DNA damage tolerance (DDT) and homologous recombination (HR) stabilize replication forks (RFs). RAD18/UBC13/three prime repair exonuclease 2 (TREX2)-mediated proliferating cell nuclear antigen (PCNA) ubiquitination is central to DDT, an error-prone lesion bypass pathway. RAD51 is the recombinase for HR. The RAD51 K133A mutation increased spontaneous mutations and stress-induced RF stalls and nascent strand degradation. Here, we report in RAD51K133A cells that this phenotype is reduced by expressing a TREX2 H188A mutation that deletes its exonuclease activity. In RAD51K133A cells, knocking out RAD18 or overexpressing PCNA reduces spontaneous mutations, while expressing ubiquitination-incompetent PCNAK164R increases mutations, indicating DDT as causal. Deleting TREX2 in cells deficient for the RF maintenance proteins poly(ADP-ribose) polymerase 1 (PARP1) or FANCB increased nascent strand degradation that was rescued by TREX2H188A, implying that TREX2 prohibits degradation independent of catalytic activity. A possible explanation for this occurrence is that TREX2H188A associates with UBC13 and ubiquitinates PCNA, suggesting a dual role for TREX2 in RF maintenance.

Department of Biology Masaryk University Kamenice 5 A7 625 00 Brno Czech Republic

Department of Genetics Albert Einstein College of Medicine Yeshiva University Bronx NY 10461 USA

Department of Molecular Medicine and Institute of Biotechnology University of Texas Health San Antonio San Antonio TX 78245 USA

Department of Molecular Medicine and Institute of Biotechnology University of Texas Health San Antonio San Antonio TX 78245 USA; Sam and Ann Barshop Institute for Longevity and Aging Studies University of Texas Health San Antonio San Antonio TX 78245 USA

Department of Molecular Medicine and Institute of Biotechnology University of Texas Health San Antonio San Antonio TX 78245 USA; The Cancer Therapy and Research Center University of Texas Health San Antonio San Antonio TX 78229 USA; Sam and Ann Barshop Institute for Longevity and Aging Studies University of Texas Health San Antonio San Antonio TX 78245 USA

International Clinical Research Center Center for Biomolecular and Cellular Engineering St Anne's University Hospital Brno Pekarska 53 656 91 Brno Czech Republic

National Centre for Biomolecular Research Masaryk University Kamenice 5 A7 625 00 Brno Czech Republic; Department of Biology Masaryk University Kamenice 5 A7 625 00 Brno Czech Republic; International Clinical Research Center Center for Biomolecular and Cellular Engineering St Anne's University Hospital Brno Pekarska 53 656 91 Brno Czech Republic

National Centre for Biomolecular Research Masaryk University Kamenice 5 A7 625 00 Brno Czech Republic; International Clinical Research Center Center for Biomolecular and Cellular Engineering St Anne's University Hospital Brno Pekarska 53 656 91 Brno Czech Republic

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RAD51 separation of function mutation disables replication fork maintenance but preserves DSB repair