Helicase Subunit Cdc45 Targets the Checkpoint Kinase Rad53 to Both Replication Initiation and Elongation Complexes after Fork Stalling

Can, Geylani; Kauerhof, A. Christine; Macak, Dominik; Zegerman, Philip

doi:10.1016/j.molcel.2018.11.025

Cited by 27 publications

(46 citation statements)

References 52 publications

(92 reference statements)

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“…Nonetheless, even in the absence of SML1, Rad53 mutants are exquisitely sensitive to chemical agents that damage DNA or stall DNA replication forks (Allen et al, 1994;Sanchez et al, 1999;Gunjan & Verreault, 2003). This sensitivity to DNA damaging agents has been primarily attributed to Rad53's role in stabilizing and restarting stalled replication forks (reviewed in detail here: Segurado & Tercero, 2009 (Can et al, 2018). Identification of additional key substrates and recruiting mechanisms will be necessary to deconstruct Rad53's functions in maintaining fork stability, which may involve a range of redundant phosphorylation events in more than one essential replisome protein.…”

Section: Of 21mentioning

confidence: 99%

“…However, precisely how DNA damage signaling maintains replication fork integrity is unknown and represents a fundamental knowledge gap in the field. Recent work has revealed that Rad53 phosphorylates the replicative helicase component Cdc45, which in turn recruits and stabilizes Rad53 at replication complexes (Can et al , ). Identification of additional key substrates and recruiting mechanisms will be necessary to deconstruct Rad53's functions in maintaining fork stability, which may involve a range of redundant phosphorylation events in more than one essential replisome protein.…”

Section: Introductionmentioning

confidence: 99%

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DNA damage kinase signaling: checkpoint and repair at 30 years

2019

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From bacteria to mammalian cells, damaged DNA is sensed and targeted by DNA repair pathways. In eukaryotes, kinases play a central role in coordinating the DNA damage response. DNA damage signaling kinases were identified over two decades ago and linked to the cell cycle checkpoint concept proposed by Weinert and Hartwell in 1988. Connections between the DNA damage signaling kinases and DNA repair were scant at first, and the initial perception was that the importance of these kinases for genome integrity was largely an indirect effect of their roles in checkpoints, DNA replication, and transcription. As more substrates of DNA damage signaling kinases were identified, it became clear that they directly regulate a wide range of DNA repair factors. Here, we review our current understanding of DNA damage signaling kinases, delineating the key substrates in budding yeast and humans. We trace the progress of the field in the last 30 years and discuss our current understanding of the major substrate regulatory mechanisms involved in checkpoint responses and DNA repair.

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Section: Of 21mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

DNA damage kinase signaling: checkpoint and repair at 30 years

2019

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“…Therefore, chromosome maintenance requires that DNA synthesis, chromosome cohesion establishment and the ability to activate the S phase checkpoint must occur simultaneously at the replication fork. Strikingly deletions of a single component of the replisome often can cause considerable defects in DNA synthesis, in chromosome cohesion (Borges et al ., 2013) and activation of the S phase checkpoint kinase Rad53 in response to fork stalling (Alcasabas et al ., 2001; Can et al ., 2019), thus highlighting how DNA synthesis is closely intertwined with other processes necessary for the maintenance of genome stability. The replicative factor Ctf18-Dcc1-Ctf8-Rfc2-5 complex (Ctf18-RFC) exemplifies these overlapping functions at forks.…”

Section: Introductionmentioning

confidence: 99%

A stable leading strand polymerase/clamp loader complex is required for normal and perturbed eukaryotic DNA replication

Stokes

Winczura

Song

et al. 2019

Preprint

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The eukaryotic replisome must faithfully replicate DNA and cope with replication fork blocks and stalling, while simultaneously promoting sister chromatid cohesion. Ctf18-RFC is an alternative PCNA loader that links all these processes together by an unknown mechanism. Here, we use integrative structural biology combined with yeast genetics and biochemistry to highlight the specific functions that Ctf18-RFC plays within the leading strand machinery via an interaction with the catalytic domain of DNA Pol ε. We show that a large and unusually flexible interface enables this interaction to occur constitutively throughout the cell cycle and regardless of whether forks are replicating or stalled. We reveal that, by being anchored to the leading strand polymerase, Ctf18-RFC can rapidly signal fork stalling to activate the S phase checkpoint. Moreover, we demonstrate that, independently of checkpoint signaling or chromosome cohesion, Ctf18-RFC functions in parallel to Chl1 and Mrc1 to protect replication forks and cell viability.

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“…ChIP-seq was performed as previously described (Can et al 2019). Antibodies for IP were anti-H2A (39945, Actif motif), IgG (AB27478, Abcam), or anti-γH2A (AB15083, Abcam) or anti-GFP (3h9, Chromotek).…”

Section: Chromatin Immunoprecipitation-sequencing (Chip-seq)mentioning

confidence: 99%

Checkpoint inhibition of origin firing prevents DNA topological stress

et al. 2019

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A universal feature of DNA damage and replication stress in eukaryotes is the activation of a checkpoint-kinase response. In S-phase, the checkpoint inhibits replication initiation, yet the function of this global block to origin firing remains unknown. To establish the physiological roles of this arm of the checkpoint, we analyzed separation of function mutants in the budding yeast Saccharomyces cerevisiae that allow global origin firing upon replication stress, despite an otherwise normal checkpoint response. Using genetic screens, we show that lack of the checkpoint-block to origin firing results in a dependence on pathways required for the resolution of topological problems. Failure to inhibit replication initiation indeed causes increased DNA catenation, resulting in DNA damage and chromosome loss. We further show that such topological stress is not only a consequence of a failed checkpoint response but also occurs in an unperturbed S-phase when too many origins fire simultaneously. Together we reveal that the role of limiting the number of replication initiation events is to prevent DNA topological problems, which may be relevant for the treatment of cancer with both topoisomerase and checkpoint inhibitors.

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Helicase Subunit Cdc45 Targets the Checkpoint Kinase Rad53 to Both Replication Initiation and Elongation Complexes after Fork Stalling

Cited by 27 publications

References 52 publications

DNA damage kinase signaling: checkpoint and repair at 30 years

DNA damage kinase signaling: checkpoint and repair at 30 years

A stable leading strand polymerase/clamp loader complex is required for normal and perturbed eukaryotic DNA replication

Checkpoint inhibition of origin firing prevents DNA topological stress

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