Identification of multiprotein complexes containing DNA replication factors by native immunoblotting of HeLa cell protein preparations with T-antigen-dependent SV40 DNA replication activity

Tom, Timothy D.; Malkas, Linda H.; Hickey, Robert J.

doi:10.1002/(sici)1097-4644(19961201)63:3<259::aid-jcb1>3.0.co;2-w

Cited by 26 publications

(16 citation statements)

References 24 publications

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“…39 It has been estimated that approximately 35 individual proteins make up this complex, which migrates as a single species on native polyacrylamide gels. 33,40 To determine whether DNMT1 is associated with the human cell DNA synthesome, the human breast cancer cell line MDA MB468 was fractionated as outlined in Figure 1, and the resulting purification fractions were analyzed for DNMT1 protein. Western blot analysis revealed that the 190kDa DNMT1 protein was present in the replication-competent NE/S3, P4, and Qs fractions from MDA MB468 cells ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…30 The synthesome migrates as a discreet high molecular weight band on native polyacrylamide gel electrophoresis and retains SV40 replication activity following electroelution. 33,40 In addition, the immunoprecipitation of individual components of the synthesome co-precipitates some, but not all of the other proteins in the complex, suggesting that the interactions within the complex are specific. Such experiments indicate that DNA polymerase α and δ, RF-C, DNA primase and PCNA appear to tightly associate with one another.…”

Section: Discussionmentioning

confidence: 99%

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DNMT1 is a Component of a Multiprotein DNA Replication Complex

Vertino¹,

Sekowski²,

Coll³

et al. 2002

Cell Cycle

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DNA methylation is a major determinant of epigenetic inheritance and plays an important role in genome stability. The accurate propagation of DNA methylation patterns with cell division requires that methylation be closely coupled to DNA replication, however the precise molecular determinants of this interaction have not been defined. In the present study, we show that the predominant DNA methyltransferase species in somatic cells, DNMT1, is a component of a multiprotein DNA replication complex termed the DNA synthesome that fully supports semi-conservative DNA replication in a cell-free system. DNMT1 protein and activity were found to co-purify with the human DNA synthesome through a series of subcellular fractionation and chromatography steps, resulting in an enrichment of methyltransferase specific activity from two human cell lines. DNA methyltransferase activity co-eluted with in vitro replication activity and DNA polymerase α activity on sucrose density gradients suggesting that DNMT1 is a tightly bound, core component of the replication complex. The synthesome-associated pool of DNA methyltransferase exhibited both maintenance and de novo methyltransferase activity and the ratio of the two was similar to that observed in whole cell lysates and for recombinant DNMT1. These data indicate that interactions within the synthesome complex do not influence the intrinsic preference of DNMT1 for hemimethylated DNA, but suggest that newly replicated DNA may be subject to low level de novo methylation. The data indicate that DNA methylation is tightly coupled to replication through physical interaction of DNMT1 and core components of the replication machinery. The definition of the molecular interactions between DNMT1 and other proteins in the replication complex in normal and neoplastic cells will provide further insight into the regulation of DNA methylation and the mechanisms underlying the alteration of DNA methylation patterns during carcinogenesis.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

DNMT1 is a Component of a Multiprotein DNA Replication Complex

Vertino¹,

Sekowski²,

Coll³

et al. 2002

Cell Cycle

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“…One of the critical regulatory points controlling human cell proliferation occurs at the level of DNA replication. Our laboratory successfully isolated, extensively purified, and characterized an intact mammalian cell multiprotein DNA-replication complex from a variety of human cell types that was both stable and fully functional (21)(22)(23)(24)(25)(26)(27)(28)(29). We proposed a model to represent the mammalian multiprotein DNA replication complex or DNA synthesome (22,23).…”

Section: Discussionmentioning

confidence: 99%

“…N͞D, not determined. *The SV40 in vitro DNA replication assay was performed according to our procedures (21)(22)(23)(24)(25)(26)(27)(28)(29). † The DNA polymerase ␦ assay was performed according to the procedures of Han et al (35).…”

Section: Discussionmentioning

confidence: 99%

A cancer-associated PCNA expressed in breast cancer has implications as a potential biomarker

Malkas

Herbert

Abdel-Aziz

et al. 2006

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

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105

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Two isoforms of proliferating cell nuclear antigen (PCNA) have been observed in breast cancer cells. Commercially available antibodies to PCNA recognize both isoforms and, therefore, cannot differentiate between the PCNA isoforms in malignant and nonmalignant breast epithelial cells and tissues. We have developed a unique antibody that specifically detects a PCNA isoform (caPCNA) associated with breast cancer epithelial cells grown in culture and breast-tumor tissues. Immunostaining studies using this antibody suggest that the caPCNA isoform may be useful as a marker of breast cancer and that the caPCNA-specific antibody could potentially serve as a highly effective detector of malignancy. We also report here that the caPCNA isoform functions in breast cancer-cell DNA replication and interacts with DNA polymerase ␦. Our studies indicate that the caPCNA isoform may be a previously uncharacterized detector of breast cancer. mass spectrometry ͉ pathology ͉ posttranslational modification ͉ DNA replication ͉ genome stability

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“…We have demonstrated that PARP is tightly associated with the core proteins of the puri®ed MRC (SimbulanRosenthal et al, 1996) that support viral DNA replication in vitro and migrates as discrete, high molecular weight complexes on native polyacrylamide gel electrophoresis (Tom et al, 1996). PARP is thought to play a regulatory role in these complexes because 15 of the *40 polypeptides of the MRC, including pol a, topoisomerase I and PCNA, undergo poly(ADPribosyl)ation by PARP (Simbulan-Rosenthal et al, Figure 2 Flow cytometric analysis of wild-type and PARP7/7 mouse ®broblasts (a) and PARP7/7(+PARP) cells (b) at various times after release from serum deprivation.…”

Section: Discussionmentioning

confidence: 99%

Poly(ADP-ribose) polymerase upregulates E2F-1 promoter activity and DNA pol α expression during early S phase

et al. 1999

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E2F-1, a transcription factor implicated in the activation of genes required for S phase such as DNA pol a, is regulated by interactions with Rb and by cell-cycle dependent alterations in E2F-1 abundance. We have shown that depletion of poly(ADP-ribose) polymerase (PARP) by antisense RNA expression downregulates pol a and E2F-1 expression during early S phase. To examine the role of PARP in the regulation of pol a and E2F-1 gene expression, we utilized immortalized mouse ®broblasts derived from wild-type and PARP knockout (PARP7/7) mice as well as PARP7/7 cells stably transfected with PARP cDNA [PARP7/7(+PARP)]. After release from serum deprivation, wild-type and PARP7/7(+PARP) cells, but not PARP7/7 cells, exhibited a peak of cells in S phase by 16 h and had progressed through the cell cycle by 22 h. Whereas [ 3 H]thymidine incorporation remained negligible in PARP7/7 cells, in vivo DNA replication maximized after 18 h in wild-type and PARP7/7(+PARP) cells. To investigate the e ect of PARP on E2F-1 promoter activity, a construct containing the E2F-1 gene promoter fused to a luciferase reporter gene was transiently transfected into these cells. E2F-1 promoter activity in control and PARP7/7(+PARP) cells increased eightfold after 9 h, but not in PARP7/7 cells. PARP7/7 cells did not show the marked induction of E2F-1 expression during early S phase apparent in control and PARP7/7(+PARP) cells. RT ± PCR analysis and pol a activity assays revealed the presence of pol a transcripts and a sixfold increase in activity in both wildtype and PARP7/7(+PARP) cells after 20 h, but not in PARP7/7 cells. These results suggest that PARP plays a role in the induction of E2F-1 promoter activity, which then positively regulates both E2F-1 and pol a expression, when quiescent cells reenter the cell cycle upon recovery from aphidicolin exposure or removal of serum.

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Identification of multiprotein complexes containing DNA replication factors by native immunoblotting of HeLa cell protein preparations with T-antigen-dependent SV40 DNA replication activity

Cited by 26 publications

References 24 publications

DNMT1 is a Component of a Multiprotein DNA Replication Complex

DNMT1 is a Component of a Multiprotein DNA Replication Complex

A cancer-associated PCNA expressed in breast cancer has implications as a potential biomarker

Poly(ADP-ribose) polymerase upregulates E2F-1 promoter activity and DNA pol α expression during early S phase

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