NFBD1, a novel nuclear protein with signature motifs of FHA and BRCT, and an internal 41-amino acid repeat sequence, is an early participant in DNA damage response.
Efficient repair of DNA double-strand breaks depends on the intact signaling cascade, comprising molecules involved in DNA damage signal pathways and checkpoints. Budding yeast Rad9 (scRad9) is required for activation of scRad53 (mammalian homolog Chk2) and transduction of the signal further downstream in this pathway. In the search for a mammalian homolog, three proteins in the human data base, including BRCA1, 53BP1, and nuclear factor with BRCT domains protein 1 (NFBD1), were found to share significant homology with the BRCT motifs of scRad9. Because BRCA1 and 53BP1 are involved in DNA damage responses, a similar role for NFBD1 was tested. We show that NFBD1 is a 250-kDa nuclear protein containing a forkhead-associated motif at its N terminus, two BRCT motifs at its C terminus, and 13 internal repetitions of a 41-amino acid sequence. Five minutes after gamma-irradiation, NFBD1 formed nuclear foci that colocalized with the phosphorylated form of H2AX and Chk2, two phosphorylation events known to be involved in early DNA damage response. NFBD1 foci are also detected in response to camptothecin, etoposide, and methylmethanesulfonate treatments. Deletion of the forkhead-associated motif or the internal repeats of NFBD1 has no effect on DNA damage-induced NFBD1 foci formation. Conversely, deletion of the BRCT motifs abrogates damage-induced NFBD1 foci. Ectopic expression of the BRCT motifs reduced damage-induced NFBD1 foci and compromised phosphorylated Chk2- and phosphorylated H2AX-containing foci. These results suggest that NFBD1, like BRCA1 and 53BP1, participates in the early response to DNA damage.
Shang
Yong Lei
YL
Department of Molecular Medicine and Institute of Biotechnology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78245, USA.
Bodero
Amanda J
AJ
Chen
Phang-Lang
PL
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D002453:Cell Cycle / Q000502:physiology; D018797:Cell Cycle Proteins; D002460:Cell Line; D004249:DNA Damage / Q000528:radiation effects; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism* / Q000528:radiation effects; D005720:Gamma Rays; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism* / Q000528:radiation effects; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators / Q000737:chemistry* / Q000378:metabolism* / Q000528:radiation effects; D014162:Transfection
D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C413163:MDC1 protein, human; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators