A redundancy of processes that cause replication fork stalling enhances recombination at two distinct sites in yeast rDNA

Abstract

DNA recombination was investigated by monitoring integration at the rDNA of a circular minichromosome containing a 35S minigene and a replication fork barrier (RFB). The effects of replication fork stalling on integration were studied in wild-type, FOB1 Delta, SIR2 Delta and the double mutant FOB1 Delta SIR2 Delta cells. The results obtained confirmed that Sir2p represses and replication fork stalling enhances integration of the minichromosome. This integration, however, only took place at two distinct sites: the RFB and the 3' end of the 35S gene. For integration to take place at the 35S gene, replication fork stalling must occur at the 3' end of the gene in both the minichromosome and the chromosomal repeats. Integration at the RFB, on the other hand, occurred readily in FOB1 Delta cells, indicating that more than a single mechanism triggers homologous recombination at this site. Altogether, these observations strongly suggest that the main role for replication fork stalling at the rDNA locus is to promote homologous recombination rather than just to prevent head-on collision of transcription and replication as originally thought.