Rad27/FEN1 prevents accumulation of unprocessed Okazaki fragments and ribosomal DNA copy number changes

DNA copy number changes are the most frequent genomic alterations in cancer cells. The ribosomal DNA (rDNA) region is particularly vulnerable to such changes due to its repetitive nature. Here, we demonstrate that Rad27/FEN-1, a structure-specific nuclease in budding yeast, plays a crucial role in maintaining rDNA stability. The production of extrachromosomal rDNA circles and severe chromosomal rDNA instability are observed in the rad27 Δ mutant, independently of Fob1-mediated DNA replication fork arrest and DNA double-strand break (DSB) formation in the rDNA. The rad27 Δ mutant accumulates unprocessed Okazaki fragments in the rDNA region, without inducing DSB formation. Similar rDNA instability is observed in DNA ligase ^Cdc9 -deficient cells. Furthermore, we show that Exonuclease 1 and PCNA can compensate for the loss of Rad27 function in the rDNA stabilization. These findings highlight the importance of proper Okazaki fragment processing in preventing non-DSB-induced rDNA copy number changes.