Cdc45 is limiting for replication initiation in humans

Köhler, Carsten; Koalick, Dennis; Fabricius, Anja; Parplys, Ann Christin; Borgmann, Kerstin; Pospiech, Helmut; Grosse, Frank

doi:10.1080/15384101.2016.1152424

Cited by 58 publications

(60 citation statements)

References 55 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Other reports suggest that checkpoint inhibition can cause replication catastrophe in a few hours, but those reports rely on cell lines that are sensitive to single-agent CHK1i (e.g. U2OS) (23,24,27,51). The U2OS cells are similar to AsPC-1 (Fig.…”

Section: Chk1 Inhibition and Replication Catastrophementioning

confidence: 99%

Inhibition of checkpoint kinase 1 following gemcitabine-mediated S phase arrest results in CDC7- and CDK2-dependent replication catastrophe

Warren

Eastman

2019

Journal of Biological Chemistry

View full text Add to dashboard Cite

Edited by Patrick SungCombining DNA-damaging drugs with DNA checkpoint inhibitors is an emerging strategy to manage cancer. Checkpoint kinase 1 inhibitors (CHK1is) sensitize most cancer cell lines to DNA-damaging drugs and also elicit single-agent cytotoxicity in 15% of cell lines. Consequently, combination therapy may be effective in a broader patient population. Here, we characterized the molecular mechanism of sensitization to gemcitabine by the CHK1i MK8776. Brief gemcitabine incubation irreversibly inhibited ribonucleotide reductase, depleting dNTPs, resulting in durable S phase arrest. Addition of CHK1i 18 h after gemcitabine elicited cell division cycle 7 (CDC7)-and cyclin-dependent kinase 2 (CDK2)-dependent reactivation of the replicative helicase, but did not reinitiate DNA synthesis due to continued lack of dNTPs. Helicase reactivation generated extensive singlestrand (ss)DNA that exceeded the protective capacity of the ssDNA-binding protein, replication protein A. The subsequent cleavage of unprotected ssDNA has been termed replication catastrophe. This mechanism did not occur with concurrent CHK1i plus gemcitabine treatment, providing support for delayed administration of CHK1i in patients. Alternative mechanisms of CHK1i-mediated sensitization to gemcitabine have been proposed, but their role was ruled out; these mechanisms include premature mitosis, inhibition of homologous recombination, and activation of double-strand break repair nuclease (MRE11). In contrast, single-agent activity of CHK1i was MRE11-dependent and was prevented by lower concentrations of a CDK2 inhibitor. Hence, both pathways require CDK2 but appear to depend on different CDK2 substrates. We conclude that a small-molecule inhibitor of CHK1 can elicit at least two distinct, context-dependent mechanisms of cytotoxicity in cancer cells. . 2 The abbreviations used are: CHK1i, checkpoint kinase 1 inhibitor; ssDNA, single-strand DNA; TBS, Tris-buffered saline; FBS, fetal bovine serum; HRP, horseradish peroxidase; RPA, replication protein A; ␥H2AX, pSer-139 histone 2AX; PCNA, proliferating cell nuclear antigen; CDK, cyclin-dependent kinase; ATR, ATM and Rad3-related; DAPI, 4Ј,6-diamidino-2-phenylindole.Figure 11. Proposed mechanism of replication catastrophe from gemcitabine plus delayed CHK1i. The normal firing of early replication origins involves CDC7-dependent phosphorylation of the MCM2-7 DNA helicase core and CDK2-dependent loading of the helicase co-factor, CDC45. Gemcitabine arrests replication by depleting dNTPs. Accumulation of RPA at stalled replication forks activates the DNA-damage response through ATR and CHK1. Over time, replication machinery dissociates from stalled replication forks, preventing their restart. CHK1 prevents firing of dormant origins. Inhibition of CHK1 activates CDK2 which, in concert with CDC7, leads to CDC45 loading and dormant origin firing. Active helicases unwind DNA, but in the continued absence of dNTPs, extensive single-strand DNA is produced that exceeds the ability of replication protein A to p...

show abstract

Section: Chk1 Inhibition and Replication Catastrophementioning

confidence: 99%

Inhibition of checkpoint kinase 1 following gemcitabine-mediated S phase arrest results in CDC7- and CDK2-dependent replication catastrophe

Warren

Eastman

2019

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…> 380 (track length) and > 600 (origin frequency) fibers obtained from 4 (track length) or 3 (origin frequency) independent experiments were measured for each condition. CDC45 excess provokes fork asymmetry (Srinivasan et al, 2013;Kohler et al, 2016) and Polg recruitment to replication foci (Kurashima et al, 2018); surplus CDC45 might therefore generate the obstacles that block the replisome in Chk1-depleted cells. Track lengths from > 290 fibers obtained from three independent experiments were measured for each condition.…”

Section: Of 16mentioning

confidence: 99%

“…The EMBO Journal 38: e101284 | 2019 ª 2019 The Authors whose increased loading onto chromatin following treatment with Chk1 and replication inhibitors triggers apoptosis in HCT116 cells (Syljuasen et al, 2005;Rodriguez et al, 2008;Zuazua-Villar et al, 2015). CDC45 excess provokes fork asymmetry (Srinivasan et al, 2013;Kohler et al, 2016) and Polg recruitment to replication foci (Kurashima et al, 2018); surplus CDC45 might therefore generate the obstacles that block the replisome in Chk1-depleted cells. In support of this hypothesis, chromatin-bound levels of CDC45 increased in Chk1-depleted cells, independently of roscovitine, which in fact functions downstream of replication barrier formation according to our model ( Fig 8A).…”

Section: Of 16mentioning

confidence: 99%

Chk1 loss creates replication barriers that compromise cell survival independently of excess origin firing

et al. 2019

View full text Add to dashboard Cite

The effectiveness of checkpoint kinase 1 (Chk1) inhibitors at killing cancer cells is considered to be fully dependent on their effect on DNA replication initiation. Chk1 inhibition boosts origin firing, presumably limiting the availability of nucleotides and in turn provoking the slowdown and subsequent collapse of forks, thus decreasing cell viability. Here we show that slow fork progression in Chk1‐inhibited cells is not an indirect effect of excess new origin firing. Instead, fork slowdown results from the accumulation of replication barriers, whose bypass is impeded by CDK‐dependent phosphorylation of the specialized DNA polymerase eta (Polη). Also in contrast to the linear model, the accumulation of DNA damage in Chk1‐deficient cells depends on origin density but is largely independent of fork speed. Notwithstanding this, origin dysregulation contributes only mildly to the poor proliferation rates of Chk1‐depleted cells. Moreover, elimination of replication barriers by downregulation of helicase components, but not their bypass by Polη, improves cell survival. Our results thus shed light on the molecular basis of the sensitivity of tumors to Chk1 inhibition.

show abstract

“…In fact, over-expression of these limiting factors results in too many origins firing synchronously during S phase, depletion of nucleotide pools and reduction in cell viability (Köhler et al, 2016; Mantiero et al, 2011; Toledo et al, 2013). As a result, initiation in all eukaryotes is extended over the course of S phase.…”

Section: A Bird’s Eye View Of Dna Replication In Eukaryotesmentioning

confidence: 99%

Replicating Large Genomes: Divide and Conquer

Rivera-Mulia

Gilbert

2016

Molecular Cell

View full text Add to dashboard Cite

Complete duplication of large metazoan chromosomes requires thousands of potential initiation sites, only a small fraction of which are selected in each cell. Assembly of the replication machinery is highly conserved and tightly regulated during cell cycle, but the sites of initiation are highly flexible their temporal order of firing is regulated at the level of large-scale multi-replicon domains. Additionally, the number of replication forks needs to be quickly adjusted in response to stress to ensure complete replication and prevent genome instability. Here we argue that large genomes are divided into domains for exactly this reason. Domain structure abrogates the need for precise initiation sites and creates a scaffold for the evolution of other chromosome functions.

show abstract

Cdc45 is limiting for replication initiation in humans

Cited by 58 publications

References 55 publications

Inhibition of checkpoint kinase 1 following gemcitabine-mediated S phase arrest results in CDC7- and CDK2-dependent replication catastrophe

Inhibition of checkpoint kinase 1 following gemcitabine-mediated S phase arrest results in CDC7- and CDK2-dependent replication catastrophe

Chk1 loss creates replication barriers that compromise cell survival independently of excess origin firing

Replicating Large Genomes: Divide and Conquer

Contact Info

Product

Resources

About