRCA1 and pol β were found to interact in immunoprecipitation assays, yet in vivo and in vitro assays for a BER role of BRCA1 were negative. An alternate approach with the human cells of immunofluorescence imaging and laser-induced DNA damage revealed negligible BRCA1 recruitment during the first 60 s after irradiation, the period typical of recruitment of pol β and other BER factors. Instead, 15 min after irradiation, BRCA1 recruitment was strong and there was γ-H2AX co-localization, consistent with DSBs and repair. The rapid recruitment of pol β was similar in BRCA1 positive and negative cells. However, a fraction of pol β initially recruited remained associated with damage sites much longer in BRCA1 positive than negative cells. Interestingly, pol β expression was required for BRCA1 recruitment, suggesting a partnership between these repair factors in DSB repair.
authors.affiliation: Laboratory of Structural Biology, NIEHS, National Institutes of Health, North Carolina, United States of America.
authors.forename: Aya
authors.initials: A
authors.lastname: Masaoka
authors.forename: Natalie R
authors.initials: NR
authors.lastname: Gassman
authors.forename: Julie K
authors.initials: JK
authors.lastname: Horton
authors.forename: Padmini S
authors.initials: PS
authors.lastname: Kedar
authors.forename: Kristine L
authors.initials: KL
authors.lastname: Witt
authors.forename: Cheryl A
authors.initials: CA
authors.lastname: Hobbs
authors.forename: Grace E
authors.initials: GE
authors.lastname: Kissling
authors.forename: Keizo
authors.initials: K
authors.lastname: Tano
authors.forename: Kenjiro
authors.initials: K
authors.lastname: Asagoshi
authors.forename: Samuel H
authors.initials: SH
authors.lastname: Wilson
mesh_terms: D000477:Alkylating Agents / Q000633:toxicity; D000818:Animals; D019313:BRCA1 Protein / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002645:Chickens; D053903:DNA Breaks, Double-Stranded; D019951:DNA Polymerase beta / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair / Q000502:physiology*; D005455:Fluorescent Antibody Technique; D055785:Gene Knockdown Techniques; D006657:Histones / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D034741:RNA, Small Interfering
chemical_list: D000477:Alkylating Agents; D019313:BRCA1 Protein; C492913:BRCA1 protein, human; C468783:H2AX protein, human; D006657:Histones; D034741:RNA, Small Interfering; D019951:DNA Polymerase beta