Elsevier

DNA Repair

Volume 7, Issue 4, 2 April 2008, Pages 558-571
DNA Repair

The genetic consequences of ablating helicase activity and the Top3 interaction domain of Sgs1

https://doi.org/10.1016/j.dnarep.2007.12.010Get rights and content

Abstract

Sgs1, the RecQ helicase homolog, and Top3, the type-IA topoisomerase, physically interact and are required for genomic stability in budding yeast. Similarly, topoisomerase III genes physically pair with homologs of SGS1 in humans that are involved in the cancer predisposition and premature aging diseases Bloom, Werner, and Rothmund–Thompson syndromes. In the absence of Top1 activity, sgs1 mutants are severely growth impaired. Here, we investigate the role of Sgs1 helicase activity and its N-terminal Top3 interaction domain by using an allele-replacement technique to integrate mutant alleles at the native SGS1 genomic locus. We compare the phenotype of helicase-defective (sgs1-hd) and N-terminal deletion (sgs1-NΔ) strains to wild-type and sgs1 null strains. Like the sgs1 null, sgs1-hd mutations suppress top3 slow growth, cause a growth defect in the absence of Srs2 helicase, and impair meiosis. However, for recombination and the synthetic interaction with top1Δ mutations, loss of helicase activity exhibits a less severe phenotype than the null. Interestingly, deletion of the Top3 interaction domain of Sgs1 causes a top3-like phenotype, and furthermore, this effect is dependent on helicase activity. These results suggest that the protein–protein interaction between these two DNA-metabolism enzymes, even in the absence of helicase activity, is important for their function in catalyzing specific changes in DNA topology.

Introduction

The Saccharomyces cerevisiae SGS1 gene belongs to a family of DNA helicases first defined by the E. coli RecQ helicase [1]. All RecQ homologs studied to date are important for genomic integrity (reviewed in [2], [3]). In humans, mutations in three of the five known RecQ/Sgs1 homologs are involved in tumor suppression and disease syndromes: Bloom syndrome (BS) and Werner syndrome (WS), caused by mutations in the BLM and WRN genes, respectively, and three syndromes: Rothmund–Thompson (RTS), RAPADALINO, and Baller–Gerold (BGS), caused by mutations in the RECQ4 gene [4], [5], [6], [7], [8]. These syndromes variously display features of premature aging, cancer predisposition, developmental abnormalities, and genomic instability (for recent review see [9]).

The Sgs1 helicase was discovered by both genetic and physical interactions with Top3, a prokaryotic-like type-I topoisomerase [10]. Strains mutant for top3 have a pleiotropic phenotype including a severe growth defect caused by a cell-cycle delay in late S/G2, hyper-recombination at multiple loci, increased chromosome nondisjunction and sensitivity to the DNA damaging agents MMS and HU, meiotic defects, and an impaired intra S-phase checkpoint [10], [11], [12], [13], [14], [15]. The phenotype of sgs1 mutants resembles that of top3 mutants but in each case the sgs1 defects are less severe and, for the most part, sgs1 is epistatic to top3 [10], [16], [17]. For example, mutation of sgs1 suppresses the slow growth of top3 mutants to the rate of an sgs1 mutant.

Recently, Rmi1, a third member of the Sgs1–Top3 complex, was discovered [18], [19]. Biochemical studies suggest that the Sgs1–Top3–Rmi1 complex plays a role in processing HR intermediates, restarting failed replication forks, and activating S-phase checkpoint arrest [17], [18], [19], [20], [21], [22], [23], [24], [25]. Rmi1 may promote binding specifically to branched DNA structures and/or stimulate Top3 strand passage [28], [29]. In humans, a conserved complex of the homologous BLM-hTOPO IIIα-BLAP75/RMI1 functions in similar processes [26], [27], [28], [29], [30].

These functional roles for Sgs1–Top3–Rmi1 in yeast are evidenced by synthetic sickness or lethality with other genes involved in replication and recombination, such as srs2, rrm3, slx1, slx4, mus81, mms4 and top1 [12], [19], [31], [32], [33], [34], [35], [36], [40]. These synthetic interactions, along with many of the defects in sgs1 and top3 mutant cells are likely a result of toxic or unresolved HR events, since they can be suppressed by mutation of genes involved in the early steps of HR such as RAD51, RAD52, RAD54, RAD55, and RAD57 [18], [19], [21], [37], [38], [39].

The RecQ, Blm, Wrn, and Sgs1 proteins all possess enzymatic helicase activity with a 3′–5′ polarity [1], [40], [42], [43]. Yet the signature helicase domain of the eukaryotic genes only covers about a third of the length of the respective proteins, approximately 400 amino acids (AA) (Fig. 1A). For example, helicase activity has been demonstrated for Sgs1 in a fragment from AA 400–1268, indicating that much of the protein is dispensable for helicase function [44]. Furthermore, loss of the enzymatic helicase activity of Sgs1 is responsible for some, but not all aspects of the sgs1 mutant phenotype including chromosome loss and missegregation as well as synthetic lethality with srs2Δ [47].

Sgs1 interacts physically with Top3, Top2, Rad16, Rad51 and Rmi1 as well as other proteins [10], [13], [16], [18], [19], [48]. Genetic and physical evidence suggest that the interaction between eukaryotic RecQ and topoisomerase III homologs is of central importance for the function of both proteins in DNA metabolism. In humans, the BLM interacts with hTOPO IIIα and stimulates its strand-passage activity, and, together with RMI1, this complex can function as a double Holliday junction (dHJ) dissolvase [28], [29], [49], [50]. In yeast, fusion of Sgs1 and Top3 into a single peptide complements several aspects of the sgs1 phenotype [51]. The Sgs1–Top3 interaction is important in the absence of srs2 and top1, as well as for complementation of MMS sensitivity and suppression of HR [46], [52].

Here, we investigate the roles of Sgs1 helicase activity and the Sgs1 interaction with Top3 by analyzing mutant alleles that disrupt these functions. In contrast to many previous studies, we only studied alleles integrated at their native genomic loci [53], [54], [55]. This permits an assessment of the true mutant phenotype and eliminates problems associated with plasmid-based complementation studies as well as position effects that may occur as a result of integration at non-native sites. We find that point mutations that inactivate the Sgs1 helicase result in many, but not all of the defects seen in sgs1 null mutants. For example, helicase-defective mutants and the null mutant all exhibit the same top3 slow growth suppression as well as synthetic interaction with srs2. However, helicase mutants do not parallel the null allele for their effects on recombination at the SUP4-o locus or their growth defects in combination with top1 mutations. To investigate what other function of the Sgs1 protein may be responsible for these differences, we focused on the Sgs1 interaction with Top3 by deleting a region of the Sgs1 N terminus necessary for physical interaction with Top3. The phenotype of this deletion mimics the loss of Top3 when helicase activity is intact. However, when both the Top3 interaction domain and helicase activity are eliminated, an sgs1 null phenotype results. These data add support to the notion that Sgs1 acts upstream of Top3 in DNA metabolism, and its helicase activity creates an intermediate DNA structure that requires the recruitment of Top3 for efficient resolution. Furthermore, these observations suggest that the physical interaction between the two proteins is required to coordinate their activities.

Section snippets

S. cerevisiae strains and genetic methods

The W1588 segregants of W303-1A (MATa ade2-1 can1-100 his3-11,15 leu2-3,112 ura3-1 trp1-1 RAD5) and their derivatives used are listed in Table 1 [56], [57]. The crosses, growth, and transformation of strains were performed by standard methods [58]. Media were prepared as described [59], except twice the amount of leucine was used. Sporulation medium was prepared as described [60]. Standard procedures were used for mating, sporulation, and dissection [59]. Cells were grown at 30 °C.

The

Mutant sgs1 alleles integrated at the genomic locus

A number of studies report the effect of various sgs1 mutations on different aspects of its phenotype. In most of these reports, alleles expressed from plasmids or integrated at non-native loci were used to study complementation [40], [41], [45], [46], [52], [64], [65], [66], [67], [68]. To avoid the ambiguities inherent in interpreting growth or lack of growth in cells transformed with a plasmid as well as to avoid potential position effects for loci integrated elsewhere, all mutant alleles

Model for the function of Sgs1 helicase activity and Top3 interaction

Here, using a variety of assays summarized in Table 6, we compare the phenotypic effects of mutations that remove the enzymatic helicase and Top3 interaction functions of Sgs1. All constructs used in this study are under control of the wild-type SGS1 promoter and expressed from the native genomic SGS1 locus. We expand on previous studies to show that point mutation of the SGS1 helicase resembles an sgs1 null allele for suppression of top3 slow growth, for meiotic defects and for synthetic

Acknowledgments

We thank Uffe Mortensen, Frederic Foucault, Greg Freyer, Morten Dunø, Michael Lisby, Erika Shor, Naz Erdeniz, Robert Reid, Marisa Wagner, and Lorraine Symington for helpful discussions concerning this work. We especially thank Kara Bernstein for careful reading of the manuscript, Qi Feng for her assistance in preparation of this manuscript and Ivana Sunjaveric for technical support. J.W. is particularly indebted to Shan Sockanathan, Floria Lupu, and Jonathan Eggenschweiler for providing

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