The N-terminal region of Sgs1, which interacts with Top3, is required for complementation of MMS sensitivity and suppression of hyper-recombination in sgs1 disruptants

Abstract

The SGS1 gene of Saccharomyces (cerevisiae is a homologue of the genes affected in Bloom's syndrome, Werner's syndrome, and Rothmund-Thomson's syndrome. Disruption of the SGS1 gene is associated with high sensitivity to methyl methanesulfonate (MMS) and hydroxyurea (HU), and with hyper-recombination phenotypes, including interchromosomal recombination between heteroalleles. SGS1 encodes a protein which has a helicase domain similar to that of Escherichia coli RecQ. A comparison of amino acid sequences among helicases of the RecQ family reveals that Sgs1,WRN, and BLM share a conserved region adjacent to the C-terminal part of the helicase domain (C-terminal conserved region). In addition, Sgs1 contains two highly charged acidic regions in its N-terminal region and the HRDC (helicase and RNaseD C-terminal) domain at its C-terminal end. These regions were also found in BLM and WRN, and in Rqh1 from Schizosaccharomyces pombe. In this study, we demonstrate that the C-terminal conserved region, as well as the helicase motifs, of Sgs1 are essential for complementation of MMS sensitivity and suppression of hyper-recombination in sgs1 mutants. In contrast, the highly charged acidic regions, the HRDC domain, and the C-terminal 252 amino acids were dispensable for the complementation of these phenotypes. Surprisingly, the N-terminal 45 amino acids of Sgs1 were absolutely required for the suppression of the above phenotypes. Introduction of missense mutations into the region encoding amino acids 4-13 abolished the ability of Sgsl to complement MMS sensitivity and suppress hyper-recombination in sgs1 mutants, and also prevented its interaction with Top3, indicating that interaction with Top3 via the N-terminal region of Sgs1 is involved in the complementation of MMS sensitivity and the suppression of hyper-recombination.

An aromatic-rich loop couples DNA binding and ATP hydrolysis in the PriA DNA helicase

“…In the RecQ subfamily of SF2 enzymes, the ARL interacts with ssDNA to structurally couple DNA-binding with ATP hydrolysis in bacteria enzymes; the same loop has been implicated in helicase function in the S. cerevisiae RecQ protein, Sgs1 and human RecQ proteins, RECQ1 and BLM (13,16,17,35,36). In the DEAD-box subfamily of RNA helicases, motif IIa (also referred to as ‘post-II’) forms a highly conserved RNA binding surface that appears to clash with dsRNA and may force strand separation (15,21,54).…”

“…Interestingly, in a few SF2 enzymes, an element upstream of motif III and immediately C-terminal to motif II has been shown to directly bind ssRNA/DNA (13–15). This segment in RecQ DNA helicases was termed an aromatic-rich loop (ARL) or motif IIa, and it has been shown to couple DNA-binding with ATP hydrolysis (13,16,17). Similarly positioned segments have been implicated in nucleic acid binding and/or ATPase activities in a small number of other SF2 subfamily helicases (18–21), but whether ARL/motif IIa elements are generally involved in the helicase mechanisms of other SF2 enzymes is not known.…”

Multimerization domains are associated with apparent strand exchange activity in BLM and WRN DNA helicases

“…Although the specific role of multimerization is unknown, genetic studies presented here clearly reveal an in vivo function for the Sgs1 coiled coil. It should be pointed out however, that the coiled coil cannot be required for all Sgs1 functions since internal deletions eliminating this domain do not generate null phenotypes such as sensitivity to DNA damaging agents [26]. On the other hand, structure-function studies suggest that it is required for complementation of top3 Δ slow-growth suppression (Fig.…”

“…BLM is conserved in most species including the yeast S. cerevisiae where it is known as Sgs1. Like human BLM, Sgs1 forms an “STR” complex with its cognate Top3 and Rmi1 subunits [19–26]. The physical interaction between BLM/Sgs1 and the Top3-Rmi1 complex requires a 100 aa domain (TR) at the extreme N-terminus of the helicases (Fig.…”

Role of Elg1 protein in double strand break repair

“…As described previously (26), logarithmically growing cells were inoculated onto SC-His plates and YPAD plates containing various concentrations of MMS to evaluate the incidence of interchromosomal HR and colony forming cells (CFCs), respectively.…”