Available online 30 March 2023, 103488

Author links open overlay panel, ABSTRACT

The genome must be monitored to ensure its duplication is completed accurately to prevent genome instability. In Saccharomyces cerevisiae, the 5’ to 3’ DNA helicase Rrm3, a member of the conserved PIF1 family, facilitates replication fork progression through an unknown mechanism. Disruption of Rrm3 helicase activity leads to increased replication fork pausing throughout the yeast genome. Here, we show that Rrm3 contributes to replication stress tolerance in the absence of the fork reversal activity of Rad5, defined by its HIRAN domain and DNA helicase activity, but not in the absence of Rad5’s ubiquitin ligase activity. The Rrm3 and Rad5 helicase activities also interact in the prevention of recombinogenic DNA lesions, and DNA lesions that accumulate in their absence need to be salvaged by a Rad59-dependent recombination pathway. Disruption of the structure-specific endonuclease Mus81 leads to accumulation of recombinogenic DNA lesions and chromosomal rearrangements in the absence of Rrm3, but not Rad5. Thus, at least two mechanisms exist to overcome fork stalling at replication barriers, defined by Rad5-mediated fork reversal and Mus81-mediated cleavage, and contribute to the maintenance of chromosome stability in the absence of Rrm3.

Section snippetsINTRODUCTION

DNA bound proteins and DNA structures need to be removed to facilitate progression of the replication fork and maintain genome stability. Thus, cells have developed mechanisms to deal with different kinds of barriers encountered during replication that restart blocked or collapsed replication forks and to repair or bypass damaged DNA. In Saccharomyces cerevisiae, the 5’ to 3’ DNA helicase Rrm3 ensures replication fork progression through nonhistone DNA-bound proteins. In the absence of Rrm3,

Yeast strains and media

Yeast strains were derived from S288C-derived strain KHSY802 (MATa, ura3-52, trp1Δ63, his3Δ200, leu2Δ1, lys2-Bgl, hom3-10, ade2Δ1, ade8, hxt13::URA3). RAD5 point mutations that disrupt the HIRAN domain (rad5-K194E) [20], ubiquitin-ligase activity (rad5-C914/917 A) [12], or helicase activity (rad5-Q1106D) [22] were introduced into plasmid pR5-28 [25] (gift from L. Prakash) by site-directed mutagenesis and confirmed by sequencing. These rad5 point mutations have been described and characterized

Integrity of the HIRAN and helicase domains of Rad5 is required for tolerance of replication stress in the absence of Rrm3

Deletion of RAD5 (rad5∆) causes hypersensitivity to DNA damage and, to a lesser extent, to replication stress, whereas deletion of RRM3 (rrm3∆) does not cause hypersensitivity to these agents [8], [12], [19]. Deleting RRM3 in the rad5∆ mutant (rad5∆ rrm3∆) leads to a further increase in sensitivity to replication stress induced by hydroxyurea (HU) (Fig. 1B). To determine which of the multiple Rad5 domains are required for replication stress tolerance in the rrm3∆ mutant we replaced the

DISCUSSION

The ATPase/helicase and HIRAN domains of Rad5 collaborate to reverse replication forks in vitro, with the ATPase/helicase thought to power leading strand unwinding to initiate four-way junction formation and the HIRAN domain capturing the 3’ OH at the nascent leading strand end to allow ATP-hydrolysis-driven branch migration of the four-way-junction, including reversal of the regressed fork back to a three-way junction [19], [20]. Although mapping to opposite ends of Rad5, the HIRAN and

Funding information

This work was supported by National Institutes of Health grant R01GM134396 to K.H.S.

Author contributions

JCM and KHS conceived the study. JCM performed experiments. JCM and KHS analyzed the data. JCM and KHS wrote the manuscript.

CRediT authorship contribution statement

Julius Muellner: Conceptualization, Investigation, Formal analysis, Validation, Visualization, Writing - original draft. Kristina Schmidt: Conceptualization, Formal analysis, Visualization, Resources, Supervision, Funding acquisition, Writing - review & editing.

Conflict of interest

The authors declare no conflict of interest.

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© 2023 Published by Elsevier B.V.