Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Different pri1 and pri2 conditional mutants of Saccharomyces cerevisiae altered, respectively, in the small (p48) and large (p58) subunits of DNA primase, show an enhanced rate of both mitotic intrachromosomal recombination and spontaneous mutation, to an extent which is correlated with the severity of their defects in cell growth and DNA synthesis. These effects might be attributable to the formation of nicked and gapped DNA molecules that are substrates for recombination and error-prone repair, due to defective DNA replication in the primase mutants. Furthermore, pri1 and pri2 mutations inhibit sporulation and affect spore viability, with the unsporulated mutant cells arresting with a single nucleus, suggesting that DNA primase plays a critical role during meiosis. The observation that all possible pairwise combinations of two pri1 and two pri2 alleles are lethal provides further evidence for direct interaction of the primase subunits in vivo. Immunopurification and immunoprecipitation studies on wild-type and mutant strains suggest that the small subunit has a major role in determining primase activity, whereas the large subunit directly interacts with DNA polymerase alpha, and either mediates or stabilizes association of the p48 polypeptide in the DNA polymerase alpha-primase complex.
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PMID:Conditional mutations in the yeast DNA primase genes affect different aspects of DNA metabolism and interactions in the DNA polymerase alpha-primase complex. 843 68

DNA polymerase alpha-primase consists of four subunits, p180, p68, p58, and p48, and comprises two essential enzymatic functions. To study the primase activity of the complex, we expressed cDNAs encoding for the human p58 and p48 subunits either as single proteins or together using Escherichia coli expression vectors. Co-expression of both primase subunits allowed the purification of a heterodimer in high yields that revealed stable primase activity. Purified recombinant p48 subunit showed enzyme activity, whereas purified p58 did not. In contrast to the heterodimer, the primase activity of p48 was unstable. The activity of p48 could be stabilized by the addition of the divalent cations Mg2+ and Mn2+ but not Zn2+. On a poly(dC) template the primase activity was hardly influenced by the monovalent cation potassium. However, by using poly(dT) as a template the recombinant p48 activity was sensitive to salt, whereas recombinant p58-p48 and the bovine DNA polymerase alpha-primase purified from thymus were less sensitive to the addition of monovalent cations. A complex of bacterially expressed primase and baculovirus-expressed p180 and p68 was assembled in vitro and shown to support replication of simian virus 40 DNA in a cell-free system.
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PMID:Primase activity of human DNA polymerase alpha-primase. Divalent cations stabilize the enzyme activity of the p48 subunit. 970 92

DNA polymerase alpha-primase (pol-prim, consisting of p180-p68-p58-p48), and primase p58-p48 (prim(2)) synthesize short RNA primers on single-stranded DNA. In the SV40 DNA replication system, only pol-prim is able to start leading strand DNA replication that needs unwinding of double-stranded (ds) DNA prior to primer synthesis. At high concentrations, pol-prim and prim(2) indistinguishably reduce the unwinding of dsDNA by SV40 T antigen (Tag). RNA primer synthesis on ssDNA in the presence of replication protein A (RPA) and Tag has served as a model system to study the initiation of Okazaki fragments on the lagging strand in vitro. On ssDNA, Tag stimulates whereas RPA inhibits the initiation reaction of both enzymes. Tag reverses and even overcompensates the inhibition of primase by RPA. Physical binding of Tag to the primase subunits and RPA, respectively, is required for these activities. Each subunit of the primase complex, p58 and p48, performs physical contacts with Tag and RPA independently of p180 and p68. Using surface plasmon resonance, the dissociation constants of the Tag/pol-prim and Tag/primase interactions were 1.2 x 10(-8) m and 1.3 x 10(-8) m, respectively.
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PMID:Protein-protein interactions of the primase subunits p58 and p48 with simian virus 40 T antigen are required for efficient primer synthesis in a cell-free system. 1074 50

DNA polymerase alpha-primase (pol-prim), a complex consisting of four subunits, is the major species-specific factor for mouse polyomavirus (PyV) and simian virus 40 (SV40) DNA replication. Although p48 is the most conserved subunit of pol-prim, it is required for in vitro PyV DNA replication but can inhibit cell-free SV40 DNA replication. Production of chimeric human-mouse p48 revealed that different regions of p48 are involved in supporting PyV DNA replication and inhibiting SV40 DNA replication. The N and C-terminal parts of p48 do not have species-specific functions in cell-free PyV DNA replication, but the central part (amino acids [aa] 129 to 320) controls PyV DNA replication in vitro. However, PyV T antigen physically binds to mouse, human, and chimeric pol-prim complexes independently, whether they support PyV DNA replication or not. In contrast to the PyV system, the inhibitory effects of mouse p48 on SV40 DNA replication are mediated by N- and C-terminal regions of p48. Thus, a chimeric p48 containing human aa 1 to 128, mouse aa 129 to 320, and human aa 321 to 418 is active in both PyV and SV40 DNA replication in vitro.
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PMID:Different regions of primase subunit p48 control mouse polyomavirus and simian virus 40 DNA replication in vitro. 1116 Jun 73

In the evolution of life, DNA replication is a fundamental process, by which species transfer their genetic information to their offspring. DNA polymerases, including bacterial and eukaryotic replicases, are incapable of de novo DNA synthesis. DNA primases are required for this function, which is sine qua non to DNA replication. In Escherichia coli, the DNA primase (DnaG) exists as a monomer and synthesizes a short RNA primer. In Eukarya, however, the primase activity resides within the DNA polymerase alpha-primase complex (Pol alpha-pri) on the p48 subunit, which synthesizes the short RNA segment of a hybrid RNA-DNA primer. To date, very little information is available regarding the priming of DNA replication in organisms in Archaea. Available sequenced genomes indicate that the archaeal DNA primase is a homolog of the eukaryotic p48 subunit. Here, we report investigations of a p48-like DNA primase from Pyrococcus furiosus, a hyperthermophilic euryarchaeote. P. furiosus p48-like protein (Pfup41), unlike hitherto-reported primases, does not catalyze by itself the synthesis of short RNA primers but preferentially utilizes deoxynucleotides to synthesize DNA fragments up to several kilobases in length. Pfup41 is the first DNA polymerase that does not require primers for the synthesis of long DNA strands.
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PMID:Archaeal primase: bridging the gap between RNA and DNA polymerases. 1130 Dec 57

Although p48 is the most conserved subunit of mammalian DNA polymerase alpha-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.
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PMID:Amino acids 257 to 288 of mouse p48 control the cooperation of polyomavirus large T antigen, replication protein A, and DNA polymerase alpha-primase to synthesize DNA in vitro. 1150 2

We characterized the primase complex of the hyperthermophilic archaeon, Pyrococcus furiosus. The two proteins, Pfup41 and Pfup46, have similar sequences to the p48 and p58 subunits, respectively, of the eukaryotic DNA polymerase alpha-primase complex. Unlike previously reported primases, the Pfup41 preferentially utilizes deoxyribonucleotides for its de novo synthesis, and moreover, it synthesizes up to several kilobases in length in a template-dependent manner (Bocquier, A., Liu, L., Cann, I., Komori, K., Kohda, D., and Ishino, Y. (2001) Curr. Biol. 11, 452-456). The p41-p46 complex showed higher DNA binding activity than the catalytic p41 subunit alone. In addition, the amount of DNA synthesized by the p41-p46 complex was much more abundant and shorter in length than that by Pfup41 alone. The activity for RNA primer synthesis, which was not detected with Pfup41, was observed from the reaction using the p41-p46 complex in vitro. The in vitro replication of M13 single-stranded DNA by the P. furiosus proteins was stimulated by ATP. Observation of the labeled primers by using [gamma-(32)P]ATP in the substrates suggests ATP as the preferable initiating nucleotide for the p41-p46 complex. These results show that the primer synthesis activity of Pfup41 is regulated by Pfup46, and the p41-p46 complex may function as the primase in the DNA replication machinery of P. furiosus, in a similar fashion to the eukaryotic polymerase alpha-primase complex.
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PMID:The archaeal DNA primase: biochemical characterization of the p41-p46 complex from Pyrococcus furiosus. 1158 1

DNA polymerase alpha-primase (pol-prim) is a heterotetramer with DNA polymerase and primase activities. The polymerase (p180) and primase (p48 and p58) subunits synthesize primers and extend them, but the function of the remaining subunit (p68) is poorly understood. Genetic studies in yeast suggested an essential role for the p68 ortholog in early S phase prior to the hydroxyurea-sensitive step, possibly a regulatory role in initiation of DNA replication, but found no evidence for an essential function of p68 later in S phase. To investigate whether the human p68 subunit has an essential role in DNA replication, we examined the ability of a purified trimeric human pol-prim lacking p68 to initiate simian virus 40 DNA replication in vitro and to synthesize and elongate primers on single-stranded DNA in the presence of T antigen and replication protein A (RPA). Both activities of trimeric pol-prim were defective, but activity was recovered upon addition of separately purified p68. Phosphorylation of p68 by cyclin A-dependent protein kinase also inhibited both activities of pol-prim. The data strongly suggest that the p68 subunit is required for priming activity of pol-prim in the presence of RPA and T antigen, both during initiation at the origin and during lagging strand replication.
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PMID:Role of the p68 subunit of human DNA polymerase alpha-primase in simian virus 40 DNA replication. 1213 79

DNA polymerase alpha-primase is a heterotetrameric complex essential for simian vacuolating virus 40 (SV40) DNA replication. We show that the C-terminal 67 amino acid residues of the human p180 subunit are essential for SV40 DNA replication as they are required for binding of the p68 subunit and play a role in the interaction with the primase subunits, p48 and p58. Furthermore, we demonstrate that exchanging these residues to those of mouse origin can only partially rescue the SV40 DNA replication activity of DNA polymerase alpha-primase.
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PMID:Control of complex formation of DNA polymerase alpha-primase and cell-free DNA replication by the C-terminal amino acids of the largest subunit p180. 1222 Jun 50

Eukaryotic DNA polymerase (pol) alpha is a complex of four subunits. The subunits in the yeast Saccharomyces cerevisiae are: 167, 79, 62 and 48 kDa polypeptides. The p79 subunit has no known enzymatic functions, but it is essential for growth and chromosomal DNA replication. We have analyzed the interaction between the subunits of yeast pol alpha, particularly p167 and p79, using a yeast two-hybrid screen and deletion analysis. We have identified the interaction sites in each of these two subunits leading to p167.p79 complex formation, and correlated our results with the available genetic data. A detailed two-hybrid analysis, using the p79 gene as the bait and a yeast genomic DNA library, identified two independent groups of positive clones. One group that displayed strong positive interaction (delta1) with p79 represented a fusion of the p167 open reading frame at 3502 bp (Ile1168), and the second group, displaying weak positive interaction (delta2) with p79, had a fusion at 3697 bp (Asn1233) with the DNA-binding domain of the yeast Gal4 transcription factor. A detailed deletion analysis of the downstream region indicated the existence of two subdomains that interact with p79. Subdomain I encompasses a 65 amino acid segment between Ile1168 and Phe1232, and subdomain II is a 25 amino acid segment between Glu1259 and Leu1283. Deletion and two-hybrid interaction analysis of the p79 subunit of pol alpha revealed a complementary region with two subdomains: a 67 amino acid segment between Asn189 and Gln255 (I) and a 68 amino acid segment between Glu256 and Met323 (II). The p79 subdomains I and II appeared to interact with the p167 subdomains I and II, respectively. Analysis of interaction between p62 and various deletion clones of p167 did not result in an unambiguous and stable positive interaction in the two-hybrid screen between these two subunits. A strong interaction between p167 and p62 would probably require the presence of either p79 or p48 in the complex.
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PMID:Subunit interactions in the assembly of Saccharomyces cerevisiae DNA polymerase alpha. 1268 56


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