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.
Highlights
It is well known that inactivation of the product of the breast cancer 1 (BRCA1) gene is linked to susceptibility to early-onset breast and ovarian cancer [1,2], and inactivating mutations in the gene confer high lifetime risk of cancer
We examine a role for BRCA1 in cellular protection against alkylating agent methyl methanesulfonate (MMS)-induced stress as a function of pol b expression
In the present study we explored the possibility that BRCA1 might be involved, along with pol b, in base excision repair (BER) repair of alkylation base damage
Summary
It is well known that inactivation of the product of the breast cancer 1 (BRCA1) gene is linked to susceptibility to early-onset breast and ovarian cancer [1,2], and inactivating mutations in the gene confer high lifetime risk of cancer. The human BRCA1 gene is organized into 24 exons and encodes a 220-kDa protein. The BRCA1 protein is generally considered to be both a tumor suppressor and a DNA repair factor involved in multiple DNA repair and genome stability processes [3,4]. BRCA1 is found in the nucleus and has a single conserved amino-terminal RING domain, and 2 tandem BRCT domains at the carboxy-terminus. The BRCT domains of BRCA1 are responsible for phosphorylationdependent localization, and these domains are thought to regulate multiple pathways, including those responsible for tumor suppression
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