Functional analysis of FHA and BRCT domains of NBS1 in chromatin association and DNA damage responses

Abstract

Rad50/Mre11/NBS1 (R/M/N) is a multi-functional protein complex involved in DNA repair, cell cycle checkpoint activation, DNA replication and replication block-induced responses. Ionizing radiation (IR) induces the phosphorylation of NBS1 and nuclear foci formation of the complex. Although it has been suggested that the R/M/N complex is associated with DNA damage sites, we present here biochemical evidence for chromatin association of the complex. We show that the chromatin association of R/M/N is independent of IR and ataxia telangiectasia mutated (ATM). We also demonstrate that optimal chromatin association of the Rad50/Mre11/NBS1 proteins requires both the conserved forkhead-associated (FHA) and breast cancer C-terminus (BRCT) domains of NBS1. Moreover, both these domains of NBS1 are required for its phosphorylation on Ser343 but not on Ser278. Importantly, both the FHA and BRCT domains are essential for IR-induced foci (IRIF) formation of R/M/N and S phase checkpoint activation, but only the BRCT domain is needed for cell survival after IR. These data demonstrate that the FHA and BRCT domains of NBS1 are crucial for the functions of the R/M/N complex.

Independent Roles for Nibrin and Mre11-Rad50 in the Activation and Function of Atm

“…Our finding that Mre11-Rad50 can stimulate Atm activation in the absence of full-length nibrin raises the possibility that the initial localization of the MRN complex to DNA damage may be dependent on Mre11-Rad50 binding and processing the DNA breaks. Subsequent accumulation of the MRN complex at the sites of DNA damage appears to be mediated via interactions of the nibrin FHA and BRCT domains with ␥H2AX and presumably other DNA damage response proteins that also accumulate in radiation-induced foci (30,(37)(38)(39).…”

MDC1 regulates intra-S-phase checkpoint by targeting NBS1 to DNA double-strand breaks

“…Previous reports indicate that retention of NBS1 at DSBs, after DNA damage, is mediated by its interaction with MDC1 (15)(16)(17)(18)(19). Moreover, the FHA and BRCT domains of NBS1 are required for its recruitment to DSBs (nuclear foci) after DNA damage (20)(21)(22). Despite these results, it is still unclear how exactly NBS1 is recruited to the sites of DNA damage.…”

Structure of a Second BRCT Domain Identified in the Nijmegen Breakage Syndrome Protein Nbs1 and its Function in an MDC1-Dependent Localization of Nbs1 to DNA Damage Sites

“…66 An 15 N-labeled XNbs1 sample was used to collect 2-D 1 H-15 N HSQC spectra and 15 N-edited NOE spectroscopy (NOESY) spectra with 120 ms mixing time and to measure backbone relaxation parameters: R 1 , R 2 , and 1 H-15 N NOE at two magnetic field strengths (600 and 700 MHz, 1 H frequency). 15 N R 1 relaxation rates were measured with 10 different relaxation delays: 10, 35, 80, 120, 170, 240, 350, 500, 720, and 1200 ms, while for 15 N R 2 relaxation rates, 14 delays were used: 4,12,16,22,40,60,80,120,160,192,240,288,336, and 400 ms. For both R 1 and R 2 measurements, a recycle delay of 1 s was used. In the measurement of 1 H-15 N NOE, spectra were collected with a 2-s relaxation delay followed by a 3-s proton saturation.…”

“…10,11 MRN is known to play a key role in sensing DNA strand breaks and then amplifying initial signal and transducing it to downstream effector proteins that regulate cell cycle checkpoint and DNA repair. [12][13][14][15] In the MRN complex, Nbs1 regulates the catalytic nucleotide-dependent DNA binding and ATP-dependent DNA unwinding functions of Mre11 and Rad50. 16,17 It is also required for the localization of Mre11 and Rad50 into the nucleus as well as for the activation of ataxia-telangiectasia mutated (ATM) kinase.…”