Identification of Proteins at Active, Stalled, and Collapsed Replication Forks Using Isolation of Proteins on Nascent DNA (iPOND) Coupled with Mass Spectrometry

Sirbu, Bianca M.; McDonald, W. Hayes; Dungrawala, Huzefa; Badu-Nkansah, Akosua; Kavanaugh, Gina M.; Chen, Yaoyi; Tabb, David L.; Chen, David

doi:10.1074/jbc.m113.511337

Cited by 227 publications

(254 citation statements)

References 47 publications

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“…The two important concerns were sensitivity and nonspecific binding (103). The recovery of TOP2A and TOP2B associated with nascent DNA from cells infected with wild-type virus but not from cells infected with the VACV DNA ligase mutant supports findings from the Evans laboratory (37).…”

Section: Discussionsupporting

confidence: 62%

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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Section: Discussionsupporting

confidence: 62%

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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“…Indeed, γH2AX was localized at stalled replication forks at early time points preceding the appearance of DSBs by iPOND. 30 Recent work has implicated RAD51, a protein involved in DNA repair by homologous recombination, in the stabilization of stalled forks and protection from MRE11-mediated cleavage of the nascent DNA strand contributing to the restoration of a functional replisome. 31 Therefore we analyzed the formation of RAD51-positive foci in APH-treated cells.…”

Section: Resultsmentioning

confidence: 99%

The human oncoprotein and chromatin architectural factor DEK counteracts DNA replication stress

et al. 2014

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DNA replication stress is a major source of DNA strand breaks and genomic instability, and a hallmark of precancerous lesions. In these hyperproliferative tissues, activation of the DNA damage response results in apoptosis or senescence preventing or delaying their development to full malignancy. In cells, in which this antitumor barrier is disabled by mutations (for example, in p53), viability and further uncontrolled proliferation depend on factors that help to cope with replication-associated DNA damage. Replication problems preferentially arise in chromatin regions harboring complex DNA structures. DEK is a unique chromatin architectural factor which binds to non-B-form DNA structures, such as cruciform DNA or four-way junctions. It regulates DNA topology and chromatin organization, and is essential for the maintenance of heterochromatin integrity. Since its isolation as part of an oncogenic fusion in a subtype of AML, DEK has been consistently associated with tumor progression and chemoresistance. How DEK promotes cancer, however, is poorly understood. Here we show that DEK facilitates cellular proliferation under conditions of DNA replication stress by promoting replication fork progression. DEK also protects from the transmission of DNA damage to the daughter cell generation. We propose that DEK counteracts replication stress and ensures proliferative advantage by resolving problematic DNA and/or chromatin structures at the replication fork. INTRODUC110NDEK is a biochemically and structurally unique non-histone chromatin protein which is conserved in all higher eukaryotes. It binds to non-8-form DNA structures, such as cruciform DNA or four-way junctions, regulates DNA topology and chromatin organization, and is essential for the maintenance of heterochromatin integrity. •2 At the cellular level, it displays pleiotropic functions and has been shown to influence differentiation, apoptosis, senescence and maintenance of cell stemness. -6Since its isolation as part of an oncogenic fusion in a subtype of acute myeloid leukemia, 7 DEK has been consistently associated with tumor progression and chemoresistance.s-10 Several lines of evidence, such as DEK-dependent formation of papilloma in a mouse model of skin cancerogenesis, 10 have led to its classification as a bona fide oncogene. In melanomas, high levels of DEK expression correlate positively with metastatic potential. chemoresistance and poor treatment outcome. Significantly, DEK expression levels can distinguish benign nevi from malignant melanoma, raising the possibility of using DEK as a tumor marker. • 9We and others have shown that DEK is involved in DNA repair and the response to DNA damage: cells with downregulated DEK expression are hypersensitive to genotoxic insults and show increased susceptibility to sublethal doses of DNA damaging agents. •12 DNA repair is affected by DEK ablation, as DNA strand breaks (DSBs) are repaired less efficiently and nonhomologous end-joining appears to be defective. 12 · 13 DEK is also a substrate for covalent and n...

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“…26 In addition, using a method called iPOND (isolation of proteins on nascent DNA), the Cortez group recently discovered that FANCD2 and FANCI, in addition to ATR, MRE11 and other proteins, are highly enriched at stalled and collapsed replication forks following depletion of deoxyribonucleotide pools. 112 Interestingly, FANCI, and not FANCD2, was also shown to accumulate at active replication forks prior to fork staling, suggesting common and independent functions for these proteins. 112 In addition, the Vaziri group has recently shown that FANCD2 is necessary for efficient initiation of DNA replication in primary human fibroblasts.…”

Section: Fancd2 and Fanci Functionmentioning

confidence: 99%

“…112 Interestingly, FANCI, and not FANCD2, was also shown to accumulate at active replication forks prior to fork staling, suggesting common and independent functions for these proteins. 112 In addition, the Vaziri group has recently shown that FANCD2 is necessary for efficient initiation of DNA replication in primary human fibroblasts. 113 An unbiased proteomics screen of chromatinbound FANCD2 immune complexes revealed that FANCD2 physically associates with the minichromosome maintenance 2-7 (MCM2-MCM7) replicative helicases.…”

Section: Fancd2 and Fanci Functionmentioning

confidence: 99%

The Fanconi anemia ID2 complex: Dueling saxes at the crossroads

Boisvert

Howlett

2014

Cell Cycle

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Identification of Proteins at Active, Stalled, and Collapsed Replication Forks Using Isolation of Proteins on Nascent DNA (iPOND) Coupled with Mass Spectrometry

Cited by 227 publications

References 47 publications

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

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

The human oncoprotein and chromatin architectural factor DEK counteracts DNA replication stress

The Fanconi anemia ID2 complex: Dueling saxes at the crossroads

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