DNA damage and mutagenesis by radiomimetic DNA-cleaving agents: bleomycin, neocarzinostatin and other enediynes

Povirk, Lawrence F.

doi:10.1016/0027-5107(96)00023-1

Cited by 394 publications

(292 citation statements)

References 98 publications

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“…As a control, a concomitant decrease in DNA-PK kinase activity was observed in extracts prepared from cells treated with RA as compared to untreated controls, the amplitude of the e ect being maximal after 5 days of incubation with RA (data not shown). Finally, in accordance to the previously described role of the Ku-DNA-PK complex in DSBs repair, the sensitivity of di erentiated cells to neocarcinostatin (a drug that acts as a radiomimetic, (Povirk, 1996)) was increased by *3- Figure 1 Decrease of Ku activity without modi®cation of Ku expression during exposure of HL60 cells to RA and DMSO. (a) Two mg of protein extracts obtained from cells treated, at time indicated, with either all-trans RA (1 mM) or DMSO (1.3% vol/ vol) were incubated with 4 ng 32 P-labeled DNA probe in the presence of 1 mg of closed circular plasmid DNA, as a non speci®c competitor.…”

Section: Resultssupporting

confidence: 72%

Inhibition of Ku heterodimer DNA end binding activity during granulocytic differentiation of human promyelocytic cell lines

et al. 2001

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The heterodimeric Ku protein (composed of the Ku 86 and Ku 70 sub-units) is a nuclear protein which binds to DNA termini without sequence speci®city. Ku is the DNA-targeting component of the large catalytic sub-unit of the DNA-dependent protein kinase complex that is required for the repair of DNA double-strand breaks in mammalian cells. We studied the expression and function of Ku/DNA-PK during granulocytic di erentiation of two human promyelocytic cell lines, HL60 and NB4, a process associated to decreased radiation resistance. After 3 days exposure to di erentiating agents (either all-trans-retinoic acid or DMSO), Ku binding to double stranded (ds)-DNA ends declined dramatically whereas Ku protein levels remain unchanged. The nuclear, but not cytoplasmic, fraction of di erentiated HL60 cells extracts exhibited a heat-sensitive inhibitory activity towards DNA binding of recombinant Ku heterodimer. We further demonstrate that immunoprecipitation of Ku is impaired in extracts from di erentiated cells by using two antibodies that recognize epitopes within the Cterminus DNA binding domains of Ku 70 and Ku 86 proteins. These results favor the hypothesis of a protein interacting with Ku that would prevent DNA binding of heterodimerized Ku protein by steric hindrance. Oncogene (2001) 20, 4373 ± 4382.

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Section: Resultssupporting

confidence: 72%

Inhibition of Ku heterodimer DNA end binding activity during granulocytic differentiation of human promyelocytic cell lines

et al. 2001

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“…Similar defects were observed in the presence of low doses of bleomycin (BLM), which generates both nicks and double-strand breaks ( Fig. 2B; Povirk 1996). Like MMS, exposure to BLM in S phase causes replication stress and activation of the S-phase checkpoint (D'Amours and Jackson 2001).…”

Section: Asf1 Promotes S-phase Progression In the Presence Of Dna Dammentioning

confidence: 61%

Histone deposition protein Asf1 maintains DNA replisome integrity and interacts with replication factor C

Franco¹,

Lam²,

Burgers³

et al. 2005

Genes Dev.

128

125

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Chromatin assembly and DNA replication are temporally coupled, and DNA replication in the absence of histone synthesis causes inviability. Here we demonstrate that chromatin assembly factor Asf1 also affects DNA replication. In budding yeast cells lacking Asf1, the amounts of several DNA replication proteins, including replication factor C (RFC), proliferating cell nuclear antigen (PCNA), and DNA polymerase (Pol ), are reduced at stalled replication forks. In contrast, DNA polymerase ␣ (Pol ␣) accumulates to higher than normal levels at stalled forks in asf1⌬ cells. Using purified, recombinant proteins, we demonstrate that RFC directly binds Asf1 and can recruit Asf1 to DNA molecules in vitro. We conclude that histone chaperone protein Asf1 maintains a subset of replication elongation factors at stalled replication forks and directly interacts with the replication machinery.[Keywords: Chromatin assembly; histone deposition; DNA replication; genome stability] Supplemental material is available at http://www.genesdev.org. In eukaryotes, DNA is assembled into a nucleoprotein complex called chromatin. The fundamental repeating unit of chromatin is the nucleosome, which consists of 146 bp of DNA wrapped around an octamer of core histone proteins, comprised of two H2A/H2B dimers flanking an inner (H3/H4) 2 tetramer. In vivo, histone deposition can occur by DNA replication-coupled or replication-independent mechanisms (for review, see Franco and Kaufman 2004). In either case, histone deposition is mediated by specialized assembly proteins. The best characterized replication-coupled chromatin assembly factor is chromatin assembly factor-1 (CAF-1), an evolutionarily conserved protein complex that binds histone H3 and H4 and delivers them to replicating DNA through an interaction with proliferating cell nuclear antigen (PCNA), the DNA polymerase processivity protein (for review, see Asf1, another evolutionarily conserved histone chaperone, functions during both replication-coupled and replication-independent chromatin assembly. Asf1 bound to histones H3/H4 stimulates histone deposition by CAF-1 in vitro (Tyler et al. 1999;Sharp et al. 2001), and Asf1 physically interacts with the p60/Cac2 subunit of CAF-1 (Tyler et al. 2001;Krawitz et al. 2002;Mello et al. 2002). In yeast, Asf1 and the Hir proteins directly interact and function together to promote heterochromatic gene silencing (Kaufman et al. 1998;Sharp et al. 2001;Sutton et al. 2001;Krawitz et al. 2002). Consistent with a role in multiple deposition pathways, Asf1 copurifies with both CAF-1 and Hir protein complexes in human cell extracts (Tagami et al. 2004).Asf1 also contributes to genome stability during S phase in a manner distinct from both CAF-1 and the Hir proteins. Unlike yeast cells lacking CAF-1 or Hir proteins, cells lacking Asf1 are sensitive to the DNA synthesis inhibitor hydroxyurea (HU) and the DNA topoisomerase I inhibitor camptothecin (CPT), and display synthetic genetic interactions with DNA synthesis genes including the initiation/elongation factor C...

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“…We investigated the response of two human glioblastoma cell lines M059K and M059J to downregulation of the individual CK2 subunits in the presence or absence of DNA damage induced by incubation of cells with the radiomimetic drug neocarzinostatin (NCS; Povirk, 1996) for 24 h. As shown in Figure 1a, transfection of cells with small interfering RNAs (siRNAs) against the individual CK2 subunits led to a marked reduction of the CK2 protein levels in both cell lines. Additionally, the sole depletion of CK2a resulted in the downregulation of CK2b, an effect that has been previously reported (Canton et al, 2001), whereas downregulation of CK2b negatively affected the expression of CK2a 0 .…”

Section: Downregulation Of Protein Kinase Ck2 Induces Cell Death In Gmentioning

confidence: 99%

Regulation of DNA-dependent protein kinase by protein kinase CK2 in human glioblastoma cells

2010

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The DNA-dependent protein kinase (DNA-PK) is a nuclear serine/threonine protein kinase composed of a large catalytic subunit (DNA-PKcs) and a heterodimeric DNA-targeting subunit Ku. DNA-PK is a major component of the nonhomologous end-joining pathway of DNA double-strand breaks repair. Although DNA-PK has been biochemically characterized in vitro, relatively little is known about its functions in the context of DNA repair and how its kinase activity is precisely regulated in vivo. Here, we report that cellular depletion of the individual catalytic subunits of protein kinase CK2 by RNA interference leads to significant cell death in M059K human glioblastoma cells expressing DNA-PKcs, but not in their isogenic counterpart, that is M059J cells, devoid of DNA-PKcs. The lack of CK2 results in enhanced DNA-PKcs activity and strongly inhibits DNA damageinduced autophosphorylation of DNA-PKcs at S2056 as well as repair of DNA double-strand breaks. By the application of the in situ proximity ligation assay, we show that CK2 interacts with DNA-PKcs in normal growing cells and that the association increases upon DNA damage. These results indicate that CK2 has an important role in the modulation of DNA-PKcs activity and its phosphorylation status providing important insights into the mechanisms by which DNA-PKcs is regulated in vivo.

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DNA damage and mutagenesis by radiomimetic DNA-cleaving agents: bleomycin, neocarzinostatin and other enediynes

Cited by 394 publications

References 98 publications

Inhibition of Ku heterodimer DNA end binding activity during granulocytic differentiation of human promyelocytic cell lines

Inhibition of Ku heterodimer DNA end binding activity during granulocytic differentiation of human promyelocytic cell lines

Histone deposition protein Asf1 maintains DNA replisome integrity and interacts with replication factor C

Regulation of DNA-dependent protein kinase by protein kinase CK2 in human glioblastoma cells

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