Beamlines | MX Case Study

The DNA-damage response

Breaks in DNA cause a cascade of intercellular responses including the activation of the Mre11/Rad50/Nbs1 (MRN) complex. This complex attaches itself to the strand break point and holds the broken ends in close proximity to one another ready for rejoining.

The subunits Mre11 and Rad50 of the complex have previously been well characterised, however the Nbs1 subunit and its molecular functions are less well known. Scientists have been using Diamond to study the crystal structure of Nbs1, which shows a conserved architecture of the N-terminal FHA/BRCT-repeat region. This conserved architecture and its potential interactions in cell DNA damage resistance was published in the journal Cell.

In Detail

 

A new scaffold for the DNA-damage response
DNA lesions such as double stranded breaks will initiate complex intracellular responses, and central to these processes are proteins with forkhead-associated (FHA) and Brca1 C-terminal (BRCT)-repeat domains. These FHA- and BRCT- repeat domains interact with target motifs after their phosphorylation by DNA damage activated serine/threonine kinases.

Human Nbs1 is a subunit of the MRN complex, which binds to double stranded breaks and holds the DNA breaks in close proximity to one another to allow the rejoining of the broken strands. The N-terminal region of the Nbs1 subunit contains FHA and BRCT motifs which are common in DNA-damage response proteins. Through these domains, Nbs1 forms phospho-dependent protein-protein interactions with molecules such as mediator of the DNA-damage checkpoint 1 (Mdc1).

Here crystallographic analysis of N-terminal region of fission yeast Nbs1 showed a conserved structure in which the FHA- and BRCT-repeat domains may have the potential to interact with conserved di-phospho motifs within Mdc1. The research suggests that mutations of phospho-binding residues in either the FHA- or BRCT repeat domain is enough to hinder interactions with Mdc1 in human cells. The research also indicated that a fixed spatial relationship between the regions could be of importance to FHA- and BRCT- domain activity.

It was also observed that there was a Mdc1-like binding site motif within Ctp1, the fission yeast ortholog of human CtlP. Nbs1 sequestration of Ctp1 promotes resistance to double strand breaks. The human and yeast Nbs1 also showed an increased affinity for diphosphorylated sequences rather than singly phosphorylated motifs suggesting an ability to respond to varying levels of phosphorylation and interacting partners.

“Our crystallographic analysis has revealed a conserved supra-modular FHA-BRCT architecture that, along with our supporting biochemical data, explains the apparent functional inter-dependence of the Nbs1 FHA and BRCT motifs first noted some time ago. Further, our observations that the binding activities of fission yeast and human Nbs1 fragments show a similar FHA-domain specificity for pSDpTD-like CK2 motifs has allowed us to identify related spNbs1 binding sites within S.pombe Ctp1 that mediate interactions necessary for resistance to DNA damage.”

Dr Steve Smerdon from the MRC National Institute for Medical Research in London

A Supramodular FHA/BRCT-Repeat Architecture Mediates Nbs1 Adaptor Function in Response to DNA Damage, Janette Lloyd, Ross Chapman, Julie Clapperton, Lesley Haire, Edgar Hartsuiker, Jiejin Li, Anthony Carr, Stephen Jackson, Stephen Smerdon, Cell, VOL 139 (1) , PAGES 100 - 111 October 2009
DOI: 10.1016/j.cell.2009.07.043