Abstract
The RecQ-like DNA helicase family is essential for the maintenance of genome stability in all organisms. Sgs1, a member of this family in Saccharomyces cerevisiae, regulates early and late steps of double-strand break repair by homologous recombination. Using nuclear magnetic resonance spectroscopy, we show that the N-terminal 125 residues of Sgs1 are disordered and contain a transient α-helix that extends from residue 25 to 38. Based on the residue-specific knowledge of transient secondary structure, we designed proline mutations to disrupt this α-helix and observed hypersensitivity to DNA damaging agents and increased frequency of genome rearrangements. In vitro binding assays show that the defects of the proline mutants are the result of impaired binding of Top3 and Rmi1 to Sgs1. Extending mutagenesis N-terminally revealed a second functionally critical region that spans residues 9–17. Depending on the position of the proline substitution in the helix functional impairment of Sgs1 function varied, gradually increasing from the C- to the N-terminus. The multiscale approach we used to interrogate structure/function relationships in the long disordered N-terminal segment of Sgs1 allowed us to precisely define a functionally critical region and should be generally applicable to other disordered proteins.
Highlights
The maintenance of genome stability is essential for organismal survival
Mutations in BLM, WRN and RecQL4 are associated with Bloom syndrome, Werner syndrome and Rothmund–Thompson syndrome, respectively, which are characterized by elevated levels of aberrant recombination events, chromosome instability and extraordinary predisposition to cancer development early in life [1]
Using multidimensional heteronuclear nuclear magnetic resonance (NMR) spectroscopy, we have identified a short segment within the first 125 residues of the intrinsically disordered N-terminus of unbound Sgs1 that has transient a-helical structure whose integrity is essential for Sgs1 function in vivo
Summary
The maintenance of genome stability is essential for organismal survival. A complex and diverse system of proteins has evolved to accomplish this function. Saccharomyces cerevisiae cells that lack Sgs exhibit several phenotypes that are similar to those of cells from persons with Bloom syndrome, most notably dysregulated homologous recombination, hypersensitivity to DNAdamaging agents, meiotic defects and cell cycle delay [2,3] These defects are caused when the helicase activity of Sgs is inactivated by mutations in the ATPase domain or the RecQ C-terminal domain, which together make up the helicase core. Despite the fact that Sgs and BLM bind Top and its human homolog Topo IIIa, respectively, there is little primary sequence similarity between the N-terminal regions where these interactions are predicted to occur Both N-termini are predicted to be intrinsically disordered [20], which may help explain their level of sequence divergence [21,22]. Some of these mutations eliminate Top binding to Sgs, cause DNA damage hypersensitivity and induce spontaneous chromosomal rearrangements
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