Coordinated Degradation of Replisome Components Ensures Genome Stability upon Replication Stress in the Absence of the Replication Fork Protection Complex

Roseaulin, Laura; Noguchi, Chiaki; Martı́nez, Esteban; Ziegler, Melissa A.; Toda, Takashi; Noguchi, Eishi

doi:10.1371/journal.pgen.1003213

Cited by 31 publications

(51 citation statements)

References 98 publications

(142 reference statements)

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“…Mrc1 also functions in coordination of polymerase and helicase functions at the fork 51 . As in the case of Dia2, we found that Pof3 is involved in Mrc1 degradation 26 . It is widely known that SCF-type ubiquitin ligases recognize phosphodegrons 39 .…”

Section: Regulation Of Mrc1 Degradation By Scfpof3 and Phosphorylationsupporting

confidence: 61%

“…pof3 ∆ cells also displayed a cell elongation phenotype, which is normally caused by accumulation of DNA damage or replication failure. Consistently, pof3 ∆ cells showed a delay in replication recovery after a low-dose CPT treatment, which did not affect replication recovery in wild-type cells 26 . Interestingly, Pol2 overexpression is deleterious to the cells (data not shown).…”

Section: Role Of Scfpof3-dependent Pol2 Degradation In Faithful Dna Rmentioning

confidence: 60%

“…Strikingly, major replicative DNA polymerases (Pol2 and Pol3) and MCM (minichromosome maintenance) helicase subunits (Mcm2/4/6) underwent degradation in swi1 ∆ cells, when cells were treated with cycloheximide, which inhibits new protein synthesis. In contrast, PCNA and Orc1, which are also essential for DNA replication, were stable 26 . These results suggest a role for Swi1 in preventing rapid degradation of replisome components.…”

Section: Role Of the Fpc In Replisome Stabilizationmentioning

confidence: 82%

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Proteasome-dependent degradation of replisome components regulates faithful DNA replication

Roseaulin

Noguchi²,

Noguchi³

2013

Cell Cycle

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The replication machinery, or the replisome, collides with a variety of obstacles during the normal process of DNA replication. In addition to damaged template DNA, numerous chromosome regions are considered to be difficult to replicate owing to the presence of DNA secondary structures and DNA-binding proteins. Under these conditions, the replication fork stalls, generating replication stress. Stalled forks are prone to collapse, posing serious threats to genomic integrity. It is generally thought that the replication checkpoint functions to stabilize the replisome and replication fork structure upon replication stress. This is important in order to allow DNA replication to resume once the problem is solved. However, our recent studies demonstrated that some replisome components undergo proteasome-dependent degradation during DNA replication in the fission yeast Schizosaccharomyces pombe. Our investigation has revealed the involvement of the SCFPof3 (Skp1-Cullin/Cdc53-F-box) ubiquitin ligase in replisome regulation. We also demonstrated that forced accumulation of the replisome components leads to abnormal DNA replication upon replication stress. Here we review these findings and present additional data indicating the importance of replisome degradation for DNA replication. Our studies suggest that cells activate an alternative pathway to degrade replisome components in order to preserve genomic integrity.

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Section: Regulation Of Mrc1 Degradation By Scfpof3 and Phosphorylationsupporting

confidence: 61%

Section: Role Of Scfpof3-dependent Pol2 Degradation In Faithful Dna Rmentioning

confidence: 60%

Section: Role Of the Fpc In Replisome Stabilizationmentioning

confidence: 82%

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Proteasome-dependent degradation of replisome components regulates faithful DNA replication

Roseaulin

Noguchi²,

Noguchi³

2013

Cell Cycle

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“…Indeed, a more recent study has identified additional novel pathways that lead to replication factor degradation in response to replisome instability (Roseaulin et al 2013). These effects on replisome integrity suggest that replication fork collapse in cells deficient in the Mec1-Rad53 (S. cerevisiae), Rad3-Cds1 (S. pombe), or ATR-CHK1 (metazoans) pathways may be caused initially by the targeted disengagement of replisome components.…”

Section: Failure To Restart Replication In Atr-deficient Cells Coincimentioning

confidence: 99%

RNF4 and PLK1 are required for replication fork collapse in ATR-deficient cells

et al. 2013

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The ATR-CHK1 axis stabilizes stalled replication forks and prevents their collapse into DNA double-strand breaks (DSBs). Here, we show that fork collapse in Atr-deleted cells is mediated through the combined effects the sumo targeted E3-ubiquitin ligase RNF4 and activation of the AURKA-PLK1 pathway. As indicated previously, Atrdeleted cells exhibited a decreased ability to restart DNA replication following fork stalling in comparison with control cells. However, suppression of RNF4, AURKA, or PLK1 returned the reinitiation of replication in Atrdeleted cells to near wild-type levels. In RNF4-depleted cells, this rescue directly correlated with the persistence of sumoylation of chromatin-bound factors. Notably, RNF4 repression substantially suppressed the accumulation of DSBs in ATR-deficient cells, and this decrease in breaks was enhanced by concomitant inhibition of PLK1. DSBs resulting from ATR inhibition were also observed to be dependent on the endonuclease scaffold protein SLX4, suggesting that RNF4 and PLK1 either help activate the SLX4 complex or make DNA replication fork structures accessible for subsequent SLX4-dependent cleavage. Thus, replication fork collapse following ATR inhibition is a multistep process that disrupts replisome function and permits cleavage of the replication fork.

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“…This method can be used to amplify specific chromosomal loci, in order to measure the quantity of DNA precipitated with the antibodies. Examples of this analysis are shown in our previous studies [37–39].

Mix the following (for 1 sample):

2 μL of template DNA (input or ChIP DNA) ( see Note 23 ).

5 μL of iQ SYBR Green Supermix.

0.3 μL of forward primer (10 μM) ( see Note 7 ).

0.3 μL of reverse primer (10 μM) ( see Note 7 ).

2.4 μL of H 2 O.

Perform PCR using the following program.

95 °C for 2 min.

95 °C for 10 s.

55–60 °C for 30 s.

72 °C for 30 s (perform SYBR Green detection).

Repeat above steps ( b )–( d ) for 39 cycles.

72 °C for 7 min.

(Optional) Melting curve analysis: 55–95 °C.

Obtain cycle threshold (Ct) values for each sample.

Calculate % precipitated DNA using ∆Ct values between WCE and ChIP samples using the following equations:

Calculate dilution factor.

…”

Section: Methodsmentioning

confidence: 99%

Chromatin Immunoprecipitation to Detect DNA Replication and Repair Factors

Gadaleta

Iwasaki

Noguchi

et al. 2015

Methods in Molecular Biology

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DNA replication is tightly coupled with DNA repair processes in order to preserve genomic integrity. During DNA replication, the replication fork encounters a variety of obstacles including DNA damage/adducts, secondary structures, and programmed fork-blocking sites, which are all difficult to replicate. The replication fork also collides with the transcription machinery, which shares the template DNA with the replisome complex. Under these conditions, replication forks stall, causing replication stress and/or fork collapse, ultimately leading to genomic instability. The mechanisms to overcome these replication problems remain elusive. Therefore, it is important to investigate how DNA repair and replication factors are recruited and coordinated at chromosomal regions that are difficult to replicate. In this chapter, we describe a chromatin immunoprecipitation method to locate proteins required for DNA repair during DNA replication in the fission yeast Schizosaccharomyces pombe. This method can also easily be adapted to study replisome components or chromatin-associated factors.

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Coordinated Degradation of Replisome Components Ensures Genome Stability upon Replication Stress in the Absence of the Replication Fork Protection Complex

Cited by 31 publications

References 98 publications

Proteasome-dependent degradation of replisome components regulates faithful DNA replication

Proteasome-dependent degradation of replisome components regulates faithful DNA replication

RNF4 and PLK1 are required for replication fork collapse in ATR-deficient cells

Chromatin Immunoprecipitation to Detect DNA Replication and Repair Factors

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