The yeast checkpoint kinases Mec1 and Rad53 are required for genomic stability in the presence of replicative stress. When replication forks stall, the stable maintenance of replisome components requires the ATR kinase Mec1/Ddc2 and the RecQ helicase Sgs1. It was unclear whether either Mec1 or Sgs1 action requires the checkpoint effector kinase, Rad53. By combining sgs1⌬ with checkpoint-deficient alleles, we can now distinguish the role of Mec1 at stalled forks from that of Rad53. We show that the S-phase-specific mec1-100 allele, like the sgs1⌬ mutation, partially destabilizes DNA polymerases at stalled forks, yet combining the mec1-100 and sgs1⌬ mutations leads to complete disassociation of the replisome, loss of RPA, irreversible termination of nucleotide incorporation, and compromised recovery from hydroxyurea (HU) arrest. These events coincide with a dramatic increase in both spontaneous and HU-induced chromosomal rearrangements. Importantly, in sgs1⌬ cells, RPA levels at stalled forks do not change, although Ddc2 recruitment is compromised, explaining the partial Sgs1 and Mec1 interdependence. Loss of Rad53 kinase, on the other hand, does not affect the levels of DNA polymerases at arrested forks, but leads to MCM protein dissociation. Finally, confirming its unique role during replicative stress, Mec1, and not Tel1, is shown to modify fork-associated histone H2A.[Keywords: Replicative stress; checkpoint; DNA polymerases; Mec1; Sgs1; chromosome instability] Supplemental material is available at http://www.genesdev.org. Intact S-phase checkpoint mechanisms are essential for cell survival and proliferation in the presence of DNA replicative stress, which can be caused by the stalling of replication forks at DNA lesions, at DNA-bound protein complexes (Ivessa et al. 2003), or as a result of reduced nucleotide levels induced by the addition of hydroxyurea (HU). Importantly, DNA replication defects and genomic instability are both hallmarks of oncogenic transformation. Indeed, cancer cells appear to persist in a state of perpetual replicative stress, which correlates with low but continuous signs of an activated DNA damage response, such as histone H2AX and CHK2 phosphorylation (Halazonetis 2004). In budding yeast, the ATR kinase homolog Mec1 and its downstream effector kinase Rad53, the hCHK2 homolog, are both central to the DNA damage checkpoint signaling cascade.A role for ATM-related kinases in the cellular response to replication fork stalling is conserved in all eukaryotes. The affinity of the mammalian ATRIP for replication protein A (RPA) suggests a model in which ATR-ATRIP is recruited to sites of damage or to abnormal structures generated at stalled replication forks that contain extended regions of RPA-bound single-stranded DNA (ssDNA) (Zou and Elledge 2003). Mec1 requires a cofactor Ddc2, the counterpart to mammalian ATRIP, and loss of either subunit abrogates the checkpoint-dependent phosphorylation of Rad53 and Pds1 proteins, precluding a checkpoint response (Paciotti et al. 2000). Once recruited,...
Previous experiments have identified a 657‐bp restriction fragment in the non‐transcribed region of the Drosophila histone gene cluster that is specifically associated with the histone‐depleted nuclear scaffold. The remaining fragments of the 5‐kb histone repeat were shown to be readily released from the scaffold; hence it was proposed that the tandemly repeated cluster of histone genes forms a series of 5‐kb loops restrained by a nuclear substructure at the sites of attachment. Here we show that the attachment fragment is tightly associated with protease‐sensitive material, whereas the solubilized fragments are relatively protein‐free. Exonuclease III digestion has been used to map the location of protein complexes on the attachment fragment. We have defined two regions of ∼200 bp whose borders provide kinetic barriers to exonuclease III degradation. They are separated by a nucleaseaccessible region of ∼100 bp. The protected regions are sufficient to mediate association of the fragment with the histonedepleted nuclei. Sequence analysis reveals an enrichment for sequences closely related to the topoisomerase II cleavage consensus in these two domains.
The decatenation activity of DNA topoisomerase II is essential for viability as eukaryotic cells traverse mitosis. Phosphorylation has been shown to stimulate topoisomerase II activity in vitro. Here we show that topoisomerase II is a phosphoprotein in yeast and that the level of incorporated phosphate is significantly higher at mitosis than in G1. Comparison of tryptic phosphopeptide maps reveals that the major phosphorylation sites in vivo are targets for casein kinase II. Incorporation of phosphate into topoisomerase II is nearly undetectable at the non‐permissive temperature in a conditional casein kinase II mutant. The sites modified by casein kinase II are located in the extreme C‐terminal domain of topoisomerase II. This domain is absent in prokaryotic and highly divergent among eukaryotic type II topoisomerases, and may serve to regulate functions of topoisomerase II that are unique to eukaryotic cells.
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