Arg304 of Human DNA Primase Is a Key Contributor to Catalysis and NTP Binding:  Primase and the Family X Polymerases Share Significant Sequence Homology

Kirk, Brian W.; Kuchta, Robert D.

doi:10.1021/bi990247c

Cited by 53 publications

(66 citation statements)

References 33 publications

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“…The location of the binding site for the initiating NTP has not been defined yet. It has been shown that the triphosphate group of the initiating nucleotide is important for dinucleotide synthesis, and it was proposed that it interacts with Arg-304 of p49 (35). However, recent structural data indicate that Arg-304 is buried inside the molecule and, accordingly, most likely plays a purely structural role; this explains the serious defect in primase activity when it is changed to another amino acid (12,13).…”

Section: Discussionmentioning

confidence: 99%

Insight into the Human DNA Primase Interaction with Template-Primer

Baranovskiy¹,

Zhang

Suwa³

et al. 2016

Journal of Biological Chemistry

100

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DNA replication in almost all organisms depends on the activity of DNA primase, a DNA-dependent RNA polymerase that synthesizes short RNA primers of defined size for DNA polymerases. Eukaryotic and archaeal primases are heterodimers consisting of small catalytic and large accessory subunits, both of which are necessary for the activity. The mode of interaction of primase subunits with substrates during the various steps of primer synthesis that results in the counting of primer length is not clear. Here we show that the C-terminal domain of the large subunit (p58 C ) plays a major role in template-primer binding and also defines the elements of the DNA template and the RNA primer that interact with p58 C . The specific mode of interaction with a template-primer involving the terminal 5-triphosphate of RNA and the 3-overhang of DNA results in a stable complex between p58 C and the DNA/RNA duplex. Our results explain how p58 C participates in RNA synthesis and primer length counting and also indicate that the binding site for initiating NTP is located on p58 C . These findings provide notable insight into the mechanism of primase function and are applicable for DNA primases from other species.The four-subunit primase-polymerase ␣ (Prim-Pol␣) 3 complex possessing DNA primase and DNA polymerase active sites is important for genome replication in eukaryotes (1, 2). PrimPol␣ synthesizes the chimeric RNA-DNA primers for replicative DNA polymerases ⑀ and ␦ (3, 4). In humans, the primase heterodimer contains a small catalytic subunit (p49; also known as p48, PRIM1, Pri1, and PriS) and a large regulatory subunit (p58; also known as PRIM2, Pri2, and PriL). Pol␣ is composed of a large catalytic subunit (p180) and a small accessory subunit (p70). p58 and p70 are connected with p180 through the interaction with a small C-terminal domain (p180 C ) that defines the tight coordination of the RNA-and DNA-polymerizing activities (5-7).Eukaryotic primases have a minimal specific recognition site on DNA and only require a pyrimidine to template the 5Ј-terminal nucleotide of the primer (8, 9). RNA primer synthesis begins with a rate-limiting initiation step that includes binding of the DNA template and two NTPs followed by dinucleotide synthesis (10). Subsequently, primase elongates the generated dinucleotide to the unit-length primer (8 -10-mer) and terminates synthesis. This unique counting ability of DNA primases, which results in RNA primers that are optimal for extension by Pol␣, has a complex mechanism that is currently unclear. Recent structural data revealed that the primase active site located on p49 uses the common mechanism of nucleic acids synthesis, where the catalytic aspartates coordinate two divalent ions and the triphosphate moiety of the incoming NTP (11,12).The large subunit of human primase is composed of two separate domains connected with a long linker, which indicates a significant conformational flexibility of this subunit (13). The N-terminal domain (p58 N ) provides a platform for interactions with p49 and P...

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Section: Discussionmentioning

confidence: 99%

Insight into the Human DNA Primase Interaction with Template-Primer

Baranovskiy¹,

Zhang

Suwa³

et al. 2016

Journal of Biological Chemistry

100

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“…Because this mutation eliminated the ability of LEF-1 to function in transient-replication assays (13), the essential function of LEF-1 in baculovirus replication is directly connected with the primase activity. Interestingly, the three aspartates in mouse primase (D109, D111, and D306) align precisely with similar residues in the 31-kDa domain of DNA polymerase ␤, suggesting that Pri-type primases are probably members of the Pol X polymerase family (24). The three aspartates (D95, D97, and D280) in the archaeon Pyrococcus furiosus (Pfu) primase could be superimposed with the activesite residues of four different DNA polymerases, including human DNA polymerase ␤, indicating similar three-dimensional arrangements in all five structures (3).…”

Section: Vol 76 2002mentioning

confidence: 86%

“…This motif is also found in primases from a number of herpesviruses, phage T7, Saccharomyces cerevisiae, archaea, and mammals (3,24). Experimental data suggest that the two proximal aspartates in this motif are important for enzymatic activity.…”

Section: Vol 76 2002mentioning

confidence: 93%

Baculovirus Replication Factor LEF-1 Is a DNA Primase

Mikhailov

Rohrmann

2002

J Virol

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The baculovirus replication factors LEF-1 and LEF-2 of the Autographa californica multinucleocapsid nucleopolyhedrovirus were overexpressed as fusions containing a hemagglutinin (HA) epitope and a HIS 6 tag using recombinant baculoviruses. LEF-1 was purified to near homogeneity and found to have primase activity in an indirect assay employing Escherichia coli DNA polymerase I (Klenow enzyme) and poly(dT) template. The LEF-1 primase products were also directly characterized by electrophoresis in 20% polyacrylamide-8 M urea gels and agarose gels. Primer synthesis was time dependent, and products of several hundred nucleotides or more were observed from the M13 single-stranded DNA (ssDNA) template. The LEF-1 primase was absolutely dependent on divalent cations (Mg 2؉ ), and optimal activity was supported by 10 mM MgCl 2 . An alkaline pH (8.8 to 9.4) was optimal, whereas monovalent salt (KCl) was inhibitory. Mutation of an invariant aspartic acid in a putative primase domain caused LEF-1 activity to be abolished. Upon ultracentrifugation in glycerol gradients, LEF-1 was found to have a sedimentation coefficient of 3S that is consistent with its being present as a monomer. Elution profiles of LEF-1 and LEF-2 from ssDNA-cellulose and DEAE resin suggested that LEF-2 may bind to both DNA and LEF-1.The Baculoviridae are a large and diverse family of rodshaped, enveloped, occluded viruses that are pathogenic for invertebrates, particularly members of the Insecta. They have been reported from over 600 species, most of which are members of the Lepidoptera, Diptera, and Hymenoptera (34). Two genera of baculoviruses have been characterized, and they include the nucleopolyhedroviruses (NPVs) (47), which have numerous virions within large polyhedron-shaped occlusion bodies, and the granuloviruses (52), which commonly have a single virion within small granular occlusion bodies. Baculovirus genomes consist of double-stranded, circular, supercoiled DNA of 100 to 180 kb, depending on the strain of virus (20). Although evidence suggests that baculovirus genomes may replicate via a rolling-circle intermediate (31,42), the mechanisms of initiation, elongation, processing, and maturation have not been determined.Baculovirus DNA replication has been shown to be associated with discrete replication factories in the nuclei of infected cells (41). In addition, a conserved set of genes that are essential or highly stimulatory for transient DNA replication have been identified for Autographa californica multinucleocapsid NPV (AcMNPV) (26, 33), Orgyia pseudotsugata MNPV (OpMNPV) (reviewed in reference 1), and Lymantria dispar MNPV (44). These include genes encoding a DNA polymerase homolog, a DNA helicase homolog, ie-1, a transactivator of early gene transcription, and late expression factors (LEFs) encoded by lef-1, -2, and -3. The DNA polymerase and DNA helicase homologs were subsequently shown to have activities associated with these enzymes (35,36). In addition, lef-3 encodes a product with the properties of a single-stranded DNA (SSB) bi...

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“…The DNA replication of ⌽X174 ssDNA was carried out in a reaction mixture containing 66 ng of ⌽X174 ssDNA (New England Biolabs); 20 mM Tris-acetate (pH 7.3); 5 mM magnesium acetate; 20 mM potassium acetate; 1 mM dithiothreitol; 0.1 mg of bovine serum albumin (BSA)/ml; 1 mM ATP; 0.1 mM (each) CTP, GTP, UTP, dATP, dCTP, dGTP, and TTP; and 0.1 mM [␣- 32 (70,71). The reaction mixture was assembled on ice, and the reaction was started by the addition of pol-prim.…”

Section: Methodsmentioning

confidence: 99%

“…The replication of PyV DNA in vitro was performed according to methods described previously (6,57). Briefly, the assay contained 1.2 g of PyV Tag, 250 ng of pUC-Py1 DNA (PyV origin DNA [52]), and 200 g of S100 or depleted S100 extract in 30 mM HEPES-KOH (pH 7.8); 1 mM dithiothreitol; 7 mM magnesium acetate; 1 mM EGTA (pH 7.8); 4 mM ATP; 0.3 mM (each) CTP, GTP, and UTP; 0.1 mM (each) dATP and dGTP; 0.05 mM (each) dCTP and dTTP; 40 mM creatine phosphate; 80 g of creatine kinase/ml, and 5 Ci each of [␣ 32 P]dCTP and [␣ 32 P]dTTP (3,000 Ci/mmol; Amersham Pharmacia Biotech); pol-prim was added as indicated. The incorporation of radioactive dNMP was measured by acid precipitation of DNA and scintillation counting.…”

Section: Methodsmentioning

confidence: 99%

Amino Acids 257 to 288 of Mouse p48 Control the Cooperation of Polyomavirus Large T Antigen, Replication Protein A, and DNA Polymerase α-Primase To Synthesize DNA In Vitro

Kautz¹,

Weißhart²,

Schneider³

et al. 2001

J Virol

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Although p48 is the most conserved subunit of mammalian DNA polymerase ␣-primase (pol-prim), the polypeptide is the major species-specific factor for mouse polyomavirus (PyV) DNA replication. Human and murine p48 contain two regions (A and B) that show significantly lower homology than the rest of the protein.Chimerical human-murine p48 was prepared and coexpressed with three wild-type subunits of pol-prim, and four subunit protein complexes were purified. All enzyme complexes synthesized DNA on single-stranded (ss) DNA and replicated simian virus 40 DNA. Although the recombinant protein complexes physically interacted with PyV T antigen (Tag), we determined that the murine region A mediates the species specificity of PyV DNA replication in vitro. More precisely, the nonconserved phenylalanine 262 of mouse p48 is crucial for this activity, and pol-prim with mutant p48, h-S262F, supports PyV DNA replication in vitro. DNA synthesis on RPA-bound ssDNA revealed that amino acid (aa) 262, aa 266, and aa 273 to 288 are involved in the functional cooperation of RPA, pol-prim, and PyV Tag.The small DNA tumor viruses simian virus 40 (SV40) and mouse polyomavirus (PyV) have provided excellent model systems to study the mechanisms and regulation of eukaryotic DNA replication (reviewed in references 7, 9, 55, and 67). Their DNA replication in vivo and in vitro largely depends on the replication apparatus of their hosts, as only one protein, namely, the multifunctional viral large T antigen (Tag), is supplied by the virus (20,26,49).The establishment of cell-free SV40 and PyV DNA replication systems was a major step towards the purification and characterization of essential host replication factors (67). These studies allowed the understanding of the molecular mechanisms involved in the assembly and progression of replication forks (7,28,67). Along with genetic and other biochemical approaches, these DNA replication systems provided insights into the processes of initiation and elongation (67). Interestingly, several parallels between eukaryotic and prokaryotic DNA duplication emerged which show that central processes of DNA metabolism are in part conserved in all kingdoms (33,60,61). The studies of the polyomaviral systems have allowed the development of a general model for eukaryotic DNA replication. In an initial step, Tag recognizes the replication origin (ori) and forms a double hexameric complex, which causes specific distortions of the double-stranded (ds) DNA (4,15,20,37,49). Subsequently, dsDNA is unwound, and multiprotein complexes are assembled by Tag and host replication proteins, such as eukaryotic single-stranded (ss) DNA-binding protein (replication protein A [RPA]), topoisomerase I (topo I), and DNA polymerase ␣-primase (polprim), are recruited (10,11,17,23,38,53,54). After binding and distortion of the dsDNA, the helicase function of Tag melts and unwinds the ds ori DNA (4, 59). For this function, Tag depends on RPA as a cofactor to stabilize the ssDNA resulting from the helicase activity of Tag (7,29,72). Th...

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Arg304 of Human DNA Primase Is a Key Contributor to Catalysis and NTP Binding: Primase and the Family X Polymerases Share Significant Sequence Homology

Cited by 53 publications

References 33 publications

Insight into the Human DNA Primase Interaction with Template-Primer

Insight into the Human DNA Primase Interaction with Template-Primer

Baculovirus Replication Factor LEF-1 Is a DNA Primase

Amino Acids 257 to 288 of Mouse p48 Control the Cooperation of Polyomavirus Large T Antigen, Replication Protein A, and DNA Polymerase α-Primase To Synthesize DNA In Vitro

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