Rif1 is a global regulator of timing of replication origin firing in fission yeast

Abstract

One of the long-standing questions in eukaryotic DNA replication is the mechanisms that determine where and when a particular segment of the genome is replicated. Cdc7/Hsk1 is a conserved kinase required for initiation of DNA replication and may affect the site selection and timing of origin firing. We identified rif1D, a null mutant of rif1 + , a conserved telomere-binding factor, as an efficient bypass mutant of fission yeast hsk1. Extensive deregulation of dormant origins over a wide range of the chromosomes occurs in rif1D in the presence or absence of hydroxyurea (HU). At the same time, many early-firing, efficient origins are suppressed or delayed in firing timing in rif1D. Rif1 binds not only to telomeres, but also to many specific locations on the arm segments that only partially overlap with the prereplicative complex assembly sites, although Rif1 tends to bind in the vicinity of the late/dormant origins activated in rif1D. The binding to the arm segments occurs through M to G1 phase in a manner independent of Taz1 and appears to be essential for the replication timing program during the normal cell cycle. Our data demonstrate that Rif1 is a critical determinant of the origin activation program on the fission yeast chromosomes.

Rif1 controls DNA replication by directing Protein Phosphatase 1 to reverse Cdc7-mediated phosphorylation of the MCM complex

“…Here we demonstrated that budding yeast Rif1 is involved in regulating DNA replication genome-wide, consistent with findings in fission yeast and mammalian cells and suggesting that DNA replication control by Rif1 is conserved through eukaryotes (Cornacchia et al 2012;Hayano et al 2012;Yamazaki et al 2012). Rif1 restrains replication by counteracting the action of DDK, and loss of Rif1 partially compensates for impaired DDK function in a cdc7-1 mutant strain.…”

“…Previous investigations showed that deleting S. pombe rif1 + suppresses mutations in DDK and that S. pombe and mammalian Rif1 affect genome-wide replication timing (Cornacchia et al 2012;Hayano et al 2012;Yamazaki et al 2012). A previous study of the S. cerevisiae rif1D replication program showed a shortened interval between replication of early and late sequences, with telomeric origins in particular replicating aberrantly early (Lian et al 2011).…”

“…The effect of S. pombe Rif1 on origins is related to the proximity of chromosomal binding sites (Hayano et al 2012), but intranuclear spatial localization may also be important (Yamazaki et al 2012). S. cerevisiae Rif1 is reported to be localized at the nuclear periphery through association with peripherally localized telomere clusters (Gotta et al 1996;Hiraga et al 2008) and by palmitoylationmediated nuclear envelope anchoring (Park et al 2011).…”

“…Deleting the S. pombe rif1 + gene suppresses the block to replication caused by mutations in DDK subunits (Hayano et al 2012). Removal of S. pombe or mammalian Rif1 also derails the replication temporal program, affecting the order in which origins initiate replication (Cornacchia et al 2012;Hayano et al 2012;Yamazaki et al 2012). Rif1 binds most strongly to telomeric regions within the fission yeast genome but can also be detected at hundreds of internal sites.…”

“…Recent studies have implicated the fission yeast (Schizosaccharomyces pombe) and mammalian Rif1 proteins in the regulation of DNA replication genome-wide. Deleting the S. pombe rif1 + gene suppresses the block to replication caused by mutations in DDK subunits (Hayano et al 2012). Removal of S. pombe or mammalian Rif1 also derails the replication temporal program, affecting the order in which origins initiate replication (Cornacchia et al 2012;Hayano et al 2012;Yamazaki et al 2012).…”