Activation Of intra-S Checkpoint Research Articles

REV3 promotes cellular tolerance to 5-fluorodeoxyuridine by activating translesion DNA synthesis and intra-S checkpoint.

The drug floxuridine (5-fluorodeoxyuridine, FUdR) is an active metabolite of 5-Fluorouracil (5-FU). It converts to 5-fluorodeoxyuridine monophosphate (FdUMP) and 5-fluorodeoxyuridine triphosphate (FdUTP), which on incorporation into the genome inhibits DNA replication. Additionally, it inhibits thymidylate synthase, causing dTMP shortage while increasing dUMP availability, which induces uracil incorporation into the genome. However, the mechanisms underlying cellular tolerance to FUdR are yet to be fully elucidated. In this study, we explored the mechanisms underlying cellular resistance to FUdR by screening for FUdR hypersensitive mutants from a collection of DT40 mutants deficient in each genomic maintenance system. We identified REV3, which is involved in translesion DNA synthesis (TLS), to be a critical factor in FUdR tolerance. Replication using a FUdR-damaged template was attenuated in REV3-/- cells, indicating that the TLS function of REV3 is required to maintain replication on the FUdR-damaged template. Notably, FUdR-exposed REV3-/- cells exhibited defective cell cycle arrest in the early S phase, suggesting that REV3 is involved in intra-S checkpoint activation. Furthermore, REV3-/- cells showed defects in Chk1 phosphorylation, which is required for checkpoint activation, but the survival of FUdR-exposed REV3-/- cells was further reduced by the inhibition of Chk1 or ATR. These data indicate that REV3 mediates DNA checkpoint activation at least through Chk1 phosphorylation, but this signal acts in parallel with ATR-Chk1 DNA damage checkpoint pathway. Collectively, we reveal a previously unappreciated role of REV3 in FUdR tolerance.

Persistent Acetylation of Histone H3 Lysine 56 Compromises the Activity of DNA Replication Origins.

In Saccharomyces cerevisiae, newly synthesized histones H3 are acetylated on lysine 56 (H3 K56ac) by the Rtt109 acetyltransferase prior to their deposition on nascent DNA behind replication forks. Two deacetylases of the sirtuin family, Hst3 and Hst4, remove H3 K56ac from chromatin after S phase. hst3Δ hst4Δ cells present constitutive H3 K56ac, which sensitizes cells to replicative stress via unclear mechanisms. A chemogenomic screen revealed that DBF4 heterozygosity sensitizes cells to NAM-induced inhibition of sirtuins. DBF4 and CDC7 encode subunits of the Dbf4-dependent kinase (DDK), which activates origins of DNA replication during S phase. We show that (i) cells harboring the dbf4-1 or cdc7-4 hypomorphic alleles are sensitized to NAM, and that (ii) the sirtuins Sir2, Hst1, Hst3, and Hst4 promote DNA replication in cdc7-4 cells. We further demonstrate that Rif1, an inhibitor of DDK-dependent activation of origins, causes DNA damage and replication defects in NAM-treated cells and hst3Δ hst4Δ mutants. cdc7-4 hst3Δ hst4Δ cells are shown to display delayed initiation of DNA replication, which is not due to intra-S checkpoint activation but requires Rtt109-dependent H3 K56ac. Our results suggest that constitutive H3 K56ac sensitizes cells to replicative stress in part by negatively influencing the activation of origins of DNA replication.

Warsaw breakage syndrome DDX11 helicase acts jointly with RAD17 in the repair of bulky lesions and replication through abasic sites

Warsaw breakage syndrome, a developmental disorder caused by mutations in the DDX11/ChlR1 helicase, shows cellular features of genome instability similar to Fanconi anemia (FA). Here we report that DDX11-deficient avian DT40 cells exhibit interstrand crosslink (ICL)-induced chromatid breakage, with DDX11 functioning as backup for the FA pathway in regard to ICL repair. Importantly, we establish that DDX11 acts jointly with the 9-1-1 checkpoint clamp and its loader, RAD17, primarily in a postreplicative fashion, to promote homologous recombination repair of bulky lesions, but is not required for intra-S checkpoint activation or efficient fork progression. Notably, we find that DDX11 also promotes diversification of the chicken Ig-variable gene, a process triggered by programmed abasic sites, by facilitating both hypermutation and homeologous recombination-mediated gene conversion. Altogether, our results uncover that DDX11 orchestrates jointly with 9-1-1 and its loader, RAD17, DNA damage tolerance at sites of bulky lesions, and endogenous abasic sites. These functions may explain the essential roles of DDX11 and its similarity with 9-1-1 during development.

H2B mono-ubiquitylation facilitates fork stalling and recovery during replication stress by coordinating Rad53 activation and chromatin assembly.

The influence of mono-ubiquitylation of histone H2B (H2Bub) on transcription via nucleosome reassembly has been widely documented. Recently, it has also been shown that H2Bub promotes recovery from replication stress; however, the underling molecular mechanism remains unclear. Here, we show that H2B ubiquitylation coordinates activation of the intra-S replication checkpoint and chromatin re-assembly, in order to limit fork progression and DNA damage in the presence of replication stress. In particular, we show that the absence of H2Bub affects replication dynamics (enhanced fork progression and reduced origin firing), leading to γH2A accumulation and increased hydroxyurea sensitivity. Further genetic analysis indicates a role for H2Bub in transducing Rad53 phosphorylation. Concomitantly, we found that a change in replication dynamics is not due to a change in dNTP level, but is mediated by reduced Rad53 activation and destabilization of the RecQ helicase Sgs1 at the fork. Furthermore, we demonstrate that H2Bub facilitates the dissociation of the histone chaperone Asf1 from Rad53, and nucleosome reassembly behind the fork is compromised in cells lacking H2Bub. Taken together, these results indicate that the regulation of H2B ubiquitylation is a key event in the maintenance of genome stability, through coordination of intra-S checkpoint activation, chromatin assembly and replication fork progression.

Cyclobutane Pyrimidine Dimer Density as a Predictive Biomarker of the Biological Effects of Ultraviolet Radiation in Normal Human Fibroblast

This study compared biological responses of normal human fibroblasts (NHF1) to three sources of ultraviolet radiation (UVR), emitting UVC wavelengths, UVB wavelengths, or a combination of UVA and UVB (solar simulator; emission spectrum, 94.3% UVA and 5.7% UVB). The endpoints measured were cytotoxicity, intra-S checkpoint activation, inhibition of DNA replication and mutagenicity. Results show that the magnitude of each response to the indicated radiation sources was best predicted by the density of DNA cyclobutane pyrimidine dimers (CPD). The density of 6-4 pyrimidine-pyrimidone photoproducts was highest in DNA from UVC-irradiated cells (14% of CPD) as compared to those exposed to UVB (11%) or UVA-UVB (7%). The solar simulator source, under the experimental conditions described here, did not induce the formation of 8-oxo-7,8-dihydroguanine in NHF1 above background levels. Taken together, these results suggest that CPD play a dominant role in DNA damage responses and highlight the importance of using endogenous biomarkers to compare and report biological effects induced by different sources of UVR.

The Intra-S Phase Checkpoint Targets Dna2 to Prevent Stalled Replication Forks from Reversing

When replication forks stall at damaged bases or upon nucleotide depletion, the intra-S phase checkpoint ensures they are stabilized and can restart. In intra-S checkpoint-deficient budding yeast, stalling forks collapse, and ∼10% form pathogenic chicken foot structures, contributing to incomplete replication and cell death (Lopes et al., 2001; Sogo et al., 2002; Tercero and Diffley, 2001). Using fission yeast, we report that the Cds1(Chk2) effector kinase targets Dna2 on S220 to regulate, both in vivo and in vitro, Dna2 association with stalled replication forks in chromatin. We demonstrate that Dna2-S220 phosphorylation and the nuclease activity of Dna2 are required to prevent fork reversal. Consistent with this, Dna2 can efficiently cleave obligate precursors of fork regression-regressed leading or lagging strands-on model replication forks. We propose that Dna2 cleavage of regressed nascent strands prevents fork reversal and thus stabilizes stalled forks to maintain genome stability during replication stress.

Mcm10 Plays a Role in Functioning of the Eukaryotic Replicative DNA Helicase, Cdc45-Mcm-GINS

Eukaryotic DNA replication is initiated at multiple origins of replication, where many replication proteins assemble under the control of the cell cycle [1]. A key process of replication initiation is to convert inactive Mcm2-7 to active Cdc45-Mcm-GINS (CMG) replicative helicase [2]. However, it is not known whether the CMG assembly would automatically activate its helicase activity and thus assemble the replisome. Mcm10 is an evolutionally conserved essential protein required for the initiation of replication [3, 4]. Although the roles of many proteins involved in the initiation are understood, the role of Mcm10 remains controversial [5-9]. To characterize Mcm10 in more detail, we constructed budding yeast cells bearing a degron-fused Mcm10 protein that can be efficiently degraded in response to auxin. In the absence of Mcm10, a stable CMG complex was assembled at origins. However, subsequent translocation of CMG, replication protein A loading to origins, and the intra-S checkpoint activation were severely diminished, suggesting that origin unwinding is defective. We also found that Mcm10 associates with origins during initiation in an S-cyclin-dependent kinase- and Cdc45-dependent manner. Thus, Mcm10 plays an essential role in functioning of the CMG replicative helicase independent of assembly of a stable CMG complex at origins.

MDC1 regulates intra-S-phase checkpoint by targeting NBS1 to DNA double-strand breaks

The product of the Nijmegen breakage syndrome gene (NBS1) plays crucial roles in DNA damage response through its association with many proteins, including MRE11 and RAD50. However, it remains to be determined exactly how NBS1 accumulates at or near DNA double-strand breaks. Here we report that MDC1 directly binds to NBS1 and targets NBS1 to the sites of DNA damage. The MDC1-NBS1 interaction occurs through a specific region (residues 200-420) of MDC1, which contains multiple consensus casein kinase 2 (CK2) phosphorylation sites. In addition, this interaction requires both the forkhead-associated (FHA) and tandem BRCA1 C-terminal (BRCT) domains of NBS1. Disruption of the MDC1-NBS1 interaction results in failure of NBS1 accumulation at DNA double-strand breaks and impairment of intra-S checkpoint activation. These studies provide important mechanistic insights as to how MDC1 regulates NBS1 and the intra-S-phase checkpoint in response to DNA damage.