Primase from Escherichia coli primes single-stranded templates in the absence of single-stranded DNA-binding protein or other auxiliary proteins. Template sequence requirements based on the bacteriophage G4 complementary strand origin and Okazaki fragment initiation sites

Swart, John R.; Griep, Mark A.

doi:10.1016/s0021-9258(18)31481-9

Cited by 46 publications

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“…Other primases have shown template-specific activity and changes in specificity depending on the presence of various subunits (18,22,23,36,39,40). We found, however, that neither the UL8 component of helicase-primase nor the ICP8 single-stranded DNA-binding protein altered the preferential site of initiation on the </>X174 DNA molecule (11).…”

Section: Discussionmentioning

confidence: 93%

“…17). Th ese include E. coli dnaG primase recognition of t he seque nce 3'-GTC-5' with synt hesis of a primer in itia tin g wit h 5' -pppAG-3' (18,19), T4 primase recogni tion of 3'-TIG-5' or 3 '-TCG -5' with synthesis of a primer ini tiating with 5 '-pppAC-3' or 5 '-ppp GC-3' (35,36), T7 primase recogni tion of3 '-CTGG(T/G) or 3'-CTGTG-5 ' wit h sy nthesis of a primer ini tiating with 5'-pppACC(N C)-3' or 5'-pppA CAC (22,37), a nd DNA polyrne rase-o-primase recognition of 3' -CPyPy-5' (where Py is pyrimidine) wit h synthesis of a primer ini tiating with purine residues (23).…”

Section: Discussionmentioning

confidence: 99%

“…Thi s G residue , in the case of HSV primase, could not be t ru ncated or su bstit ute d by an A or T residue. Th e fact th at a n oligonucleotid e exte nding only 2 bases past thi s 3' -G res idue was abl e to se rve as a template distingui sh ed HSV prim ase from E. coli a nd T7 prim ases, which req uire at least six nucl eotid es 3 ' to t he 3 '-GTC-5 ' ini ti ati on si te (18,22).…”

Section: Discussionmentioning

confidence: 99%

“…17). On the other hand, the primases of Escherichia coli (18)(19)(20), T4 (21) and T7 (22), as well as the mammalian polymerase-o-prirnase complex (23) have all been shown to invoke some level of template specificity for their initiation. Here we identify the predominant HSV primase initiation site within the q.,X174 virion DNA template and use DNA oligonucleotides to define the size and sequence requirements for primer synthesis.…”

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confidence: 99%

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Sequence-dependent Primer Synthesis by the Herpes Simplex Virus Helicase-Primase Complex

Tenney

Sheaffer

Hurlburt

et al. 1995

Journal of Biological Chemistry

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The herpes simplex virus helicase-primase complex, a heterotrimer of the UL5, UL8, and UL52 proteins, displays a single predominant site of primer synthesis on phi X174 virion DNA (Tenney, D. J., Hurlburt, W. W., Micheletti, P. M., Bifano, M., and Hamatake, R. K. (1994) J. Biol. Chem. 269, 5030-5035). This site was mapped and found to be deoxycytosine-rich, directing the synthesis of a primer initiating with several guanine residues. The size and sequence requirements for primer synthesis were determined using oligonucleotides containing variations of the predominant template. Although the efficiency of primer synthesis on oligonucleotides was influenced by template size, it was absolutely dependent on nucleotide sequence. Conversely, the ATPase activity on oligonucleotide templates was dependent on template size rather than nucleotide sequence. Furthermore, only oligonucleotides containing primase templates were inhibitory in a coupled primase-polymerase assay using phi X174 DNA as template, suggesting that primer synthesis or primase turnover is rate-limiting. Additionally, stimulation of helicase-primase by the UL8 component and that by the ICP8 protein were shown to differ mechanistically using different templates: the UL8 component stimulated the rate of primer synthesis on phi X174 virion DNA and oligonucleotide templates, while ICP8 stimulation occurred only on phi X174 virion DNA.

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Section: Discussionmentioning

confidence: 93%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Sequence-dependent Primer Synthesis by the Herpes Simplex Virus Helicase-Primase Complex

Tenney

Sheaffer

Hurlburt

et al. 1995

Journal of Biological Chemistry

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“…Primase initiates RNA polymer synthesis at the sequence d(CTG) within a ssDNA template (Swart & Griep, 1993;Yoda & Okazaki, 1991;Yoda et al, 1988). Initiation begins with ATP and GTP complementary to the thymine and cytosine and proceeds in the usual direction.…”

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confidence: 99%

The Role of Zinc and the Reactivity of Cysteines in Escherichia coli Primase

Griep

Lokey

1996

Biochemistry

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Primase is the zinc metalloenzyme responsible for synthesizing RNA primers for use during DNA synthesis. To establish whether the zinc played a catalytic or structural role, the zinc was removed and the activity of the apoprimase determined. The zinc was removed with p-(hydroxymercuri)-benzenesulfonate (PMPS), which covalently reacts with cysteine sulfhydryls, EDTA was added to chelate the zinc, DTT was added to remove the PMPS from the apoprimase, and then the apoprimase was separated from the small molecules. The resulting apoprimase was fully active, indicating that the zinc played a structural role but not one involved in thermodynamic folding/unfolding. PMPS and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) cysteine reactivities indicated that the cysteines fell into three categories: one or two were fast-reacting, three were zinc-ligating, and two or three were slow or nonreacting. The major distinction between apoprimase and natural primase was that apoprimase became inactivated during storage at 4 degrees C for 10 days. Storage-induced inactivation correlated with disulfide bond formation and could be reversed by incubation with a mild reducing agent. Apoprimase oxidation also prevented zinc reconstitution which was only achieved with freshly-reduced enzyme, indicating that the zinc-ligating cysteines participated in the inactivating disulfide bonds. The conclusion was that, in natural primase, the zinc prevented disulfide bond formation which, in turn, prevented inactivation. The zinc reconstitution studies identified a strong and a weak zinc binding site. Zinc could be prevented from binding to the weak site by the presence of magnesium, indicating that the weak site may be the catalytic magnesium site in which two of the seven cysteines were located.

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Homogenous Assays for Escherichia coli DnaB-Stimulated DnaG Primase and DnaB Helicase and Their Use in Screening for Chemical Inhibitors

Zeng

Yang

Kao

et al. 2002

Analytical Biochemistry

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Primase from Escherichia coli primes single-stranded templates in the absence of single-stranded DNA-binding protein or other auxiliary proteins. Template sequence requirements based on the bacteriophage G4 complementary strand origin and Okazaki fragment initiation sites

Cited by 46 publications

References 0 publications

Sequence-dependent Primer Synthesis by the Herpes Simplex Virus Helicase-Primase Complex

Sequence-dependent Primer Synthesis by the Herpes Simplex Virus Helicase-Primase Complex

The Role of Zinc and the Reactivity of Cysteines in Escherichia coli Primase

Homogenous Assays for Escherichia coli DnaB-Stimulated DnaG Primase and DnaB Helicase and Their Use in Screening for Chemical Inhibitors

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