Stalled replication forks generate a distinct mutational signature in yeast

Abstract

Proliferating cells acquire genome alterations during the act of DNA replication. This leads to mutation accumulation and somatic cell mosaicism in multicellular organisms, and is also implicated as an underlying cause of aging and tumorigenesis. The molecular mechanisms of DNA replication-associated genome rearrangements are poorly understood, largely due to methodological difficulties in analyzing specific replication forks in vivo. To provide an insight into this process, we analyzed the mutagenic consequences of replication fork stalling at a single, site-specific replication barrier (the Tus/ complex) engineered into the yeast genome. We demonstrate that transient stalling at this barrier induces a distinct pattern of genome rearrangements in the newly replicated region behind the stalled fork, which primarily consist of localized losses and duplications of DNA sequences. These genetic alterations arise through the aberrant repair of a single-stranded DNA gap, in a process that is dependent on Exo1- and Shu1-dependent homologous recombination repair (HRR). Furthermore, aberrant processing of HRR intermediates, and elevated HRR-associated mutagenesis, is detectable in a yeast model of the human cancer predisposition disorder, Bloom's syndrome. Our data reveal a mechanism by which cellular responses to stalled replication forks can actively generate genomic alterations and genetic diversity in normal proliferating cells.

Spontaneous replication fork collapse regulates telomere length homeostasis in wild type cells

“…additionally, this high rate of fork collapse is partly suppressed when EXO1 is deleted, consistent with the previously observed stabilization of stalled replication forks in the absence of ExoI (53)(54)(55)(56). We therefore suggest that the essential function of the t-RPA complex is to promote replication of duplex telomeric DNA, rather than to protect termini from resection.…”

Tools To Live By: Bacterial DNA Structures Illuminate Cancer

“…Moreover, the role of E. coli RecQ in prevention of reversed-fork HJs may be shared by the budding yeast RecQ homolog Sgs1. 2D-gel electrophoresis of DNAfrom sgs1-deficient cells showed an increase in X-DNA structures near an engineered replication-fork barrier, supporting a role for Sgs1 in reducing reversed forks [37].…”

Esc2 promotes telomere stability in response to DNA replication stress

“…Previously, we reported that the his2 :: URA3 -14xTus/ Ter cassette could trigger distinct types of localized mutations in URA3 (which confer resistance to 5-fluoro-orotic acid; 5-FOA) and that the Tus/ Ter -induced URA3 mutation rate was elevated ∼7-fold when the SGS1 gene was deleted (15). We therefore compared URA3 mutation rates for the his2 (non-telomeric region) and TEL06R Tus/ Ter barriers in wild-type (WT) and sgs1 mutants.…”

“…To verify that the TEL06R phenotypes were due to replication fork stalling at Tus/ Ter , we compared the effects of reversing the orientation of the 21x Ter module such that the Tus/ Ter barrier, which we demonstrated previously to act in a polar manner (15), was in the ‘permissive’ (non-arresting) configuration. In this scenario, there was no detectable increase in 5-FOA resistant colonies (Supplementary Figure S1A and B), confirming that the Tus-dependent URA3 mutagenesis is due to replication fork stalling.…”

“…This replication fork barrier can be co-engineered with a URA3 genetic reporter that allows detection of localized mutations and genome rearrangements that arise after the Tus/ Ter barrier is activated. Previously, we used the Tus/ Ter system to demonstrate that stalled replication forks can generate localized deletions and duplications at a non-telomeric locus (15). Furthermore, we demonstrated that Sgs1, a member of the evolutionarily conserved RecQ helicase family, counteracts the generation of these genome rearrangements.…”