RNA Priming Coupled with DNA Synthesis on Natural Template by Calf Thymus DNA Polymerase α-Primase

Motoshi Suzuki, Ericka Savoysky, Shunji Izuta, Masahiro Tatebe, Tetsuya Okajima, Shonen Yoshida

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

A bovine genomic DNA library was surveyed with respect to the template activity for RNA-primed DNA synthesis by calf thymus DNA polymerase a-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 oligodeoxyadenylate [(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 α-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 α-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.

Original languageEnglish
Pages (from-to)12782-12792
Number of pages11
JournalBiochemistry
Volume32
Issue number47
DOIs
Publication statusPublished - 01-01-1993

Fingerprint

DNA Primase
DNA-Directed DNA Polymerase
RNA
DNA
Substitution reactions
Pyrimidines
Electrophoretic Mobility Shift Assay
calf thymus DNA
pyrimidine
Assays
Gels
Poly T
Genomic Library
Single-Stranded DNA
Gene Library

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Suzuki, Motoshi ; Savoysky, Ericka ; Izuta, Shunji ; Tatebe, Masahiro ; Okajima, Tetsuya ; Yoshida, Shonen. / RNA Priming Coupled with DNA Synthesis on Natural Template by Calf Thymus DNA Polymerase α-Primase. In: Biochemistry. 1993 ; Vol. 32, No. 47. pp. 12782-12792.
@article{29f5e25e0a6b4e239852f42b9ee9b3c3,
title = "RNA Priming Coupled with DNA Synthesis on Natural Template by Calf Thymus DNA Polymerase α-Primase",
abstract = "A bovine genomic DNA library was surveyed with respect to the template activity for RNA-primed DNA synthesis by calf thymus DNA polymerase a-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 oligodeoxyadenylate [(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 α-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 α-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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Suzuki, M, Savoysky, E, Izuta, S, Tatebe, M, Okajima, T & Yoshida, S 1993, 'RNA Priming Coupled with DNA Synthesis on Natural Template by Calf Thymus DNA Polymerase α-Primase', Biochemistry, vol. 32, no. 47, pp. 12782-12792. https://doi.org/10.1021/bi00210a030

RNA Priming Coupled with DNA Synthesis on Natural Template by Calf Thymus DNA Polymerase α-Primase. / Suzuki, Motoshi; Savoysky, Ericka; Izuta, Shunji; Tatebe, Masahiro; Okajima, Tetsuya; Yoshida, Shonen.

In: Biochemistry, Vol. 32, No. 47, 01.01.1993, p. 12782-12792.

Research output: Contribution to journalArticle

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T1 - RNA Priming Coupled with DNA Synthesis on Natural Template by Calf Thymus DNA Polymerase α-Primase

AU - Suzuki, Motoshi

AU - Savoysky, Ericka

AU - Izuta, Shunji

AU - Tatebe, Masahiro

AU - Okajima, Tetsuya

AU - Yoshida, Shonen

PY - 1993/1/1

Y1 - 1993/1/1

N2 - A bovine genomic DNA library was surveyed with respect to the template activity for RNA-primed DNA synthesis by calf thymus DNA polymerase a-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 oligodeoxyadenylate [(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 α-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 α-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.

AB - A bovine genomic DNA library was surveyed with respect to the template activity for RNA-primed DNA synthesis by calf thymus DNA polymerase a-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 oligodeoxyadenylate [(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 α-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 α-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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