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

Abstract

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...

Role of Androgen Receptor in Progression of LNCaP Prostate Cancer Cells from G1 to S Phase

“…Progression of cells from G 1 to S phase requires not only pre-RC assembly but also the recruitment of enzymes of DNA synthesis to pre-RC. DNA synthesis requires the concerted action of a number of enzymes and proteins in megacomplexes, which are referred to as DNA replication machinery, DNA synthesome or replitase [36], [37], [38]. Pre-RC serves as a “launching pad” for the assembly of DNA replication machinery at sites of DNA replication [16].…”

Telomere Shortening Mechanisms

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