A Proteomic Characterization of Factors Enriched at Nascent DNA Molecules

López-Contreras, Andrés J.; Ruppen, Isabel; Nieto-Soler, Maria; Murga, Matilde; Rodríguez‐Acebes, Sara; Remeseiro, Silvia; Rodrigo-Perez, Sara; Rojas, Ana M.; Méndez, Juan Pablo; Muñoz, Javier; Fernández-Capetillo, Óscar

doi:10.1016/j.celrep.2013.03.009

Cited by 116 publications

(115 citation statements)

References 62 publications

(75 reference statements)

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“…In vitro studies demonstrated that hCtf4 stimulated reactions catalyzed by Pol α and Pol e and weakly interacted with these Pols (19). Furthermore, recent studies on the identification of replisome proteins revealed the association of hCtf4 with nascent DNA chains (26).…”

Section: Discussionmentioning

confidence: 99%

Interaction between human Ctf4 and the Cdc45/Mcm2-7/GINS (CMG) replicative helicase

Kang

Farina

Bermudez

et al. 2013

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

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Chromosome transmission fidelity 4 (Ctf4) is a conserved protein required for DNA replication. In this report, interactions between human Ctf4 (hCtf4) and the replicative helicase containing the cell division cycle 45 (Cdc45)/minichromosome maintenance 2-7 (Mcm2-7)/Go, Ichi, Nii, and San (GINS) (CMG) proteins [human CMG (hCMG) complex] were examined. The hCtf4-CMG complex was isolated following in vitro interaction of purified proteins (hCtf4 plus the hCMG complex), coinfection of Spodoptera frugiperda (Sf9) insect cells with viruses expressing the hCMG complex and hCtf4, and from HeLa cell chromatin after benzonase and immunoprecipitation steps. The stability of the hCtf4-CMG complex depends upon interactions between hCtf4 and multiple components of the hCMG complex. The hCtf4-CMG complex, like the hCMG complex, contains DNA helicase activity that is more salt-resistant than the helicase activity of the hCMG complex. We demonstrate that the hCtf4-CMG complex contains a homodimeric hCtf4 and a monomeric hCMG complex and suggest that the homodimeric hCtf4 acts as a platform linking polymerase α to the hCMG complex. The role of the hCMG complex as the core of the replisome is also discussed.replisome core structure | dimerization | replication initiation E ukaryotic DNA replication is a stepwise process by which protein complexes are assembled on chromatin. During the G1 phase of the cell cycle, the origin recognition complex (ORC) binds to replication origins and recruits cell division cycle 6 (Cdc6) (1). This complex leads to the association of cdc10 dependent transcript 1 (Cdt1)/Mcm2-7 with chromatin and the loading of the Mcm2-7 complex as a head-to-head dimer (2, 3). At the G1/S transition, this prereplication complex is altered further by a number of replication initiation factors whose actions are facilitated by the cyclin-dependent and cell division cycle 7 (Cdc7)-dumbbell former 4 (Dbf4) kinases (4). These replication initiation factors [which include synthetically lethal with dpb11-1 (Sld)2, Sld3, Sld7, DNA polymerase B possible subunit 11 (Dpb11), and DNA polymerase e (Pol e) in budding yeast] play critical roles resulting in the interaction of Cdc45 and GINS with the Mcm2-7 complex and the formation of the replicative DNA helicase complex containing Cdc45/Mcm2-7/GINS (CMG) (5). Other replication factors (including Mcm10 and Ctf4) contribute to the activation of the CMG helicase and association of proteins that recruit the replicative Pols to effect DNA replication (6, 7). Interactions between TopBP1-interacting, replication-stimulating protein (Treslin)

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Section: Discussionmentioning

confidence: 99%

Interaction between human Ctf4 and the Cdc45/Mcm2-7/GINS (CMG) replicative helicase

Kang

Farina

Bermudez

et al. 2013

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

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“…By labeling cells with EdU at 3.5 h after infection, we took advantage of the dramatic decrease in EdU incorporation into nuclear DNA to preferentially label viral DNA. With some modifications that further enriched viral DNA, the protocol closely followed that previously used (63)(64)(65). We chose iPOND over the alternative AniPOND procedure (83) because formaldehyde cross-linking allows stringent washing of the beads with SDS.…”

Section: Discussionmentioning

confidence: 99%

“…To address gaps in knowledge of the VACV DNA replication and transcription machinery, we adapted recently developed click chemistry methods for visualizing newly synthesized chromosomal DNA (61,62) and for isolating and identifying protein components of nuclear eukaryotic (63)(64)(65) and herpesvirus (66,67) replisomes. The method is based on the in vivo incorporation of ethynyl deoxynucleoside analogs, such as 5-ethynyl-2=-deoxyuridine (EdU), into replicating DNA.…”

mentioning

confidence: 99%

Identification of Vaccinia Virus Replisome and Transcriptome Proteins by Isolation of Proteins on Nascent DNA Coupled with Mass Spectrometry

Senkevich

Katsafanas

Weisberg

et al. 2017

J Virol

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Poxviruses replicate within the cytoplasm and encode proteins for DNA and mRNA synthesis. To investigate poxvirus replication and transcription from a new perspective, we incorporated 5-ethynyl-2=-deoxyuridine (EdU) into nascent DNA in cells infected with vaccinia virus (VACV). The EdU-labeled DNA was conjugated to fluor-or biotin-azide and visualized by confocal, superresolution, and transmission electron microscopy. Nuclear labeling decreased dramatically after infection, accompanied by intense labeling of cytoplasmic foci. The nascent DNA colocalized with the VACV single-stranded DNA binding protein I3 in multiple puncta throughout the interior of factories, which were surrounded by endoplasmic reticulum. Complexes containing EdU-biotin-labeled DNA cross-linked to proteins were captured on streptavidin beads. After elution and proteolysis, the peptides were analyzed by mass spectrometry to identify proteins associated with nascent DNA. The known viral replication proteins, a telomere binding protein, and a protein kinase were associated with nascent DNA, as were the DNA-dependent RNA polymerase and intermediate-and late-stage transcription initiation and elongation factors, plus the capping and methylating enzymes. These results suggested that the replicating pool of DNA is transcribed and that few if any additional viral proteins directly engaged in replication and transcription remain to be discovered. Among the host proteins identified by mass spectrometry, topoisomerases II␣ and II␤ and PCNA were noteworthy. The association of the topoisomerases with nascent DNA was dependent on expression of the viral DNA ligase, in accord with previous proteomic studies. Further investigations are needed to determine possible roles for PCNA and other host proteins detected. IMPORTANCE Poxviruses, unlike many well-characterized animal DNA viruses, replicate entirely within the cytoplasm of animal cells, raising questions regarding the relative roles of viral and host proteins. We adapted newly developed procedures for click chemistry and iPOND (Isolation of proteins on nascent DNA) to investigate vaccinia virus (VACV), the prototype poxvirus. Nuclear DNA synthesis ceased almost immediately following VACV infection, followed swiftly by the synthesis of viral DNA within discrete cytoplasmic foci. All viral proteins known from genetic and proteomic studies to be required for poxvirus DNA replication were identified in the complexes containing nascent DNA. The additional detection of the viral DNAdependent RNA polymerase and intermediate and late transcription factors provided evidence for a temporal coupling of replication and transcription. Further studies are needed to assess the potential roles of host proteins, including topoisomerases II␣ and II␤ and PCNA, which were found associated with nascent DNA.

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“…To identify potential nucleases and helicases required for processing replication forks after 2 hours exposure to HU, we used siRNAs to knock down enzymes known to act on collapsed forks plus those identified at unperturbed and stalled replication forks through iPOND analysis [36][37][38]. Knockdown of EXO1, BLM, and CtIP reduced phospho-CHK1 levels in response to HU whereas MRE11 knockdown did not ( Figure 3A and Supplementary Figure 4A).…”

Section: Ddk Likely Promotes Fork Processing By Regulating the Activimentioning

confidence: 99%

DDK has a primary role in processing stalled replication forks to initiate downstream checkpoint signaling

Sasi

Coquel

Lin

et al. 2017

Preprint

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Abstract:CDC7-DBF4 kinase (DDK) is required to initiate DNA replication in eukaryotes by activating the replicative MCM helicase. DDK has also been reported to have diverse and sometimes conflicting roles in the replication checkpoint response in various organisms but the underlying mechanisms are far from settled. Here we show that human DDK promotes limited resection of newly synthesized DNA at stalled replication forks or sites of DNA damage to initiate replication checkpoint signaling. DDK is also required for efficient fork restart and G2/M cell cycle arrest. DDK exhibits genetic interactions with the ssDNA exonuclease EXO1, and we show that EXO1 is also required for nascent strand degradation following exposure to HU, raising the possibility that DDK regulates EXO1 directly. Thus, DDK has a primary and previously undescribed role in the replication checkpoint to promote ssDNA accumulation at stalled forks, which is required to initiate a robust checkpoint response and cell cycle arrest to maintain genome integrity.Keywords: DNA replication checkpoint/ replication fork/ fork resection/ fork restart/ DDK peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/207266 doi: bioRxiv preprint first posted online 3 DDK is a two-subunit kinase that is essential to initiate DNA replication at individual replication origins by phosphorylating and activating the MCM2-7 replicative helicase. The regulatory subunit DBF4 binds to CDC7 and is required for its kinase activity [1]. DDK also has roles in replication checkpoint signaling that are less well understood [1]. In budding yeast, DDK is a target of the checkpoint effector kinase Rad53 that is activated following replication stress [2]. Rad53-mediated phosphorylation of Dbf4 modestly reduces DDK activity [2] and also inhibits late origin firing [3,4], but there is also evidence that DDK is required for the complete activation of Rad53 kinase [5]. In fission yeast, DDK subunits Hsk1 and Dfp1 are phosphorylated upon HU treatment by the checkpoint kinase Cds1, orthologous to budding yeast Rad53 and mammalian CHK2 [6][7][8]. Deletion of either Cds1 or its activating protein Mrc1 partially rescues the temperature sensitivity of hsk1-1312 mutants suggesting that Cds1 (like Rad53) inhibits DDK activity [8,9]. Cds1 activation in response to HU, however, was reduced significantly in hsk1(ts) strains at restrictive temperature and the cell cycle arrest was also aberrant as seen by an increased population of 'cut' cells (indicative of mitosis without complete DNA replication) [8,10]. These paradoxical results in yeast could be explained by a negative feedback loop where DDK first helps to initiate replication checkpoint activation and then the checkpoint pathway subsequently alters DDK activity to inhibit late origin firing.Initial studies in human cells showed that the replication checkpoint inhibits DDK activity, presumably to inhibit origin firing [11,...

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A Proteomic Characterization of Factors Enriched at Nascent DNA Molecules

Cited by 116 publications

References 62 publications

Interaction between human Ctf4 and the Cdc45/Mcm2-7/GINS (CMG) replicative helicase

Interaction between human Ctf4 and the Cdc45/Mcm2-7/GINS (CMG) replicative helicase

Identification of Vaccinia Virus Replisome and Transcriptome Proteins by Isolation of Proteins on Nascent DNA Coupled with Mass Spectrometry

DDK has a primary role in processing stalled replication forks to initiate downstream checkpoint signaling

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