S-phase checkpoint proteins Tof1 and Mrc1 form a stable replication-pausing complex

Abstract

The checkpoint regulatory mechanism has an important role in maintaining the integrity of the genome. This is particularly important in S phase of the cell cycle, when genomic DNA is most susceptible to various environmental hazards. When chemical agents damage DNA, activation of checkpoint signalling pathways results in a temporary cessation of DNA replication. A replication-pausing complex is believed to be created at the arrested forks to activate further checkpoint cascades, leading to repair of the damaged DNA. Thus, checkpoint factors are thought to act not only to arrest replication but also to maintain a stable replication complex at replication forks. However, the molecular mechanism coupling checkpoint regulation and replication arrest is unknown. Here we demonstrate that the checkpoint regulatory proteins Tof1 and Mrc1 interact directly with the DNA replication machinery in Saccharomyces cerevisiae. When hydroxyurea blocks chromosomal replication, this assembly forms a stable pausing structure that serves to anchor subsequent DNA repair events.

Rec8 Guides Canonical Spo11 Distribution along Yeast Meiotic Chromosomes

“…6His-3FLAG, 6His-3HA, or 6His-2myc tag was fused to the C terminus of the protein by using a cassette amplified from pU6H3FLAG, pU6H3HA, or pU6H2myc (De Antoni and Gallwitz, 2000;Katou et al, 2003). The rec8 null allele was introduced by polymerase chain reaction (PCR)-mediated gene disruption.…”

“…For mapping of DSB sites in rad50S mutants, cells at 7 h after meiotic induction were treated as described above but without formaldehyde treatment. The immunoprecipitated DNA and input DNA were amplified by random priming, fragmented, end labeled, and hybridized to two types of high-density oligonucleotide array (SC3456a520015F, P/N 520015 and rik-DACF, P/N 510636; Affymetrix, Santa Clara, CA) as described previously (Katou et al, 2003;Lengronne et al, 2004). These two chips cover the entire yeast chromosome VI (rikDACF), or chromosome III, IV, V, and the right arm of VI (SC3456a520015F), with a resolution of 300 base pairs and 100 base pairs (within 17-kb around YCR048W, chromosome III 198101 base pairs and 215100 base pairs, on SC3456a520015F), except for some repeat sequences (Ty LTR, telomeric region, rRNA and some other genes, indicated with white boxes in Supplemental Figure S1).…”

“…ChIP was performed as described previously (Katou et al, 2003), with some modifications. Nontagged strains were treated as a negative control.…”

“…In this study, we examined the distribution of Spo11 along meiotic chromosomes in wild-type budding yeast cells, and we compared it with that of Rec8, by using a high-resolution genome tiling array and chromatin immunoprecipitation (ChIP) assay (Katou et al, 2003;Lengronne et al, 2004). We demonstrate that Spo11 is first recruited to centromeric regions, and then relocalizes to arm regions, in concert with the progression of the premeiotic DNA replication.…”

Reconstruction of the Kinetochore during Meiosis in Fission Yeast Schizosaccharomyces pombe

“…Chromatin immunoprecipitation (ChIP) was performed as described previously (Jin et al, 2002, Katou et al, 2003. Cnl2-GFP-3HA cells were grown at 26°C to log phase and shifted to 18°C for 30 min.…”