The Sgs1 Helicase of Saccharomyces cerevisiae Inhibits Retrotransposition of Ty1 Multimeric Arrays

DNA helicases catalyze the unwinding of duplex DNA into individual DNA strands (42). A plethora of DNA helicases within cells is involved in DNA replication, repair, recombination, and transcription. Members of the RecQ family of DNA helicases are involved in the maintenance of genome stability in all organisms characterized, from bacteria to humans (7). Mutations in the SGS1 gene, which encodes the only RecQ homologue in Saccharomyces cerevisiae, result in elevated levels of mitotic homologous and illegitimate recombination, increased rates of chromosomal nondisjunction, and accelerated aging (21, 56, 61, 62, 65). Similarly, mutations in human genes encoding the RecQ homologues RecQL4 (35, 49), WRN (67), and BLM (18) give rise to rare hereditary disorders that are characterized by genome instability and a pronounced predisposition to cancer. Notably, expression of either WRN or BLM in yeast complements the hyperrecombination phenotypes of an sgs1 mutant (65). These findings suggest that the mechanisms by which Sgs1 preserves genetic stability in yeast will serve as a paradigm for the role of RecQ homologues in human disease. The SGS1 gene was originally isolated in a screen for genetic interaction with DNA topoisomerase III (21). Both topoisomerase III and Sgs1 repress recombination of DNA repeats, and inactivation of either Sgs1 or topoisomerase III results in S-phase-specific defects. Therefore, it was proposed that the combined activity of Sgs1 and topoisomerase III is required to initiate DNA repair events during replication (7). In support of this model, Sgs1 was shown to colocalize with the cell cycle checkpoint kinase, Rad53, in S-phase-specific foci and to participate in activation of the intra-S checkpoint (19). In the absence of Sgs1, DNA lesions that arise during DNA replication may be diverted into a homologous recombination pathway. This view is supported by recent reports that inactivation of Sgs1 and Srs2, another DNA helicase with a partially redundant role in genome maintenance, results in a severe growth defect involving a high incidence of mitotic arrest (22, 40, 46). The growth defect is suppressed by inactivation of homologous recombination proteins, including Rad51, Rad52, Rad55, and Rad57, suggesting that mitotic arrest in sgs1 srs2 mutants results from DNA lesions that arise from abortive recombination events (22, 46). Taken together, these findings suggest that DNA lesions that block the replication fork act as triggers for homologous recombination in sgs1Δ mutants. Sgs1 is critical for the stability of repeated DNA sequences such as those in the RDN1 locus. RDN1 contains 100 to 200 tandem repeats of a 9.1-kb region encoding the RNA polymerase I (Pol I)-transcribed 35S rRNA gene and the Pol III-transcribed 5S rRNA gene. Both recombination between ribosomal DNA (rDNA) repeats and Pol II-mediated transcription are inhibited in the RDN1 locus (6, 25, 57, 58). Recombination in the rDNA can give rise to extrachromosomal rDNA circles (ERCs), which accumulate in aging cells and may trigger senescence (55). Many trans-acting factors that repress recombination in rDNA have been identified. Where analyzed, genes that inhibit rDNA recombination, including SIR2, TOP1, UBC2, and ZDS2, have been found to also be required for transcriptional silencing in rDNA (6, 8, 20, 25, 52, 57). Furthermore, both SIR2 and ZDS2 forestall aging (31, 52). These and other findings have led to models in which a specific type of heterochromatin mediates the repression of recombination and transcription in the rDNA, and the integrity of this heterochromatin is a primary determinant of longevity (26, 29). In sgs1 mutants, mitotic recombination in the rDNA is increased sevenfold (21) and longevity is reduced by 60% (55, 56). Paradoxically, ERCs accumulate to the same level in the presence and absence of Sgs1 (28, 46). The role of Sgs1 in rDNA silencing has not been investigated previously, despite its potential to provide insight into the mechanism by which Sgs1 represses rDNA recombination and aging. In this study, we determined the effect of Sgs1 on the expression and transposition of Ty1 elements in rDNA and euchromatic DNA. Ty1 elements constitute one of five families of retrovirus-like transposons (Ty1 to Ty5) in yeast. Approximately 30 Ty1 elements reside at dispersed sites in the haploid genome, and the majority are transpositionally competent (12, 34). Ty1 elements consist of a central coding domain flanked by long terminal repeats (LTRs). The coding domain contains two overlapping open reading frames: TYA1, which encodes a capsid protein, and TYB1, which encodes protease, integrase (IN), and reverse transcriptase (RT). Ty1 elements are transcribed by Pol II to form a terminally redundant transcript. Ty1 RNA is translated into two proteins, TyA1 and the TyA1-TyB1 fusion protein, which assemble into virus-like particles (VLPs) that encapsulate Ty1 RNA during assembly. Maturation of VLPs occurs by protease-mediated processing of Ty1 precursor proteins, and it is required for synthesis of a full-length linear cDNA by RT, using Ty1 RNA as a template. Subsequently, the Ty1 cDNA and IN protein are transported to the nucleus, presumably as components of a preintegration complex (33, 48). IN mediates transposition of Ty1 cDNA at a nonhomologous target in the genome. Alternatively, Ty1 cDNA can recombine with genomic Ty1 elements, which occurs by IN-independent and Rad52-dependent mechanisms (53). The introduction of Ty1 cDNA into the genome by transposition or recombination is referred to as Ty1 cDNA-mediated mobility. Several host mechanisms limit the potentially mutagenic effects of Ty1 transposition. First, Ty1 elements are preferentially targeted to genomic regions that do not encode proteins, including the upstream regions of Pol III-transcribed genes (16, 30). Second, Ty1 elements that are located in the rDNA array are subject to transcriptional silencing (6). Third, transposition is inhibited by host factors that block posttranscriptional steps in Ty1 replication. For example, Fus3, a mitogen-activated protein kinase, destabilizes VLP-associated proteins via its negative regulation of the invasive growth pathway (10). In addition, Ssl2 and Rad3, two helicase components of transcription factor TFIIH, promote degradation of Ty1 cDNA, thereby repressing transposition 100-fold or more (39). Finally, members of the Rad52 epistasis group, including Rad50, Rad51, Rad52, Rad54, and Rad57, reduce Ty1 cDNA levels and transposition by inhibiting cDNA synthesis or stability (50). In this work, we demonstrate that Sgs1 inhibits the mobility of Ty1 elements by a novel mechanism. Initially, we proposed that Sgs1 might specifically repress transcription (and therefore transposition) of rDNA-Ty1 elements, since Sgs1 is required for the repression of rDNA recombination. To our surprise, we found that Sgs1 is not involved in the transcriptional silencing of Ty1 elements in the rDNA, although it is required for their mitotic stability. Furthermore, Sgs1 is a global inhibitor of Ty1 mobility that acts primarily at a step following cDNA synthesis. In the absence of Sgs1, extrachromosomal Ty1 cDNA molecules recombine at a high frequency, forming multimeric cDNA arrays that are subsequently integrated into the genome. We demonstrate that transposition of multimeric cDNA is the major cause of increased Ty1 mobility in sgs1Δ mutants.