Gemma Regan-Mochrie1,2, Timothy Hoggard3, Nikhil Bhagwat4,5, Gerard Lynch1, Neil Hunter4,5, Dirk Remus1, Catherine A. Fox3 and Xiaolan Zhao1 1Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, New York 10065, USA; 2Gerstner Sloan Kettering Graduate School of Biomedical Sciences, Memorial Sloan Kettering Cancer Center, New York, New York 10065, USA; 3Department of Biomolecular Chemistry, School of Medicine and Public Health, University of Wisconsin, Madison, Wisconsin 53706, USA; 4Howard Hughes Medical Institute, University of California at Davis, Davis, California 95616, USA; 5Department of Microbiology and Molecular Genetics, University of California at Davis, Davis, California 95616, USA Corresponding author: zhaox1mskcc.org Abstract

Sumoylation is emerging as a posttranslation modification important for regulating chromosome duplication and stability. The origin recognition complex (ORC) that directs DNA replication initiation by loading the MCM replicative helicase onto origins is sumoylated in both yeast and human cells. However, the biological consequences of ORC sumoylation are unclear. Here we report the effects of hypersumoylation and hyposumoylation of yeast ORC on ORC activity and origin function using multiple approaches. ORC hypersumoylation preferentially reduced the function of a subset of early origins, while Orc2 hyposumoylation had an opposing effect. Mechanistically, ORC hypersumoylation reduced MCM loading in vitro and diminished MCM chromatin association in vivo. Either hypersumoylation or hyposumoylation of ORC resulted in genome instability and the dependence of yeast on other genome maintenance factors, providing evidence that appropriate ORC sumoylation levels are important for cell fitness. Thus, yeast ORC sumoylation status must be properly controlled to achieve optimal origin function across the genome and genome stability.

Received April 4, 2022. Accepted July 14, 2022.