Successful and accurate completion of the replication of damage-containing DNA requires mainly recombination and RAD18-dependent DNA damage tolerance pathways. RAD18 governs at least two distinct mechanisms: translesion synthesis (TLS) and template switching (TS)-dependent pathways. Whereas TS is mainly error-free, TLS can work in an error-prone manner and, as such, the regulation of these pathways requires tight control to prevent DNA errors and potentially oncogenic transformation and tumorigenesis. In humans, the PCNA-associated recombination inhibitor (PARI) protein has recently been shown to inhibit homologous recombination (HR) events. Here, we describe a biochemical mechanism in which PARI functions as an HR regulator after replication fork stalling and during double-strand break repair. In our reconstituted biochemical system, we show that PARI inhibits DNA repair synthesis during recombination events in a PCNA interaction-dependent way but independently of its UvrD-like helicase domain. In accordance, we demonstrate that PARI inhibits HR in vivo, and its knockdown suppresses the UV sensitivity of RAD18-depleted cells. Our data reveal a novel human regulatory mechanism that limits the extent of HR and represents a new potential target for anticancer therapy.

Biotech Research and Innovation Centre University of Copenhagen 2200 Copenhagen Denmark

Department of Biochemistry and Molecular Biology New York Medical College Valhalla 10595 NY USA

Department of Biology Masaryk University 625 00 Brno Czech Republic

Department of Biology Masaryk University 625 00 Brno Czech Republic National Centre for Biomolecular Research Masaryk University 625 00 Brno Czech Republic International Clinical Research Center Center for Biomolecular and Cellular Engineering St Anne's University Hospital Brno 656 91 Brno Czech Republic

Institute of Genetics Biological Research Center Hungarian Academy of Sciences 6726 Szeged Hungary

Institute of Genetics Biological Research Center Hungarian Academy of Sciences 6726 Szeged Hungary Department of Biology Masaryk University 625 00 Brno Czech Republic

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Pro-recombination Role of Srs2 Protein Requires SUMO (Small Ubiquitin-like Modifier) but Is Independent of PCNA (Proliferating Cell Nuclear Antigen) Interaction