Human DNA Primase:  Anion Inhibition, Manganese Stimulation, and Their Effects on In Vitro Start-Site Selection

Kirk, Brian W.; Kuchta, Robert D.

doi:10.1021/bi990351u

Cited by 25 publications

(33 citation statements)

References 32 publications

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“…The same dependence has been observed for the human DNA primase: it was shown that Mn 2+ binds and stimulates the primase by decreasing the K m value for NTPs. 27 Thus, the determination of the intracellular composition in Archaea would be extremely useful in understanding the in vivo activities of the archaeal DNA primase and would contribute to understanding more precisely the length and composition of the primers synthesized. It is also possible that, in vivo, other factors are required to modulate the synthesis capability of the DNA primase.…”

Section: Discussionmentioning

confidence: 99%

“…Unfortunately, the optimal conditions fixed for measuring dNTP incorporation were insufficient to detect NTP incorporation. Because the presence of manganese improves NTP incorporation of the archaeal and eukaryotic primases, 13,17,[27][28][29] we studied the influence of Mn 2+ and Mg 2+ concentrations on RNA synthesis capability. Increased MgCl 2 concentrations only slightly enhanced NTP incorporation, in contrast to MnCl 2 (Fig.…”

Section: Rna Synthesis Capability Of the Pab Dna Primasementioning

confidence: 99%

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The Heterodimeric Primase from the Euryarchaeon Pyrococcus abyssi: A Multifunctional Enzyme for Initiation and Repair?

Breton

Henneke

Norais

et al. 2007

Journal of Molecular Biology

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We report on the characterization of the DNA primase complex of the hyperthermophilic archaeon Pyrococcus abyssi (Pab). The Pab DNA primase complex is composed of the proteins Pabp41 and Pabp46, which show sequence similarities to the p49 and p58 subunits, respectively, of the eukaryotic polymerase alpha-primase complex. Both subunits were expressed, purified, and characterized. The Pabp41 subunit alone had no RNA synthesis activity but could synthesize long (up to 3 kb) DNA strands. Addition of the Pabp46 subunit increased the rate of DNA synthesis but decreased the length of the DNA fragments synthesized and conferred RNA synthesis capability. Moreover, in our experimental conditions, Pab DNA primase had comparable affinities for ribonucleotides and deoxyribonucleotides, and its activity was dependent on the presence of Mg2+ and Mn2+. Interestingly, Pab DNA primase also displayed DNA polymerase, gap-filling, and strand-displacement activities. Genetic analyses undertaken in Haloferax volcanii suggested that the eukaryotic-type heterodimeric primase is essential for survival in archaeal cells. Our results are in favor of a multifunctional archaeal primase involved in priming and repair.

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Section: Discussionmentioning

confidence: 99%

Section: Rna Synthesis Capability Of the Pab Dna Primasementioning

confidence: 99%

The Heterodimeric Primase from the Euryarchaeon Pyrococcus abyssi: A Multifunctional Enzyme for Initiation and Repair?

Breton

Henneke

Norais

et al. 2007

Journal of Molecular Biology

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“…Eukaryal DNA primase is optimally active in the presence of manganese (15) and displays an exceptionally high rate of nucleotide misincorporation during templated RNA synthesis (16 -19). Archaeal DNA primase, too, is optimally active in the presence of manganese (20 -22).…”

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confidence: 99%

Nucleotide Misincorporation, 3′-Mismatch Extension, and Responses to Abasic Sites and DNA Adducts by the Polymerase Component of Bacterial DNA Ligase D

Yakovleva

Shuman

2006

Journal of Biological Chemistry

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DNA ligase D (LigD) participates in a mutagenic pathway of nonhomologous end joining in bacteria. LigD consists of an ATP-dependent ligase domain fused to a polymerase domain (POL) and a phosphoesterase module. The POL domain performs templated and nontemplated primer extension reactions with either dNTP or rNTP substrates. Here we report that Pseudomonas LigD POL is an unfaithful nucleic acid polymerase. Although the degree of infidelity in nucleotide incorporation varies according to the mispair produced, we find that a correctly paired ribonucleotide is added to the DNA primer terminus more rapidly than the corresponding correct deoxyribonucleotide and incorrect nucleotides are added much more rapidly with rNTP substrates than with dNTPs, no matter what the mispair configuration. We find that 3 mispairs are extended by LigD POL, albeit more slowly than 3 paired primer-templates. The magnitude of the rate effect on mismatch extension varies with the identity of the 3 mispair, but it was generally the case that mispaired ends were extended more rapidly with rNTP substrates than with dNTPs. These results lend credence to the suggestion that LigD POL might fill in short 5-overhangs with ribonucleotides when repairing double strand breaks in quiescent cells. We report that LigD POL can add a deoxynucleotide opposite an abasic lesion in the template strand, albeit slowly. Ribonucleotides are inserted more rapidly at an abasic lesion than are deoxys. LigD POL displays feeble activity in extending a preformed primer terminus opposing an abasic site, but can readily bypass the lesion by slippage of the primer 3 di-or trinucleotide and realignment to the template sequence distal to the abasic site. Covalent benzo[a]pyrene-dG and benzo[c]phenanthrene-dA adducts in the template strand are durable roadblocks to POL elongation. POL can slowly insert a dNMP opposite the adduct, but is impaired in the subsequent extension step. Bacterial DNA ligase D (LigD)2 is distinguished from all other DNA ligases insofar as its enzymatic activity is not limited to sealing DNA strands. Rather, it is a polyfunctional repair enzyme consisting of an ATP-dependent ligase domain fused to a polymerase domain (POL) and a phosphoesterase domain (1-10). Interest in LigD is driven by evidence that it collaborates with a bacterial Ku homolog in a nonhomologous end joining (NHEJ) pathway characterized by a high incidence of frameshift mutations at the sites of double strand break (DSB) repair (1-4, 9, 10). Biochemical characterization of the POL and phosphoesterase components suggests that they provide a means of remodeling the 3Ј ends of broken DNA strands prior to sealing by the ligase component (2-10).The LigD POL domain catalyzes either nontemplated single nucleotide additions to a blunt-ended duplex DNA or fill-in synthesis at a 5Ј-tailed duplex DNA; these are the molecular signatures of mutagenic mycobacterial NHEJ in vivo at bluntend and 5Ј-overhang DSBs, respectively (2, 9, 10). POL activity in vitro is optimal in the presence of manganese (...

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“…Previous studies have demonstrated that eukaryotic primase preferentially initiates within pyrimidine-rich sequences (3,32,35,40,44,71,85,89). In order for primase to initiate DNA synthesis, a stable complex must form between primase and the pyrimidine-rich initiation sites.…”

Section: Discussionmentioning

confidence: 99%

In the Simian Virus 40 In Vitro Replication System, Start Site Selection by the Polymerase α-Primase Complex Is Not Significantly Altered by Changes in the Concentration of Ribonucleotides

Purviance

Prack

Barbaro

et al. 2001

J Virol

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The simian virus 40 (SV40) in vitro replication system was previously used to demonstrate that the human polymerase (Pol) ␣-primase complex preferentially initiates DNA synthesis at pyrimidine-rich trinucleotide sequences. However, it has been reported that under certain conditions, nucleoside triphosphate (NTP) concentrations play a critical role in determining where eukaryotic primase initiates synthesis. Therefore, we have examined whether increased NTP concentrations alter the template locations at which SV40 replication is initiated. Our studies demonstrate that elevated ribonucleotide concentrations do not significantly alter which template sequences serve as initiation sites. Of considerable interest, the sequences that serve as initiation sites in the SV40 system are similar to those that serve as initiation sites for prokaryotic primases. It is also demonstrated that regardless of the concentration of ribonucleotides present in the reactions, DNA synthesis initiated outside of the core origin. These studies provide additional evidence that the Pol ␣-primase complex can initiate DNA synthesis only after a considerable amount of single-stranded DNA is generated.Studies employing the simian virus 40 (SV40) in vitro replication system have been used to establish much of what is known about the enzymology of eukaryotic DNA replication (10,40,81). This system has also been used to study the reaction mechanisms that take place during particular stages in the replication process; for instance, during the initiation of DNA replication. SV40 replication is initiated when T antigen (T-ag), the single viral protein necessary for replication, site specifically binds to the viral origin (reviewed in references 8, 11, and 31). Upon binding, T-ag assembles into a double hexamer (21,23,51,61,77) that is able to function as a helicase (22,34,66,68,84). Owing to its helicase activity, T-ag is capable of catalyzing origin-specific unwinding, provided replication protein A (RPA) and topoisomerase I are present in the reaction (15,22,27,88).Recent insights into initiation of DNA replication at the SV40 origin include images of T-ag double hexamers assembled on the viral core origin (77). Additional studies have helped to define the minimal core origin sequences necessary for T-ag double hexamer formation (41,42,67). Further insights into T-ag's interactions with the central region of the core origin, site II, were provided by the solution structure of the T-ag origin binding domain (T-ag-obd 131-260 ) (49). These studies established that a pair of loops is utilized to make sequence-specific contacts with site II (49); a similar surface is used by the DNA-binding domain of papillomavirus to bind to DNA (29). Recent experiments have also helped to unravel how T-ag's interactions with the core origin are controlled by the cell cycle machinery (5; reference 83 and references therein).The SV40 replication system has also been used to investigate the formation of nascent DNA in the vicinity of the SV40 origin following T-ag cataly...

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Human DNA Primase: Anion Inhibition, Manganese Stimulation, and Their Effects on In Vitro Start-Site Selection

Cited by 25 publications

References 32 publications

The Heterodimeric Primase from the Euryarchaeon Pyrococcus abyssi: A Multifunctional Enzyme for Initiation and Repair?

The Heterodimeric Primase from the Euryarchaeon Pyrococcus abyssi: A Multifunctional Enzyme for Initiation and Repair?

Nucleotide Misincorporation, 3′-Mismatch Extension, and Responses to Abasic Sites and DNA Adducts by the Polymerase Component of Bacterial DNA Ligase D

In the Simian Virus 40 In Vitro Replication System, Start Site Selection by the Polymerase α-Primase Complex Is Not Significantly Altered by Changes in the Concentration of Ribonucleotides

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