Abstract
<title>Abstract</title> <p>PolySUMOylation has emerged as an important regulator of genome stability beyond its well-established role in proteasomal targeting. Using Schizosaccharomyces pombe, we identify a role for SUMO chains in protecting ribosomal DNA (rDNA), a highly repetitive genomic region prone to replication stress. Cells lacking SUMO chain formation exhibit elevated spontaneous replication fork stalling at programmed rDNA barriers, instability of the rDNA array, and excessive recruitment of the homologous recombination factor Rad52. Mechanistically, polySUMOylation restrains Rad52 polyubiquitination and limits the accumulation of K63-linked polyubiquitination of Rad52, whereas fusion of an artificial SUMO chain suppresses both ubiquitination and aberrant Rad52 association with rDNA. Consistently, disruption of Ubc13-dependent K63 ubiquitination alleviates Rad52 accumulation at rDNA and partially suppresses defects associated with SUMO chain deficiency. Together, our findings identify polySUMOylation as a negative regulator of K63-linked ubiquitin signaling that protects fragile chromatin regions from replication-associated instability.</p>