Eukaryotic DNA primase

Arezi, Bahram; Kuchta, Robert D.

doi:10.1016/s0968-0004(00)01680-7

Cited by 127 publications

(140 citation statements)

References 39 publications

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“…In E. coli, the primed template is transferred from the DnaG primase to DNA polymerase III by the combined actions of the ␤ clamp, the clamp loader, and ssDNA-binding protein (40). A similar conserved mechanism occurs in eukaryotes (41). Our finding that the N terminus of Mcm10p stabilized the interaction of the pol ␣-primase complex with primed DNA templates suggests that Mcm10p may also affect this type of transfer.…”

Section: Mg2mentioning

confidence: 68%

Fission Yeast Mcm10p Contains Primase Activity

Fien

Hurwitz

2006

Journal of Biological Chemistry

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DNA replication occurs through a complex series of reactions that are mechanistically coordinated at the replication fork. Studies of model DNA replication systems indicate that origin recognition by an initiator protein permits assembly and activation of the replicative helicase at the origin. Helicases unwind duplex DNA to generate a single-strand template on which primases can initiate synthesis of RNA primers that are 4 -20 nucleotides in length and that are extended by DNA polymerases to make Okazaki fragments (1). Primases are physically coupled to the replicative DNA helicases and DNA polymerases, which translocate together through the duplex, resulting in DNA unwinding coincident with synthesis of RNA primers and their extension with deoxynucleotides.In eukaryotes, the Mcm proteins are essential replication factors that were identified as proteins required for minichromosomal maintenance in a genetic screen for mutants defective in initiation of replication (2). Six of these members are sequencerelated proteins, Mcm2-7, which interact to form a hexameric complex. Although a substantial body of data suggests that the Mcm2-7p complex acts as the replicative helicase (3), helicase activity has been detected only in the Mcm4-6-7 subcomplex (4 -6). Prior to the initiation of replication, the Mcm2-7 complex associates with the initiator at replication origins. At the G 1 /S transition, both S phase cyclin-dependent kinase and Cdc7p-Dbf4p kinase activities rise, promoting the maturation of the pre-replicative complex to the preinitiation complex (7). Phosphorylation of the chromatin-bound Mcm2-7 complex by the Cdc7-Dbf4 kinase (8) Previously, we reported that, in vitro, S. pombe Mcm10p (amino acids 1-593; SpMcm10p) binds preferentially to singlestranded DNA (ssDNA) and to the large subunit of the pol ␣-primase complex and activates its DNA synthetic activity (25). In addition, we found that Mcm10p facilitates the binding of the pol ␣-primase complex to primed DNA and forms a stable ternary complex, suggesting that Mcm10p recruits the pol ␣-primase complex to template DNA. In keeping with these in vitro findings, Mcm10p was shown recently to stabilize the large subunit of the pol ␣-primase complex in S. cerevisiae (15) as well as the chromatin association of the pol ␣-primase complex in S. pombe (26).In this study, we demonstrate that full-length SpMcm10p (amino acids 1-593) and its C-terminal fragment (amino acids 416 -593) contain primase activity that catalyzes the synthesis of oligoribonucleotides that are extended by Escherichia coli pol I. Primase activity both co-sedimented and co-eluted with these full-length and truncated proteins, although their hydro-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Section: Mg2mentioning

confidence: 68%

Fission Yeast Mcm10p Contains Primase Activity

Fien

Hurwitz

2006

Journal of Biological Chemistry

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“…Asp 109 , Asp 111 , and Asp 306 of p49 are thought to form the metal-binding core of the active site. The sequence of primase, which can be compared with that of pol ␤, consists of two discrete domains, a 31-kDa domain that contains phosphodiester bondforming activity and an 8-kDa domain that can bind DNA and contains deoxyribose lyase activity (14). We have not detected the similarity in the entire sequence of GANP molecule to these known residues as a DNA-primase member; therefore, it is necessary to determine whether GANP has a DNA-primase activity.…”

Section: Resultsmentioning

confidence: 95%

“…GANP DNA-primase would be recruited to the origin-binding complex with DNA-helicase of MCMs, which is presumably efficient for rapid cell growth. The conventional DNA-primase, composed of p49, p58, and p180 pol ␣ subunit, first synthesizes RNA primers up to 10 mers (14). However, the activity might not be sufficient to coordinate the enormous rate of proliferation that takes place in GC-B cells.…”

Section: Resultsmentioning

confidence: 99%

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Germinal center-associated nuclear protein (GANP) has a phosphorylation-dependent DNA-primase activity that is up-regulated in germinal center regions

Kuwahara

Tomiyasu

Fujimura

et al. 2001

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

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“…The p180 subunit possesses the catalytic DNA polymerase activity, and disruption of this gene is lethal (32,33). p58 and p48 form the DNA-dependent RNA polymerase (primase) activity (34,35), whereas the p68 subunit has no known catalytic activity but serves a regulatory role (36,37). Pol ␣ plays an essential role in lagging strand synthesis by first creating short (7-12 nucleotide) RNA primers followed by DNA extension.…”

mentioning

confidence: 99%

Physical Interactions between Mcm10, DNA, and DNA Polymerase α

Warren

Huang²,

Fanning³

et al. 2009

Journal of Biological Chemistry

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Mcm10 is an essential eukaryotic protein required for the initiation and elongation phases of chromosomal replication. Specifically, Mcm10 is required for the association of several replication proteins, including DNA polymerase ␣ (pol ␣), with chromatin. We showed previously that the internal (ID) and C-terminal (CTD) domains of Mcm10 physically interact with both single-stranded (ss) DNA and the catalytic p180 subunit of pol ␣. However, the mechanism by which Mcm10 interacts with pol ␣ on and off DNA is unclear. As a first step toward understanding the structural details for these critical intermolecular interactions, x-ray crystallography and NMR spectroscopy were used to map the binary interfaces between Mcm10-ID, ssDNA, and p180. The crystal structure of an Mcm10-ID⅐ssDNA complex confirmed and extended our previous evidence that ssDNA binds within the oligonucleotide/oligosaccharide binding-fold cleft of Mcm10-ID. We show using NMR chemical shift perturbation and fluorescence spectroscopy that p180 also binds to the OB-fold and that ssDNA and p180 compete for binding to this motif. In addition, we map a minimal Mcm10 binding site on p180 to a small region within the p180 N-terminal domain (residues 286 -310). These findings, together with data for DNA and p180 binding to an Mcm10 construct that contains both the ID and CTD, provide the first mechanistic insight into how Mcm10 might use a handoff mechanism to load and stabilize pol ␣ within the replication fork.

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Eukaryotic DNA primase

Cited by 127 publications

References 39 publications

Fission Yeast Mcm10p Contains Primase Activity

Fission Yeast Mcm10p Contains Primase Activity

Germinal center-associated nuclear protein (GANP) has a phosphorylation-dependent DNA-primase activity that is up-regulated in germinal center regions

Physical Interactions between Mcm10, DNA, and DNA Polymerase α

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