Immunological analysis of the polypeptide structure of calf thymus DNA polymerase-primase complex.

Holmes, Andrew M.; Cheriathundam, Elizabeth; Bollum, F. J.; Chang, Lucy M.S.

doi:10.1016/s0021-9258(18)67329-6

Cited by 43 publications

(16 citation statements)

References 24 publications

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“…Our results showing that DNA polymerase a-primase binds to poly(dG) (Figure 5) are contradictory to previous results by Holmes et al (1986) that DNA polymerase a-primase binds to neither poly(dG) nor poly(dA). The reasons for this discrepancy may be due to the different reaction conditions.…”

Section: Discussioncontrasting

confidence: 99%

RNA priming coupled with DNA synthesis on natural template by calf thymus DNA polymerase .alpha.-primase

et al. 1993

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A bovine genomic DNA library was surveyed with respect to the template activity for RNA-primed DNA synthesis by calf thymus DNA polymerase alpha-primase complex. About 7% of the single-stranded DNA clones contained distinct initiation sites consisting of pyrimidine clusters of pyrimidine-rich sequences. The initiation sites were located at or near the 3'-end of the pyrimidine clusters. One of these sequences, containing a 10-mer pyrimidine cluster with major initiation sites, was analyzed in detail. By the successive substitutions of pyrimidines in the cluster with oligodeoxydenylate [(dA)n] in the 5' to 3' direction, the minimum length of the initiation sequence was estimated to be as long as the 7-mer. In contrast, when one or three pyrimidines at the 3'-end of the cluster were replaced with (dA)n, the priming activity was largely lost, indicating that these pyrimidine residues were indispensable for priming. Furthermore, base substitutions of upstream or downstream sequences outside the pyrimidine cluster also decreased the total priming frequencies. Interestingly, the base substitutions inside or outside of the pyrimidine cluster sometimes caused a shift in the major priming sites. These results indicate that the minimum priming unit of the CTPPS1 template for RNA-primed DNA synthesis consists of a pyrimidine cluster (6-mer) with one purine at its 3'-border and that both the 3'-downstream 6-bases and the 5'-upstream 17-bases modulate the priming by enhancing the priming frequency and/or slightly shifting the sites of initiation of primer synthesis. It was also revealed that the lengths of the product RNA primers became shorter as the length of pyrimidine cluster was shortened by substitution with (dA)n. The gel retardation assay further showed that the complex formation between DNA polymerase alpha-primase and the DNA templates was strongly in competition with poly(dC), poly(dG), and poly(dT) but not with poly(dA). Furthermore, template activities as well as the pyrimidine contents of a series of base-substituted DNA correlated well with their affinities to the enzyme, as measured by both gel retardation assay and their Km values for the priming reaction. Apparently, DNA polymerase alpha-primase primarily recognizes the minimum priming unit consisting of a pyrimidine cluster with a purine at the 3'-boundary of purine, but the initiation of primer RNA synthesis can be modified by pyrimidine residues outside of the minimum priming unit.

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Section: Discussioncontrasting

confidence: 99%

RNA priming coupled with DNA synthesis on natural template by calf thymus DNA polymerase .alpha.-primase

et al. 1993

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“…Physical association might also permit "channeling" of the primer-template from the primase active site to the pol a active site for efficient DNA synthesis. However, direct mechanistic evidence supporting these hypotheses is lacking.The primase and polymerase catalytic activities have been physically separated and are associated with different polypeptides within the complex: primase2 with the 48-kDa subunit and pol a with the 180-kDa subunit (Holmes et al, 1986;Grosse & Nasheuer, 1988;Santocanale et al, 1993). Thus, the 48-and 180-kDa subunits each contain a functioning active site as well as DNA binding domains that are at least partially independent.…”

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confidence: 99%

“…The primase and polymerase catalytic activities have been physically separated and are associated with different polypeptides within the complex: primase2 with the 48-kDa subunit and pol a with the 180-kDa subunit (Holmes et al, 1986;Grosse & Nasheuer, 1988;Santocanale et al, 1993). Thus, the 48-and 180-kDa subunits each contain a functioning active site as well as DNA binding domains that are at least partially independent.…”

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confidence: 99%

Calf Thymus DNA Polymerase .alpha.-Primase: "Communication" and Primer.cntdot.Template Movement between the Two Active Sites

Sheaff¹,

Kuchta²,

Ilsley³

1994

Biochemistry

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The DNA polymerase alpha-primase complex replicates single-stranded DNA by first synthesizing a short RNA primer (primase) which is then further elongated by the incorporation of dNTPs (DNA polymerase alpha). While primase and pol alpha function independently prior to synthesis of an RNA primer, the two activities become coordinated after primer synthesis. After primase generates a primer-template, it moves from the primase active site to the pol alpha active site for further elongation without dissociating into solution. Intramolecular transfer occurs immediately after primer synthesis and is employed on both long templates such as poly(dT) and short synthetic templates (< or = 60 nucleotides). Primer-template transfer and elongation by pol alpha are rapid compared to primer synthesis. After pol alpha elongates the primer, primase reinitiates primer synthesis, and the cycle is repeated. However, if dNTPs are absent such that primer elongation cannot occur, further primase activity is inhibited after a single round of primer synthesis. This "negative regulation" of primase activity is mediated by the newly generated primer-template provided the following conditions are met: (1) Primase synthesizes the primer; (2) the primer is 7-10 nucleotides long and remains bound to the template; (3) the template is of sufficient length; (4) the primer-template dissociates slowly from the enzyme complex; and (5) the primer-template interacts with the pol alpha active site. Polymerization of multiple dNTPs by pol alpha rapidly reactivates primase; hence, negative regulation of primase activity likely ensures a new primer is not synthesized until the previous one has been elongated by pol alpha.

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“…e eucaryotic replicative enzyme DNA polymerase a is characterized by its relatively high molecular weight, complex subunit composition, specific induction during the S-phase of the cell cycle, ability to use RNA primers, and sensitivity to aphidicolin, BuPdGTP,1 and BuAdATP (Fry & Loeb, 1986). Highly purified preparations of DNA polymerase a from mammalian cells and insects as well as the analogous DNA polymerase I in yeast consist of a DNA polymerase catalytic subunit of Mt = 145000-185000 in tight association with two to three additional subunits of Mrs = 47 000-86 000 (Kaguni et al, 1983;Chang et al, 1984;Karawya et al, 1984;Wahl et al, 1984;Denhardt & Faust, 1985;Plevani et al, 1985;Vishwanatha et al, 1986;Holmes et al, 1986;Wong et al, 1986;Nasheuer & Grosse, 1987 Pausch et al, 1988). Usually DNA polymerase « preparations exhibit a tightly bound oligoribonucleotide polymerase called primase that is associated with the , = 47 000-70000 polypeptides (Plevani et al, 1985;Holmes et al, 1986;Nasheuer & Grosse, 1987;Pausch et al, 1988).…”

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A novel primase-free form of murine DNA polymerase .alpha. induced by infection with minute virus of mice

Ho¹,

Gupta²,

Faust³

1989

Biochemistry

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Two species of DNA polymerase alpha free of primase activity were identified in extracts of Ehrlich mouse cells that had been infected with minute virus of mice. Primase-free forms of DNA polymerase alpha eluted with 150 and 180 mM NaCl during ion-exchange chromatography on DEAE-cellulose columns, exhibited sedimentation coefficients of 11 S and 8.2 S, respectively, and were inhibited by aphidicolin, N2-(p-n-butylphenyl)-9-(2-deoxy-beta-D-ribofuranosyl)guanine 5'-triphosphate, and 2-(p-n-butylanilino)-9-(2-deoxy-beta-D-ribofuranosyl)adenine 5'-triphosphate. The ratio of primase-free DNA polymerase alpha to the DNA polymerase alpha-primase complex increased from 1.5 to greater than 100 during the course of infection, and free primase was produced during the MVM replicative cycle.

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Immunological analysis of the polypeptide structure of calf thymus DNA polymerase-primase complex.

Cited by 43 publications

References 24 publications

RNA priming coupled with DNA synthesis on natural template by calf thymus DNA polymerase .alpha.-primase

RNA priming coupled with DNA synthesis on natural template by calf thymus DNA polymerase .alpha.-primase

Calf Thymus DNA Polymerase .alpha.-Primase: "Communication" and Primer.cntdot.Template Movement between the Two Active Sites

A novel primase-free form of murine DNA polymerase .alpha. induced by infection with minute virus of mice

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