The level of origin firing inversely affects the rate of replication fork progression

Zhong, Yuan; Nellimoottil, Tittu Thomas; Peace, Jared M.; Knott, Simon; Villwock, Sandra K.; Yee, Janis; Jancuska, Jeffrey M.; Rege, Sanket; Tecklenburg, Marianne; Sclafani, Robert A.; Tavaré, Simon; Aparicio, Oscar M.

doi:10.1083/jcb.201208060

Cited by 96 publications

(107 citation statements)

References 42 publications

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“…Keeping in mind that fork arrest at Ter occurred in psf1-1 and WT cells but not in cdc7-1 at 37°C, the data suggested that there was no detectable correlation between the relative rate of fork progression and fork arrest at Ter. It should be noted that the psf1 and WT tracts are longer in the DNA combing profile because initiation was blocked in cdc7-1 and psf1-1 cells, and those that initiated, presumably because of less competition for existing initiation factors, cause somewhat faster fork movement (56). Key Role for DDK-Catalyzed Phosphorylation in PFA.…”

Section: Dna Combing Showed No Correspondence Between Replication Forkmentioning

confidence: 99%

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Phosphorylation of CMG helicase and Tof1 is required for programmed fork arrest

Bastia

Srivastava

Zaman

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Several important physiological transactions, including control of replicative life span (RLS), prevention of collision between replication and transcription, and cellular differentiation, require programmed replication fork arrest (PFA). However, a general mechanism of PFA has remained elusive. We previously showed that the Tof1–Csm3 fork protection complex is essential for PFA by antagonizing the Rrm3 helicase that displaces nonhistone protein barriers that impede fork progression. Here we show that mutations of Dbf4-dependent kinase (DDK) of Saccharomyces cerevisiae, but not other DNA replication factors, greatly reduced PFA at replication fork barriers in the spacer regions of the ribosomal DNA array. A key target of DDK is the mini chromosome maintenance (Mcm) 2–7 complex, which is known to require phosphorylation by DDK to form an active CMG [Cdc45 (cell division cycle gene 45), Mcm2–7, GINS (Go, Ichi, Ni, and San)] helicase. In vivo experiments showed that mutational inactivation of DDK caused release of Tof1 from the chromatin fractions. In vitro binding experiments confirmed that CMG and/or Mcm2–7 had to be phosphorylated for binding to phospho-Tof1–Csm3 but not to its dephosphorylated form. Suppressor mutations that bypass the requirement for Mcm2–7 phosphorylation by DDK restored PFA in the absence of the kinase. Retention of Tof1 in the chromatin fraction and PFA in vivo was promoted by the suppressor mcm5-bob1, which bypassed DDK requirement, indicating that under this condition a kinase other than DDK catalyzed the phosphorylation of Tof1. We propose that phosphorylation regulates the recruitment and retention of Tof1–Csm3 by the replisome and that this complex antagonizes the Rrm3 helicase, thereby promoting PFA, by preserving the integrity of the Fob1–Ter complex.

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Section: Dna Combing Showed No Correspondence Between Replication Forkmentioning

confidence: 99%

“…FACS analysis was performed on either a synchronous or asynchronous culture grown at 24°C or 37°C as described (56).…”

Section: Analysis Of Mcm4 Phosphorylationmentioning

confidence: 99%

Phosphorylation of CMG helicase and Tof1 is required for programmed fork arrest

Bastia

Srivastava

Zaman

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Loss of SIRT7 resulted in a significant reduction in DNA replication fork velocity in SirT7 −/− primary MEFs as compared with WT, which became exacerbated as cells were kept in culture (Fig 4H and I). Reduced replication rate often correlates with increased activation of dormant origins of replication, which is believe to be an attempt to rescue global replication rate after forks collapsed (Zhong et al , 2013). Consistently, we observed a remarkable increase in the presence of stalled replication forks and in the firing of new origins of replication upon replication block with HU in SirT7 −/− primary MEFs as compared with WT (Fig EV2A–C).…”

Section: Resultsmentioning

confidence: 99%

SIRT 7 promotes genome integrity and modulates non‐homologous end joining DNA repair

et al. 2016

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Sirtuins, a family of protein deacetylases, promote cellular homeostasis by mediating communication between cells and environment. The enzymatic activity of the mammalian sirtuin SIRT7 targets acetylated lysine in the N‐terminal tail of histone H3 (H3K18Ac), thus modulating chromatin structure and transcriptional competency. SIRT7 deletion is associated with reduced lifespan in mice through unknown mechanisms. Here, we show that SirT7‐knockout mice suffer from partial embryonic lethality and a progeroid‐like phenotype. Consistently, SIRT7‐deficient cells display increased replication stress and impaired DNA repair. SIRT7 is recruited in a PARP1‐dependent manner to sites of DNA damage, where it modulates H3K18Ac levels. H3K18Ac in turn affects recruitment of the damage response factor 53BP1 to DNA double‐strand breaks (DSBs), thereby influencing the efficiency of non‐homologous end joining (NHEJ). These results reveal a direct role for SIRT7 in DSB repair and establish a functional link between SIRT7‐mediated H3K18 deacetylation and the maintenance of genome integrity.

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“…It has been reported that the level of origin firing inversely affected the rate of fork progression (Zhong et al 2013) presumably because active forks compete for limiting dNTP pools, which is an important determinant of fork progression (Poli et al 2012). Since Mcm4 NSD, Sld3, and Dbf4 mutation increased the number of origins that were activated, it was possible that their effects on replication fork progression in HU were in part the consequence of competition for limiting dNTP pools or limiting DNA replication proteins.…”

Section: Discussionmentioning

confidence: 99%

Concerted activities of Mcm4, Sld3, and Dbf4 in control of origin activation and DNA replication fork progression

2016

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Eukaryotic chromosomes initiate DNA synthesis from multiple replication origins in a temporally specific manner during S phase. The replicative helicase Mcm2-7 functions in both initiation and fork progression and thus is an important target of regulation. Mcm4, a helicase subunit, possesses an unstructured regulatory domain that mediates control from multiple kinase signaling pathways, including the Dbf4-dependent Cdc7 kinase (DDK). Following replication stress in S phase, Dbf4 and Sld3, an initiation factor and essential target of Cyclin-Dependent Kinase (CDK), are targets of the checkpoint kinase Rad53 for inhibition of initiation from origins that have yet to be activated, so-called late origins. Here, whole-genome DNA replication profile analysis is used to access under various conditions the effect of mutations that alter the Mcm4 regulatory domain and the Rad53 targets, Sld3 and Dbf4. Late origin firing occurs under genotoxic stress when the controls on Mcm4, Sld3, and Dbf4 are simultaneously eliminated. The regulatory domain of Mcm4 plays an important role in the timing of late origin firing, both in an unperturbed S phase and in dNTP limitation. Furthermore, checkpoint control of Sld3 impacts fork progression under replication stress. This effect is parallel to the role of the Mcm4 regulatory domain in monitoring fork progression. Hypomorph mutations in sld3 are suppressed by a mcm4 regulatory domain mutation. Thus, in response to cellular conditions, the functions executed by Sld3, Dbf4, and the regulatory domain of Mcm4 intersect to control origin firing and replication fork progression, thereby ensuring genome stability.

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The level of origin firing inversely affects the rate of replication fork progression

Abstract: Cells with reduced origin firing have an increased rate of replication fork progression, whereas fork progression is slowed in cells with excess origins.

Cited by 96 publications

References 42 publications

Phosphorylation of CMG helicase and Tof1 is required for programmed fork arrest

Phosphorylation of CMG helicase and Tof1 is required for programmed fork arrest

SIRT 7 promotes genome integrity and modulates non‐homologous end joining DNA repair

Concerted activities of Mcm4, Sld3, and Dbf4 in control of origin activation and DNA replication fork progression

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