Deletion of ULS1 confers damage tolerance in sgs1 mutants through a Top3-dependent D-loop mediated fork restart pathway

Highlights

• Top3 is needed for the HU resistance conferred to sgs1Δ mutants by deletion of the putative SUMO-targeted ubiquitin ligase, Uls1.

• Top3 is required for coordinated breaks and D-loop formation within the rDNA following HU-induced damage.

• Uls1 absence channels repair of HU-induced damage in sgs1Δ mutants from Rad51-independent HR to Rad51-dependent HR.

• Rescue of sgs1Δ mutants by deletion of ULS1 requires the SUMO ligase activity of Mms21.

Abstract

Homologous recombination (HR)-based repair during DNA replication can apparently utilize several partially overlapping repair pathways in response to any given lesion. A key player in HR repair is the Sgs1-Top3-Rmi1 (STR) complex, which is critical for resolving X-shaped recombination intermediates formed following bypass of methyl methanesulfonate (MMS)-induced damage. STR mutants are also sensitive to the ribonucleotide reductase inhibitor, hydroxyurea (HU), but unlike MMS treatment, HU treatment is not accompanied by X-structure accumulation, and it is thus unclear how STR functions in this context. Here we provide evidence that HU-induced fork stalling enlists Top3 prior to recombination intermediate formation. The resistance of sgs1Δ mutants to HU is enhanced by the absence of the putative SUMO (Small Ubiquitin MOdifier)-targeted ubiquitin ligase, Uls1, and we demonstrate that Top3 is required for this enhanced resistance and for coordinated breaks and subsequent d-loop formation at forks stalled at the ribosomal DNA (rDNA) replication fork block (RFB). We also find that HU resistance depends on the catalytic activity of the E3 SUMO ligase, Mms21, and includes a rapid Rad51-dependent restart mechanism that is different from the slow Rad51-independent HR fork restart mechanism operative in sgs1Δ ULS1+ mutants. These data support a model in which repair of HU-induced damage in sgs1Δ mutants involves an error-prone break-induced replication pathway but, in the absence of Uls1, shifts to one that is higher-fidelity and involves the formation of Rad51-dependent d-loops.

Introduction

Maintaining genome integrity is vital for cell survival and cancer prevention. This maintenance is particularly challenging in the context of replication, where DNA repair machinery must coordinate activities with the replisome. It is well established that cells have a number of partially overlapping pathways that can repair DNA damage during replication, but how pathway choice is decided is still unclear. Recently, it has become evident that an intricate crosstalk occurring between the replication machinery, chromatin structure, and DNA repair complexes can strongly influence repair pathway choice (Reviewed in [1,2]). A better understanding of how these different pathways assist and compensate for one another to repair a given lesion could provide insight into cancer chemotherapeutic strategies.

The ribonucleotide reductase inhibitor, hydroxyurea (HU) predominantly induces replication fork stalling, causing uncoupling of the replicative MCM helicase from the polymerase machinery and generating ssDNA ahead of replicated DNA; in some DNA repair mutant backgrounds this stalling can further lead to reversal of the replication fork [[3], [4], [5], [6], [7]]. Interestingly, while fork reversal with subsequent fork restart is one of the primary responses to fork stalling in mammalian cells, likely due to the action of PARP, it is a rare event in yeast; fork restart in yeast thus must arise by other mechanisms [[8], [9], [10], [11]].

The STR complex, comprising the RecQ helicase Sgs1, the topoisomerase Top3, and the OB-fold containing protein Rmi1, is critical for genome integrity, including the HR-dependent reestablishment of damaged replication forks. Mutations in any of the STR complex members lead to increased rates of aberrant recombination, and elevated levels of X-shaped recombination intermediates during S-phase, particularly in the setting of MMS-induced DNA damage [[12], [13], [14], [15], [16], [17]]. Both in vitro and in vivo studies strongly indicate a direct role for this complex in resolving recombination intermediates, through the combined catalytic activities of the Sgs1 helicase unwinding DNA and the Top3 topoisomerase transiently nicking and mediating strand passage to decatenate joint molecules [[13], [14], [15],[18], [19], [20]]. Previous work from our own lab has shown sgs1Δ top3Δ mutants are more sensitive than sgs1Δ mutants to MMS, and that Top3 is capable of working independently from its complex members to resolve a specific type of MMS-induced X-shaped and hemicatenated template switch recombination intermediate termed a Rec-X [13].

However, STR mutants are similarly sensitive to HU, which unlike MMS, does not result in X-structure accumulation, indicating that the STR complex could have important functions outside of X-shaped recombination intermediate resolution [12,15]. Indeed, Sgs1 has also been implicated in earlier steps in the rescue of damaged forks including polymerase stabilization, Rad53 DNA damage response kinase recruitment, and 5′ DNA end resection, with Top3 and Rmi1 serving primarily to support Sgs1 in these contexts [[21], [22], [23], [24], [25]]. These functions likely aid in fork stabilization, but whether other functions of the STR complex are important at the fork and whether Top3 is capable of additional independent roles outside of Rec-X resolution are not fully understood. Interestingly, deletion of SGS1 results in increased rates of break-induced replication (BIR) and a compensatory loss of gene conversion (GC) events (i.e. synthesis dependent strand annealing and double Holliday junction (dHJ)-mediated HR events) following a double strand break (DSB), suggesting Sgs1, and possibly other STR complex members can mediate repair pathway choice in this setting [26,27]. However, how these activities might influence repair pathway choice at damaged replication forks has not been explored.

Recently it has been shown that genetic inactivation of Uls1, a Swi2/Snf2-related putative SUMO-targeted ubiquitin ligase (STUbL), provides damage tolerance to sgs1Δ mutants in a post-replicative, HR-dependent manner [28,29]. Given our previous observations that overexpression of Top3 can also provide rescue to sgs1Δ mutants on MMS, we investigated whether the rescue conferred by deletion of ULS1 required these same Top3 functions. We find that rescue of sgs1Δ mutants in the absence of Uls1, while dependent on Top3, does not involve Top3-mediated Rec-X resolution but instead utilizes a novel Top3-mediated break-induced fork restart mechanism. Furthermore, we provide evidence that repair of HU-induced stalled forks occurs predominantly through d-loop intermediates to restart replication, and that deletion of ULS1 confers resistance in sgs1Δ mutants by shifting this d-loop mediated repair from a Rad51-independent to a Rad51-dependent pathway.