Linkage between Werner Syndrome Protein and the Mre11 Complex via Nbs1

Abstract

The Werner syndrome and the Nijmegen breakage syndrome are recessive genetic disorders that show increased genomic instability, cancer predisposition, hypersensitivity to mitomycin C and ␥-irradiation, shortened telomeres, and cell cycle defects. The protein mutated in the premature aging disease known as the Werner syndrome is designated WRN and is a member of the RecQ helicase family. The Nbs1 protein is mutated in Nijmegen breakage syndrome individuals and is part of the mammalian Mre11 complex together with the Mre11 and Rad50 proteins. Here, we show that WRN associates with the Mre11 complex via binding to Nbs1 in vitro and in vivo. In response to ␥-irradiation or mitomycin C, WRN leaves the nucleoli and co-localizes with the Mre11 complex in the nucleoplasm. We detect an increased association between WRN and the Mre11 complex after cellular exposure to ␥-irradiation. Small interfering RNA and complementation experiments demonstrated convergence of WRN and Nbs1 in response to ␥-irradiation or mitomycin C. Nbs1 is required for the Mre11 complex promotion of WRN helicase activity. Taken together, these results demonstrate a functional link between the two genetic diseases with partially overlapping phenotypes in a pathway that responds to DNA double strand breaks and interstrand cross-links.DNA damage in the form of double strand breaks (DSBs) 1 or cross-links compromises the integrity of cells. Defects in repairing these lesions are associated with human chromosome fragility syndromes such as Werner syndrome (WS), Nijmegen breakage syndrome (NBS), ataxia telangiectasia (AT), and ataxia telangiectasia-like disorder (ATLD). In contrast to WS, NBS and ataxia telangiectasia-like disorder (with Mre11 mutations) are defective in checkpoint responses and show clinical features of immunodeficiency, hyperpigmentation spots, and ovarian dysgenesis. Noticeably, in addition to the aforementioned cellular similarities, both WS and NBS display graying of the hair, short stature, a "bird-like" face, increased cancer susceptibility, and reduced life span (1-6). Loss of fidelity in repairing DNA DSBs results in chromosomal rearrangements (7), a defect that is prominent in both WS and NBS. The proteins mutated in these syndromes are implicated in the repair of DNA DSBs and interstrand cross-links (ICLs) (2,4,5,8,9). Vertebrate Nbs1 is essential for repairing DNA DSBs by homologous recombination (10), and WRN participates in this repair pathway by resolving the recombinational intermediates (11). WS cells are more sensitive to DNA cross-linkers than to any other genotoxic drugs and are defective in repairing DNA ICLs in vivo (2, 8). WRN exhibits 3Ј 3 5Ј helicase, 3Ј 3 5Ј exonuclease, and ATPase activities (12, 13). Although Nbs1 has no known enzymatic activity, it is required for optimal exonuclease and endonuclease activities of the Mre11 complex and for the correct nuclear localization of Mre11 and Rad50 proteins (4, 14). The Mre11 complex exhibits clear 3Ј 3 5Ј exonuclease activity but demonstrates only limited DNA unwindin...

Understanding the molecular basis of common fragile sites instability: Role of the proteins involved in the recovery of stalled replication forks

“…56,58,59 Although how each biochemical activity of WRN contributes to its function is not fully clarified, several studies indicate specific involvement in different metabolic processes. [60][61][62][63][64][65][66][67] Moreover, WS cells have an elevated spontaneous yield of DNA breakage, which is consistent with the observed high rate of chromosomal rearrangements, 56,68 and depends on an alternative pathway, probably involving break-induced replication, to recover stalled replication forks. 69 These findings support the hypothesis that, upon replication fork stalling, WRN acts to limit DSBs and fork collapse or to promote error-free repair of DSBs.…”

The Werner syndrome protein is required for recruitment of chromatin assembly factor 1 following DNA damage

“…These results suggest that WRN may be involved in processing ionizing radiation-induced double-strand breaks. In agreement, WRN also interacts physically with the Mre11-Rad50-NBS1 complex, which functions in homologous recombination for double-strand break processing (Cheng et al, 2004). WRN may also play a role in base excision repair as WRN stimulates DNA polymerase b-strand displacement synthesis via its helicase activity (Harrigan et al, , 2006.…”

“…WRN has been shown to interact with Ku and DNA-PKcs (Li and Comai, 2000;Orren et al, 2001;Karmakar et al, 2002a, b;Karmakar and Bohr, 2005;Li et al, 2005). Consistently, WS fibroblasts transformed with Simian Virus-40 (SV40) T antigen or immortalized by expressing human telomerase reverse transcriptase (hTERT) display a mild but distinct sensitivity to ionizing radiation compared to controls (Yannone et al, 2001;Cheng et al, 2004). WS cells also display extensive deletions at non-homologous joined ends as well as nonhomologous chromosome exchanges (Oshima et al, 2002).…”

Developing master keys to brain pathology, cancer and aging from the structural biology of proteins controlling reactive oxygen species and DNA repair

“…WRN has been implicated in certain DNA repair events, as WS cells display hypersensitivity to specific DNA-damaging agents (Bohr, 2005) and show a mild, yet distinct sensitivity to ionizing radiation (Yannone et al, 2001,Cheng et al, 2004. Moreover, repair roles have been indicated by physical and functional interactions with proteins in the DNA repair pathways.…”

“…WRN links to BER include physical and functional interaction with polβ , polδ (Szekely et al, 2000), replication protein A (RPA) (Brosh et al, 1999), flap endonuclease 1 (FEN-1) (Brosh et al, 2001b), PCNA (Lebel et al, 1999) and poly(ADP-ribose)polymerase 1 (PARP-1) (von Kobbe et al, 2003a). Possible roles in the HRR pathway are suggested by interactions with the MRN complex (Cheng et al, 2004) and Rad52 (Baynton et al, 2003) and by colocalization with Rad51 in camptothecin-treated cells (Sakamoto et al, 2001). Similarly, a link to the NHEJ pathway is indicated by in interactions of WRN with the NHEJ-essential protein kinase DNA-PK (Yannone et al, 2001,Karmakar et al, 2002a,Li and Comai, 2002.…”