Human cell DNA replication is mediated by a discrete multiprotein complex

Jiang, Hai Yan; Hickey, Robert J.; Abdel-Aziz, Waleed; Tom, Timothy D.; Wills, Philip; Liu, Jianying; Malkas, Linda H.

doi:10.1002/jcb.10182

Cited by 20 publications

(22 citation statements)

References 36 publications

(76 reference statements)

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“…33,[35][36][37] These studies have shown that the replication activity present in a combined low-salt nuclear extract (NE)/ post mitochondrial cytosolic supernatant (S3), [NE/S3] partitions exclusively into the interface fraction (referred to as P4) after centrifugation on a 2M sucrose cushion. This procedure separates high molecular weight complexes that collect at the interface from soluble proteins in the supernatant fraction (designated S4) (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…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%

“…The Malkas-Hickey laboratories have previously reported on the isolation and extensive characterization of a multiprotein replication complex from human and mouse cells that fully supports origin directed, semi-conservative DNA replication in a cell-free system, termed the DNA 'synthesome'. 32,33 A number of proteins known to be required for eukaryotic DNA synthesis in vitro have been shown to copurify with the synthesome, including DNA polymerases α, δ and ε, DNA primase, replication protein A (RPA), replication factor-C (RF-C), DNA ligase I, topoisomerases I and II, and PCNA. 32,[34][35][36][37] Other proteins that function in a regulatory capacity such as poly(ADP-ribose) polymerase (PARP), have also been identified in the complex.…”

Section: Introductionmentioning

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: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

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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“…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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Characterization of RNA primers synthesized by the human breast cancer cell DNA synthesome

Dai

Liu

Malkas

et al. 2009

J of Cellular Biochemistry

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We previously reported on the purification and characterization of a functional multi-protein DNA replication complex (the DNA synthesome) from human cells and tissues. The synthesome is fully competent to carry-out all phases of the DNA replication process in vitro. In this study, DNA primase, a component of the synthesome, is examined to determine its activity and processivity in the in vitro synthesis and extension of RNA primers. Our results show that primase activity in the P4 fraction of the synthesome is 30-fold higher than that of crude cell extracts. The synthesome synthesizes RNA primers that are 7-10 ribonucleotides long and DNA primers that are 20-40 deoxyribonucleotides long using a poly(dT) template of exogenous single-stranded DNA. The synthesome-catalyzed RNA primers can be elongated by E. coli DNA polymerase I to form the complementary DNA strands on the poly(dT) template. In addition, the synthesome also supports the synthesis of native RNA primers in vitro using an endogenous supercoiled double-stranded DNA template. Gel analysis demonstrates that native RNA primers are oligoribonucleotides of 10-20 nt in length and the primers are covalently link to DNA to form RNA-primed nascent DNA of 100-200 nt. Our study reveals that the synthesome model is capable of priming and continuing DNA replication. The ability of the synthesome to synthesize and extend RNA primers in vitro elucidates the organizational and functional properties of the synthesome as a potentially useful replication apparatus to study the function of primase and the interaction of primase with other replication proteins. KeywordsPRIMASE; RNA PRIMER; DNA REPLICATION; DNA SYNTHESOME; IN VITRO DNA replication in eukaryotic cells is a multi-step and tightly coordinated process that requires a specific set of active proteins to participate and interact with one another in each reaction [Malkas, 1998;Frouin et al., 2003]. The initiation of DNA replication involves the synthesis of a short RNA primer of about 10 ribonucleotides in length that is catalyzed by DNA primase [Waga and Stillman, 1998]. RNA primers play a central role in the initiation of DNA replication on both the leading and lagging strands at the replication fork [Wang, 1991]. After the synthesis of RNA primers, DNA polymerase α(pol α) elongates the primers by polymerizing deoxyribonucleotide triphosphates (dNTPs) to generate DNA primers of about 20-40 nucleotides [Baker and Bell, 1998;Arezi and Kuchta, 2000]. Pol α and primase are thought to be dissociated from the DNA template after the synthesis of DNA primers. DNA pol δ and probably also DNA pol ε extend DNA primers for DNA replication to produce one continuous DNA fragment on the leading strand, or many discontinuous DNA fragments (Okazaki *Correspondence to: Robert J. Hickey, 1044 W. Walnut Street, R4-170 Indianapolis, IN 46202., E-mail: rohickey@iupui.edu. NIH Public AccessAuthor Manuscript J Cell Biochem. Author manuscript; available in PMC 2010 January 12. NIH-PA Author ManuscriptNIH-PA Author Manuscript NI...

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Human cell DNA replication is mediated by a discrete multiprotein complex

Cited by 20 publications

References 36 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

Characterization of RNA primers synthesized by the human breast cancer cell DNA synthesome

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