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.

Cited by 63Open access

Mechanism of primase activity human primase polymerase PrimPol

2022

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Helicase-primase inhibitors

Gerald Kleymann · Drugs of the Future · 2003

The protein family of helicases separate or unwind duplex nucleic acids such as oligonucleotides, RNA or DNA into single strands. This enzymatic activity is essential for metabolic processes of DNA and RNA replication, transcription etc. and are often associated in enzyme complexes. To multiply, many pathogens encode their own helicase and/or primase to synthesize DNA. Inhibition of this enzymatic activity abrogates microbial propagation and can lead to the development of potent drugs for treating and curing infection. Recently, helicase-primase inhibitors were reported for the treatment of herpes simplex disease. Almost 25 years after the launch of the milestone antiherpes drug aciclovir (20virax), these drug candidates represent the first compound class which outperforms the only available nucleosidic inhibitors of the herpes TK/DNA-polymerase with respect to all predinical pharmacologicai parameters. The helicase-primase enzyme complex appears to be the Achilles heel of herpes simplex viruses and is a prime target of antiinfective drug discovery. The antibiotic ciprofloxacin inhibits the bacterial topoisomerase II (DNA-gyrase) and IV and thus interferes with supercoiling of DNA. Drugs that inhibit supercoiling or unwinding of DNA seem to be the most effective antimicrobial agents. The mechanism of action of these drugs is based not only on the inhibition of a target but also on a mode of action that triggers subsequent molecular events. cascades and pathways which prevent propagation and even survival of the pathogen.

Cited by 8

The promiscuous primase

Si-Houy Lao-Sirieix, Luca Pellegrini, Stephen D. Bell · Trends in Genetics · 2005

Cited by 53

bacteriophage T4 primase

Karyn Goudie Belanger, Christine Mirzayan, Helen E. Kreuzer et al. · 1995

Cited by 0

primase-DNA-2bp

Pengzhi Wu, Niklas Senning, Frederic Allain · Biological Magnetic Resonance Bank · 2026

Cited by 0Open access

Primase (UL52) Proteins

Stacy D. Carrington-lawrence, Ra K. Weller, Stacy D. Carrington-lawrence et al. · 2002

Cited by 0

Eukaryotic DNA primase

Bahram Arezi, Robert D. Kuchta · Trends in Biochemical Sciences · 2000

Cited by 139

primase-DNA-1bp

Pengzhi Wu, Frederic Allain · Biological Magnetic Resonance Bank · 2026

Cited by 0Open access

Resolution of DNA polymerase-α-primase complex and primase free

Ira M. Simet, Satyajit Ray, Subhash Basu · Journal of Biosciences · 1987

Cited by 1

DNA Sequence Recognition by DNA Primase Using High-Throughput Primase Profiling

Stefan Ilić, Shira Cohen, Ariel Afek et al. · Journal of Visualized Experiments · 2019

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. The binding of prokaryotic DnaG-like primases to DNA occurs at a specific trinucleotide recognition sequence. It is a pivotal step in the formation of Okazaki fragments. Conventional biochemical tools that are used to determine the DNA recognition sequence of DNA primase provide only limited information. Using a high-throughput microarray-based binding assay and consecutive biochemical analyses, it has been shown that 1) the specific binding context (flanking sequences of the recognition site) influences the binding strength of the DNA primase to its template DNA, and 2) stronger binding of primase to the DNA yields longer RNA primers, indicating higher processivity of the enzyme. This method combines PBM and primase activity assay and is designated as high-throughput primase profiling (HTPP), and it allows characterization of specific sequence recognition by DNA primase in unprecedented time and scalability.

Cited by 3

DNA Primase Isolated from the Yeast DNA Primase-DNA Polymerase Complex

Marianne Su‐Ling Brooks, Lawrence B. Dumas · Journal of Biological Chemistry · 1989

We have utilized immunoaffinity chromatography as a means of efficiently isolating a stable yeast DNA primase from the DNA primase-DNA polymerase complex, allowing identification of the polypeptides associated with this DNA primase activity and comparison of its enzymatic properties with those of the larger protein complex. A mouse monoclonal antibody specifically recognizing the DNA polymerase subunit was used to purify the complex. Stable DNA primase was subsequently separated from the complex in high yield. The highly purified protein fraction which bound to the DNA polymerase antibody column consisted of polypeptides with apparent molecular masses of 180, 86, 70, 58, 49, and 47 kDa. DNA primase activity eluted with a fraction containing only the 58-, 49-, and 47-kDa polypeptides. Partial chemical cleavage analysis of these three proteins demonstrated that the 49- and 47-kDa polypeptides are structurally related while the 58-kDa protein is unrelated to the other two. A DNA primase inhibitory monoclonal antibody was able to inhibit the activity of the purified DNA primase as well as the activity of the enzyme in the larger complex. In immunoprecipitation experiments, all three polypeptides were found in the immune complex. Thus, these three polypeptides are sufficient for DNA primase activity. In reactions using ribonucleotide substrates and natural as well as synthetic DNA templates, the purified DNA primase exhibited the same precise synthesis of unit length oligomers as did the larger protein complex and was able to extend these RNA oligomers by one additional unit length. An examination of the effects of deoxynucleotides on these DNA primase-catalyzed reactions revealed that the yeast DNA primase is an RNA-polymerizing enzyme and lacks significant DNA-polymerizing activity under the conditions tested.

Cited by 23Open access

The Archaeal DNA Primase

Lidong Liu, Kayoko Komori, Sonoko Ishino et al. · Journal of Biological Chemistry · 2001

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.

Cited by 79Open access

[Pol alpha-primase complex].

Masaharu Takemura, Shonen Yoshida · PubMed · 2002

Cited by 1

DNA Primase Small Subunit

National Cancer Institute · Definitions · 2020

DNA primase small subunit (420aa, ~50 kDa) is encoded by the human PRIM1 gene. This protein is involved in the biosynthesis of RNA primers for the Okazaki fragments made during DNA replication of the lagging strand.

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