Regulation of DNA-replication origins during cell-cycle progression

Abstract

We have shown previously that chromosome VI of Saccharomyces cerevisiae contains nine origins of DNA replication that differ in initiation frequency and replicate sequentially during the S phase of the cell cycle. Here we show that there are links between activation of these multiple origins and regulation of S-phase progression. We study the effects of a DNA-damaging agent, methyl methane sulphonate (MMS), and of mutations in checkpoint genes such as rad53 on the activity of origins, measured by two-dimensional gel analysis, and on cell-cycle progression, measured by fluorescence-activated cell sorting. We find that when MMS slows down S-phase progression it also selectively blocks initiation from late origins. A rad53 mutation enhances late and/or inefficient origins and releases the initiation block by MMS. Mutation of rad53 also results in a late origin becoming early replicating. We conclude that rad53 regulates the timing of initiation of replication from late origins during normal cell growth and blocks initiation from late origins in MMS-treated cells. rad53 is, therefore, involved in the cell's surveillance of S-phase progression. We also find that orc2, which encodes subunit 2 of the origin-recognition complex, is involved in suppression of late origins.

The Yeast Ubiquitin Ligase SCFMet30Regulates Heavy Metal Response

“…Among other effects, the Mec1/ Rad53 pathway has been shown to down-regulate Cln2 expression in response to MMS exposure and consequently to delay S-phase initiation (Sidorova and Breeden, 1997). Furthermore, it has been demonstrated that Mec1-dependent activation of Rad53 inhibits firing of late replicating origins and thus to slow S-phase progression (Santocanale and Diffley, 1998;Shirahige et al, 1998). It is therefore feasible that activation of the Mec1/Rad53 pathway contributes to the delay in the G 1 /S transition and slow S-phase progression that we observed in response to cadmium exposure ( Figures 2B and 5, B and C).…”

ATM: A mediator of multiple responses to genotoxic stress

“…RAD53 might exert its control of S phase progression by blocking the ®ring from late replication origins (Shirahige et al, 1998;Santocanale and Di ey, 1998).…”

Suppression of replication fork progression in low-dose-specific p53-dependent S-phase DNA damage checkpoint

“…Suppression of late origin firing in response to DNA damage is dependent on Mec1 and Rad53 in yeast (Santocanale and Diffley, 1998;Shirahige et al, 1998), and on their respective homologues ATM and Chk2 in mammalian cells (Larner et al, 1999;Falck et al, 2001). Suppression of replication fork progression was demonstrated in a classic work of fiber autoradiography analysis of irradiated cells (Watanabe, 1974).…”