Tolerance of Sir1p/origin recognition complex-dependent silencing for enhanced origin firing at HMRa.
The HMR-E silencer is a DNA element that directs the formation of silent chromatin at the HMRa locus in Saccharomyces cerevisiae. Sir1p is one of four Sir proteins required for silent chromatin formation at HMRa. Sir1p functions by binding the origin recognition complex (ORC), which binds to HMR-E, and recruiting the other Sir proteins (Sir2p to -4p). ORCs also bind to hundreds of nonsilencer positions distributed throughout the genome, marking them as replication origins, the sites for replication initiation. HMR-E also acts as a replication origin, but compared to many origins in the genome, it fires extremely inefficiently and late during S phase. One postulate to explain this observation is that ORC's role in origin firing is incompatible with its role in binding Sir1p and/or the formation of silent chromatin. Here we examined a mutant HMR-E silencer and fusions between robust replication origins and HMR-E for HMRa silencing, origin firing, and replication timing. Origin firing within HMRa and from the HMR-E silencer itself could be significantly enhanced, and the timing of HMRa replication during an otherwise normal S phase advanced, without a substantial reduction in SIR1-dependent silencing. However, although the robust origin/silencer fusions silenced HMRa quite well, they were measurably less effective than a comparable silencer containing HMR-E's native ORC binding site.
McConnell
Kristopher H
KH
Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health, 587 MSC, 1300 University Ave., Madison, WI 53706-1532. cfox@wisc.edu.
Müller
Philipp
P
Fox
Catherine A
CA
D001665:Binding Sites; D004261:DNA Replication / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D020868:Gene Silencing; D009154:Mutation; D051738:Origin Recognition Complex / Q000235:genetics / Q000378:metabolism*; D016196:S Phase / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*
C497214:ORC1 protein, S cerevisiae; D051738:Origin Recognition Complex; C084121:SIR1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae