Association of yeast DNA topoisomerase III and Sgs1 DNA helicase: studies of fusion proteins

Abstract

The Sgs1 protein of the budding yeast Saccharomyces cerevisiae is a member of the RecQ DNA helicase family that includes the human Bloom, Werner, and Rothmund-Thompson syndrome proteins. The N-terminal region outside the central DNA helicase core of Sgs1, particularly the part containing the first 100 amino acid residues of the 1,447-residue protein, is known to be functionally important and has been implicated in Sgs1-DNA topoisomerase III (Top3) interaction. We show in this work that the functionality of a truncated Sgs1 lacking its N-terminal 106 residues can be restored by replacing the truncated region with Top3. Fusion of Top3 to a mutant Sgs1 with a Val-29 to Glu substitution, which interferes with Sgs1-Top3 interaction, similarly restores the functionality of the mutant Sgs1(V29E) protein. The Top3-Sgs1(Delta1-106) and Top3-Sgs1(V29E) fusion proteins behave like wild-type Sgs1 in complementing several aspects of the sgs1 phenotype, including the hypersensitivity of sgs1 cells to methyl methanesulfonate and hydroxyurea. Complementation by the fusion proteins required both the topoisomerase activity of Top3 and the helicase activity of the Sgs1 polypeptide. These results suggest that the sole function of the N-terminal 106 amino acid residues of Sgs1 is for Top3 binding, and that the coordinated actions of Sgs1 and Top3 are important in cellular processes such as the processing of DNA after exposure of cells to DNA-damaging agents.

Figure 1

Schematic representations of Top3, Sgs1, and the Top3-Sgs1 fusion proteins used in this study. Arrows indicate the positions of point mutations Top3(Y355F), Sgs1(V29E), and Sgs1(K706A) that abolish Top3 activity, inhibit interaction between Sgs1 and Top3, and inactivate the helicase activity of Sgs1, respectively. The helicase domain in Sgs1 is indicated by a hatched box. In the fusion protein Top3-Sgs1(Δ1–106), the C terminus of Top3 is joined to residue 107 of Sgs1.

Figure 2

Complementation of the hypersensitivity of sgs1 cells to MMS and HU by the expression of various proteins. A strain RB103 (MATα sgs1-3∷TRP1 leu2 trp1 ade2 his3 ura3) deficient in Sgs1 was transformed with various plasmids, and individual transformants were picked, resuspended at equal concentrations, and plated at 5-fold serial dilutions on YPD plates containing either 1.4 mM MMS or 100 mM HU. Cells were grown for 2 days at 30°C and photographed. (A) Row 2 shows serial dilutions of cells harboring the empty vector, and the other rows show cells expressing plasmid-borne genes encoding the following proteins: row 1, Sgs1; row 3, Sgs1(K706A); row 4, Sgs1(Δ1–106); row 5, Top3-Sgs1(Δ1–106); row 6, Top3(Y355F)-Sgs1(Δ1–106); row 7, Top3-Sgs1(Δ1–106; K706A). (B) The proteins expressed from the plasmid-borne genes were as follows: row 1, full-length Sgs1; row 3, Sgs1(V29E); row 4, Top3-Sgs1(V29E); row 5, Top3(Y355F)-Sgs1(V29E); row 6, Top3-Sgs1(V29E; K706A); row 7, Top3. Row 2 again shows serial dilutions of cells harboring the empty vector. (C) The first two rows correspond to the first two rows of A and B, and the third row shows serial dilutions of cells expressing Top3 fused to the N terminus of full-length Sgs1.

Figure 3

The topoisomerase activity of Top3 is required for complementation of the slow-growth phenotype of top1 top3 cells. Mutant cells were transformed with constructs expressing wild-type Top3, Top3 containing a point mutation at the active site (Y355F), or the empty vector control. Colonies were picked and restreaked on YPD plates, and plates were incubated at 30°C for 3 days.

Figure 4

Complementation of sgs1 and sgs1 top1 strains by the expression of Top3-Sgs1 fusion proteins. The sgs1 (RB103) and sgs1 top1 (AMR58) strains were transformed with plasmids expressing the indicated Top3-Sgs1 fusion proteins. Colonies were picked and restreaked and grown at 30°C for 2–3 days. (A) Strain sgs1 top1. (B) Strain sgs1.