Identification of sequences that target BRCA1 to nuclear foci following alkylative DNA damage.
BRCA1 is a tumor suppressor involved in the maintenance of genome integrity. BRCA1 co-localizes with DNA repair proteins at nuclear foci in response to DNA double-strand breaks caused by ionizing radiation (IR). The response of BRCA1 to agents that elicit DNA single-strand breaks (SSB) is poorly defined. In this study, we compared chemicals that induce SSB repair and observed the most striking nuclear redistribution of BRCA1 following treatment with the alkylating agent methyl methanethiosulfonate (MMTS). In MCF-7 breast cancer cells, MMTS induced movement of endogenous BRCA1 into distinctive nuclear foci that co-stained with the SSB repair protein XRCC1, but not the DSB repair protein gamma-H2AX. XRCC1 did not accumulate in foci after ionizing radiation. Moreover, we showed by deletion mapping that different sequences target BRCA1 to nuclear foci induced by MMTS or by ionizing radiation. We identified two core MMTS-responsive sequences in BRCA1: the N-terminal BARD1-binding domain (aa1-304) and the C-terminal sequence aa1078-1312. These sequences individually are ineffective, but together they facilitated BRCA1 localization at MMTS-induced foci. Site-directed mutagenesis of two SQ/TQ motif serines (S1143A and S1280A) in the BRCA1 fusion protein reduced, but did not abolish, targeting to MMTS-inducible foci. This is the first report to describe co-localization of BRCA1 with XRCC1 at SSB repair foci. Our results indicate that BRCA1 requires BARD1 for targeting to different types of DNA lesion, and that distinct C-terminal sequences mediate selective recruitment to sites of double- or single-strand DNA damage.
Au
Wendy W Y
WW
Westmead Institute for Cancer Research, University of Sydney, Westmead Millennium Institute at Westmead Hospital, Westmead NSW 2145, Australia.
Henderson
Beric R
BR
D000477:Alkylating Agents / Q000494:pharmacology*; D000595:Amino Acid Sequence; D064007:Ataxia Telangiectasia Mutated Proteins; D019313:BRCA1 Protein / Q000737:chemistry* / Q000378:metabolism*; D001943:Breast Neoplasms / Q000235:genetics; D018797:Cell Cycle Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000187:drug effects* / Q000378:metabolism* / Q000528:radiation effects; D053904:DNA Breaks, Single-Stranded / Q000528:radiation effects; D004249:DNA Damage; D004260:DNA Repair / Q000187:drug effects / Q000528:radiation effects; D004268:DNA-Binding Proteins / Q000378:metabolism; D005260:Female; D006801:Humans; D008741:Methyl Methanesulfonate / Q000031:analogs & derivatives* / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects / Q000528:radiation effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000187:drug effects / Q000528:radiation effects; D011839:Radiation, Ionizing; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D000076105:X-ray Repair Cross Complementing Protein 1
D000477:Alkylating Agents; D019313:BRCA1 Protein; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D000076105:X-ray Repair Cross Complementing Protein 1; C000619371:XRCC1 protein, human; C014674:methyl methanethiosulfonate; D008741:Methyl Methanesulfonate; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases