MRC National Institute for Medical Research, London
Science for Health
Conserved structure in DNA-damage response proteins
02 October 2009
Genomic integrity is constantly challenged by the generation of DNA lesions, of which double-stranded breaks (DSBs) are generally considered the most toxic. DSBs can arise through the actions of DNA-damaging chemicals and ionizing radiation, or a variety of other mechanisms. DNA-damage detection initiates a complex and orchestrated set of intracellular responses involving kinase-signaling cascades, cell-cycle checkpoint activation and the deployment of DNA-repair proteins. Central to these processes is a diverse group of proteins that function as platforms for the assembly of multi-protein complexes through the combinatorial activities of phosphoserine/threonine binding protein modules, such as forkhead-associated (FHA) and Brca1 C-terminal (BRCT)-repeat, which interact with specific motifs following their phosphorylation by DNA-damage activated or cell-cycle responsive serine/threonine kinases.
Nbs1 was originally identified as the gene mutated in Nijmegen breakage syndrome, a rare autosomal-recessive human disease characterized by immune disorders, microcephaly, growth retardation, hypersensitivity to ionizing radiation and predisposition to lymphoid cancers. Human Nbs1 is a subunit of the Mre11- Rad50-Nbs1 (MRN) complex, which binds to DSBs and acts as a bridge to hold the DNA ends in close proximity and promote their rejoining. Functionally, Nbs1 seems to play key roles in most or all of the DNA-damage-checkpoint signaling functions of the MRN complex through interactions with a number of proteins. In spite of the array of Nbs1 activities, little is currently known about how it mediates its diverse protein interactions at the molecular level.
Steve Smerdon (pictured), from NIMR's Division of Molecular Structure, in collaboration with colleagues at the Gurdon Institute and the University of Sussex, has described an integrated structural, biochemical, genetic and cell-biological analysis of the Nbs1 N-terminal FHA/BRCT-repeat region. They have completed a 2.3Å crystal structure of the N-terminal region of fission yeast Nbs1, revealing an unusual but conserved architecture in which FHA and BRCT-repeat domains are intimately associated rather than existing in a more modular, beads-on-string arrangement that had been predicted previously. They demonstrated that di-phosphorylated pSer-Asp-pThr-Asp motifs, recently identified as multi-copy docking sites in a human Nbs1-interacting partner called Mdc1, are evolutionarily conserved Nbs1 binding targets. They also showed that similar phospho-motifs within Ctp1, the fission yeast orthologue of the human tumour suppressor CtIP, are necessary for DNA-damage repair. Finally, they established that human Nbs1 interactions with Mdc1 occur through both its FHA and BRCT-repeat domains, suggesting how their structural and functional inter-dependence underpins Nbs1 adaptor functions in the DNA-damage response.
The Nbs1 N-terminal regulatory region
The overall architecture of the Nbs1 N-terminal regulatory region (top left) shows how the FHA and BRCT-repeat domains are structurally fused.
Although a lot is known aboout the Mre11 and Rad50 components of the MRN complex, the Nbs1 subunit has, until now, managed to evade any really detailed analysis. This work has shown us a pretty much unsuspected architecture for Nbs1 and, although surprising, it nicely explains a lot of previous biology stretching back to the original identification of the molecule in the late 1990's and the original description of Nijmegen breakage syndrome nearly 30 years ago. For us, the real key was figuring out how to make recombinant forms of the yeast and human proteins. This took us a long time, more years than I care to remember. However, when we solved the problem, the comparative biochemistry we were able to do ultimately led us to define the specificity of these molecules and identify what turns out to be a very important binding partner. The way in which the yeast and human proteins interact with their respective cellular targets turns out to be pretty well conserved, in spite of large differences in their primary sequence. This again shows how function and structure are often much better conserved than simple sequence comparisons might predict.
Steve Smerdon
Original article
The research findings are published in full in:
Janette Lloyd, J. Ross Chapman, Julie A. Clapperton, Lesley F. Haire, Edgar Hartsuiker, Jiejin Li, Antony M. Carr, Stephen P. Jackson & Stephen J. Smerdon (2009).
A supra-modular FHA/BRCT-repeat architecture mediates Nbs1 adaptor function in response to DNA-damage
Cell, 139(1): 100-11. Publisher abstract
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