Targeting of PCNA to sites of DNA replication in the mammalian cell nucleus*

Somanathan, Suryanarayan; Suchyna, Thomas M.; Siegel, Alan J.; Berezney, Ronald

doi:10.1002/1097-4644(20010401)81:1<56::aid-jcb1023>3.0.co;2-#

Cited by 58 publications

(50 citation statements)

References 56 publications

(65 reference statements)

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“…It has been shown that enzymes required for DNA replication are concentrated into similar granules during S-phase (57). One of these enzyme is PCNA, and both immunostained PCNA and EGFP-tagged PCNA have been shown to be concentrated into replication foci (58,59). As shown in Fig.…”

Section: Characterization Of Parp-1-dsred and Topo I-eyfp-tomentioning

confidence: 87%

Poly(ADP-ribosyl)ation as a DNA Damage-induced Post-translational Modification Regulating Poly(ADP-ribose) Polymerase-1-Topoisomerase I Interaction

Yung¹,

Sato

Satoh³

2004

Journal of Biological Chemistry

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Poly(ADP-ribosyl)ation is a post-translational modification that occurs immediately after exposure of cells to DNA damaging agents. In vivo, 90% of ADP-ribose polymers are attached to the automodification domain of poly(ADP-ribose) polymerase-1 (PARP-1), the main enzyme catalyzing this modification reaction. This enzyme forms complexes with transcription initiation, DNA replication, and DNA repair factors. In most known cases, the interactions occur through the automodification domain. However, functional implications of the automodification reaction on these interactions have not yet been elucidated. In the present study, we created fluorescent protein-tagged PARP-1 to study this enzyme in live cells and focused on the interaction between PARP-1 and topoisomerase I (Topo I), one of the enzymes that interacts with PARP-1 in vitro. Here, we demonstrate that PARP-1 co-localizes with Topo I throughout the cell cycle. Results from bioluminescence resonance energy transfer assays suggest that the co-localization is because of a direct protein-protein interaction. In response to DNA damage, PARP-1 de-localization and a reduction in bioluminescence resonance energy transfer signal because of the automodification reaction are observed, suggesting that the automodification reaction results in the disruption of the interaction between PARP-1 and Topo I. Because Topo I activity has been reported to be promoted by PARP-1, we then investigated the effect of the disruption of this interaction on Topo I activity, and we found that this disruption results in the reduction of Topo I activity. These results suggest that a function for the automodification reaction is to regulate the interaction between PARP-1 and Topo I, and consequently, the Topo I activity, in response to DNA damage.Poly(ADP-ribosyl)ation is a major post-translational protein modification that specifically occurs in response to DNA damage (1-3). This modification is catalyzed by an abundant nuclear enzyme, poly(ADP-ribose) polymerase-1 (PARP-1), 1 which is composed of N-terminal DNA binding, an automodification, and C-terminal catalytic domains (4). The N-terminal DNA-binding domain contains two zinc finger motifs, which have a high binding affinity for DNA breaks (5). Upon binding, the N-terminal catalytic domain initiates ADP-ribose polymer formation using NAD ϩ as a donor of ADP-ribose residues (1, 2). In vitro, various enzymes and factors have been reported to be poly(ADP-ribosyl)ated by PARP-1 (2, 6), whereas, in vivo, over 90% of ADP-ribose polymers are produced on the automodification domain of PARP-1 (7-9). Because poly(ADP-ribosyl)ation can be detected as early as 2 to 3 min after the exposure of cells to DNA damaging agents (10), modification of proteins with ADP-ribose polymers has been suggested to play roles in the early stages of DNA damage response (1, 2).Within the automodification domain, there are 15 glutamic acid residues that are potential ADP-ribose polymer attachment sites (11). The length of ADP-ribose polymers, which are highly negativel...

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Section: Characterization Of Parp-1-dsred and Topo I-eyfp-tomentioning

confidence: 87%

Poly(ADP-ribosyl)ation as a DNA Damage-induced Post-translational Modification Regulating Poly(ADP-ribose) Polymerase-1-Topoisomerase I Interaction

Yung¹,

Sato

Satoh³

2004

Journal of Biological Chemistry

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“…51,52 Remarkably, a fraction of p21 wt GFP was found to be chromatinbound, and to co-localize with PCNA recruited to detergent-insoluble chromatin. The cell cycle position of the majority of GFP-expressing cells, as determined by PI staining, showed that these cells had a G 1 -phase DNA content, thus indicating that PCNA bound to chromatin was actually at the G 1 /S phase transition, i.e., when replication proteins like PCNA, are assembled at DNA replication sites, 46,47,53 before DNA synthesis starts. This result was confirmed by the percentage of cells showing a detectable expression of cyclin A in p21GFP positive cells, in agreement with a previous study.…”

Section: Discussionmentioning

confidence: 99%

“…45 The latter is the form directly involved in the DNA replication process. 46,47 However, a direct proof that p21 can bind PCNA recruited to DNA replication sites, and the mechanism by which this interaction interferes with DNA synthesis in vivo, are still missing. In this study we have induced expression of a p21GFP fusion protein and analysed its ability to interact and co-localize with PCNA at DNA replication sites.…”

Section: Introductionmentioning

confidence: 99%

p21CDKN1A Does Not Interfere with Loading of PCNA at DNA Replication Sites, but Inhibits Subsequent Binding of DNA Polymerase D at the G1/S Phase Transition

Cazzalini¹,

Perucca²,

Riva³

et al. 2003

Cell Cycle

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The ability of the cyclin-dependent kinase (CDK) inhibitor p21 CDKN1A to interact with PCNA recruited to DNA replication sites was investigated to elucidate the relevance of this interaction in cell cycle arrest. To this end, expression of p21 protein fused to green fluorescent protein (GFP) was induced in HeLa cells. G 1 phase cell cycle arrest induced by p21GFP occurred also at the G 1 /S transition, as shown by cyclin A immunostaining of GFP-positive cells. Confocal microscopy analysis and co-immunoprecipitation studies showed that p21GFP co-localized and interacted with chromatin-bound PCNA and CDK2. GFP-p21 mutant forms unable to bind to PCNA (p21 PCNA-) or CDK (p21 CDK-) induced cell cycle arrest, although immunoprecipitation experiments showed these mutants to be unstable. Expression of HA-tagged p21wt or mutant proteins confirmed the ability of both mutants to arrest cell cycle. p21 wt HA and p21 CDK -HA, but not p21 PCNA-, co-localized and co-immunoprecipitated with chromatin-bound PCNA. Association of p21 to chromatin-bound PCNA resulted in the loss of interaction with the p125 catalytic subunit of DNA polymerase δ (pol δ). These results suggest that in vivo p21 does not interfere with loading of PCNA at DNA replication sites, but prevents, or displaces subsequent binding of pol δ to PCNA at the G 1 /S phase transition.

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“…Because end resection and HR are activated mainly after cells enter the S phase, we specifically monitored the recruitment of CtIP to DSBs in S phase cells. We co-expressed RFP-PCNA with EGFP-CtIP alleles, and S phase cells are marked by PCNA foci (31). A series of N terminus CtIP deletion mutants including CtIP 45-897, CtIP 75-897, and CtIP 135-897, which are defective in CtIP dimerization, were examined, and all of these mutants are significantly impaired in localizing to DSBs (Fig.…”

Section: Ctip Dimerization Mutants Are Impaired In Hr-mediated Dsb Rementioning

confidence: 99%

CtIP Protein Dimerization Is Critical for Its Recruitment to Chromosomal DNA Double-stranded Breaks

Wang

Shao

Shi

et al. 2012

Journal of Biological Chemistry

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Background: DNA double-stranded break (DSB) repair is critical for the maintenance of genome stability and prevention of cancer. Results: Dimerization of CtIP, a critical DSB repair protein, is important for its localization to chromosomal DSBs in mammalian cells. Conclusion: CtIP dimerization is required for DSB repair. Significance: These studies help to understand the molecular mechanisms of DSB repair in mammalian cells.

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Targeting of PCNA to sites of DNA replication in the mammalian cell nucleus*

Cited by 58 publications

References 56 publications

Poly(ADP-ribosyl)ation as a DNA Damage-induced Post-translational Modification Regulating Poly(ADP-ribose) Polymerase-1-Topoisomerase I Interaction

Poly(ADP-ribosyl)ation as a DNA Damage-induced Post-translational Modification Regulating Poly(ADP-ribose) Polymerase-1-Topoisomerase I Interaction

p21CDKN1A Does Not Interfere with Loading of PCNA at DNA Replication Sites, but Inhibits Subsequent Binding of DNA Polymerase D at the G1/S Phase Transition

CtIP Protein Dimerization Is Critical for Its Recruitment to Chromosomal DNA Double-stranded Breaks

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