DNA replication forks pause at silent origins near the HML locus in budding yeast

Chromosomal replicators in budding yeast contain an autonomously replicatin g sequence (ARS) that functions in a plasmid, but certain ARSs are silent a s replication origins in their natural chromosomal context, In chromosome m , the HML ARS cluster (ARs302-ARS303-ARS320) and ARS301 flank the transcrip tionally silent mating-type locus HML, and all of these ARSs are silent as replication origins. AFS301 and ARS302 function in transcriptional silencin g mediated by the origin recognition complex (ORC) and a heterochromatin st ructure, while the functions of ARS303 and ARS320 are not known. In this wo rk, we discovered replication fork pause sites at the HML ARS cluster and A RS301 by analyzing DNA replication intermediates from the chromosome via tw o-dimensional gel electrophoresis. The replication fork pause at the HML AR S cluster was independent of cis- and trans-acting mutations that abrogate transcriptional silencing at HML, Deletion of the HIML ARS cluster led to l oss of the pause site. Insertion of a single, heterologous ARS (ARS305) in place of the HML ARS cluster reconstituted the pause site, as did multiple copies of DNA elements (A and B1) that bind ORC. The orc2-1 mutation, known to alter replication timing at origins, did not detectably affect the paus e but activated the silent origin at the HML ARS cluster in a minority of c ells. Delaying the time of fork arrival at HML led to the elimination of th e pause sites at the HIML ARS cluster and at the copy of ARS305 inserted in place of the cluster. Loss of the pause sites was accompanied by activatio n of the silent origins in the majority of cells. Thus, replication fork mo vement near HML pauses at a silent origin which is competent for replicatio n initiation but kept silent through Orc2p, a component of the replication initiator. possible functions for replication fork pause sites in checkpoin ts, S-phase regulation, mating-type switching, and transcriptionally silent heterochromatin are discussed.