Replisome instability, fork collapse, and gross chromosomal rearrangements arise synergistically from Mec1 kinase and RecQ helicase mutations

Abstract

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,...

During Replication Stress, Non-Smc Element 5 (Nse5) Is Required for Smc5/6 Protein Complex Functionality at Stalled Forks

“…Chromatin Immunoprecipitation-ChIP/quantitative realtime PCR (qPCR) was performed as described previously (32)(33)(34). DNA quantification by real-time PCR was performed on an ABI 7900 sequence detector system at the Southern Alberta Cancer Research Institute.…”

“…One measure of replication correctness during replication stress is to monitor replisome association with stalled forks by ChIP. The recovered DNA from the ChIP was quantified by qPCR with primer pairs to two early-firing origins, ARS305 and ARS607, with late-firing ARS501 serving as a negative control (32)(33)(34). We determined the association of DNA polymerase ⑀ by monitoring Myc-Pol2 recovery with stalled forks when cells were released into S phase in the presence of HU at the indicated time points.…”

Dephosphorylation of γH2A by Glc7/Protein Phosphatase 1 Promotes Recovery from Inhibition of DNA Replication

“…Active Rad53 kinase molecules are then released from the complex and can phosphorylate downstream targets to arrest mitotic cell cycle progression. Mec1 activation is supported by independent loading onto DNA of the Ddc1-Rad17-Mec3 complex by Rad24-RFC, which enhances Mec1 ability to transmit and amplify the DNA damage signals (24).Mec1 and Tel1 also phosphorylate histone H2A on serine 129 (␥H2A) in response to DNA DSBs (12, 28, 30) and inhibition of DNA replication (7,41). Formation of ␥H2A is necessary for recruitment of DNA repair and chromatin remodeling factors to DSB sites and for efficient DSB repair (reviewed in references 1 and 37).…”

“…Mec1 and Tel1 also phosphorylate histone H2A on serine 129 (␥H2A) in response to DNA DSBs (12, 28, 30) and inhibition of DNA replication (7,41). Formation of ␥H2A is necessary for recruitment of DNA repair and chromatin remodeling factors to DSB sites and for efficient DSB repair (reviewed in references 1 and 37).…”

The checkpoint transcriptional response: Make sure to turn it off once you are satisfied

“…17,18,24,[29][30][31] Mec1 and Rad53-dependent signaling is crucial to mediate stabilization and repair of replication forks, inhibition of late origin firing, increased production of deoxyribonucleotides (dNTPs), among other responses. 1,9,10,14,28,[31][32][33][34][35][36][37][38] While the dramatic loss of viability observed in cells lacking Mec1 and/or Rad53 exposed to replication stress is related to translationindependent functions, the identification of multiple functional connections between Mec1/Rad53 signaling and transcription highlight the central importance of regulating transcription during replication stress. 30,31,39,40 …”