In vitro expansion of mammalian telomere repeats by DNA polymerase α-primase

K. Nozawa, Motoshi Suzuki, M. Takemura, S. Yoshida

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

Among the polymerases, DNA polymerase α-primase is involved in lagging strand DNA synthesis. A previous report indicated that DNA polymerase α-primase initiates primer RNA synthesis with purine bases on a single-stranded G-rich telomere repeat. In this study, we found that DNA polymerase α-primase precisely initiated with adenosine opposite the 3'-side thymidine in the G-rich telomere repeat 5'-(TTAGGG)(n)-3' under rATP-rich conditions. Then, DNA polymerase α-primase synthesized the nascent DNA fragments by extending the primer. It was remarkable that DNA polymerase α-primase further expanded the product DNA far beyond the length of the template DNA, as ladders of multiple hexanucleotides on polyacrylamide gel electrophoresis. Using an oligomer duplex 5'-A(GGGTTA)5-3'/5'-(TAACCC)5T-3' as a template-primer, we show that both the Klenow fragment of Escherichia coli DNA polymerase I and HIV reverse transcriptase could expand telomere DNA sequences as well, giving products greater than the size of the template DNA. The maximum product lengths with these polymerases were ~40-90 nt longer than the template length. Our data imply that DNA polymerases have an intrinsic activity to expand the hexanucleotide repeats of the telomere sequence by a slippage mechanism and that DNA polymerase α uses both the repeat DNA primers and the de novo RNA primers for expansion. On the other hand, a plasmid harboring a eukaryotic telomere repeat showed remarkable genetic instability in E.coli. The telomere repeats exhibited either expansions or deletions by multiple hexanucleotide repeats during culture for a number of generations, suggesting involvement of the slippage mechanism in the instability of telomeric DNA in vivo.

Original languageEnglish
Pages (from-to)3117-3124
Number of pages8
JournalNucleic Acids Research
Volume28
Issue number16
Publication statusPublished - 15-08-2000
Externally publishedYes

Fingerprint

DNA Primase
Telomere
DNA-Directed DNA Polymerase
DNA
DNA Polymerase I
Escherichia coli
HIV Reverse Transcriptase
DNA Primers
In Vitro Techniques
Adenosine
Thymidine
Polyacrylamide Gel Electrophoresis
Plasmids

All Science Journal Classification (ASJC) codes

  • Genetics

Cite this

Nozawa, K. ; Suzuki, Motoshi ; Takemura, M. ; Yoshida, S. / In vitro expansion of mammalian telomere repeats by DNA polymerase α-primase. In: Nucleic Acids Research. 2000 ; Vol. 28, No. 16. pp. 3117-3124.
@article{7b5fe1cae6f040c9b50f54642f8a5ab3,
title = "In vitro expansion of mammalian telomere repeats by DNA polymerase α-primase",
abstract = "Among the polymerases, DNA polymerase α-primase is involved in lagging strand DNA synthesis. A previous report indicated that DNA polymerase α-primase initiates primer RNA synthesis with purine bases on a single-stranded G-rich telomere repeat. In this study, we found that DNA polymerase α-primase precisely initiated with adenosine opposite the 3'-side thymidine in the G-rich telomere repeat 5'-(TTAGGG)(n)-3' under rATP-rich conditions. Then, DNA polymerase α-primase synthesized the nascent DNA fragments by extending the primer. It was remarkable that DNA polymerase α-primase further expanded the product DNA far beyond the length of the template DNA, as ladders of multiple hexanucleotides on polyacrylamide gel electrophoresis. Using an oligomer duplex 5'-A(GGGTTA)5-3'/5'-(TAACCC)5T-3' as a template-primer, we show that both the Klenow fragment of Escherichia coli DNA polymerase I and HIV reverse transcriptase could expand telomere DNA sequences as well, giving products greater than the size of the template DNA. The maximum product lengths with these polymerases were ~40-90 nt longer than the template length. Our data imply that DNA polymerases have an intrinsic activity to expand the hexanucleotide repeats of the telomere sequence by a slippage mechanism and that DNA polymerase α uses both the repeat DNA primers and the de novo RNA primers for expansion. On the other hand, a plasmid harboring a eukaryotic telomere repeat showed remarkable genetic instability in E.coli. The telomere repeats exhibited either expansions or deletions by multiple hexanucleotide repeats during culture for a number of generations, suggesting involvement of the slippage mechanism in the instability of telomeric DNA in vivo.",
author = "K. Nozawa and Motoshi Suzuki and M. Takemura and S. Yoshida",
year = "2000",
month = "8",
day = "15",
language = "English",
volume = "28",
pages = "3117--3124",
journal = "Nucleic Acids Research",
issn = "0305-1048",
publisher = "Oxford University Press",
number = "16",

}

Nozawa, K, Suzuki, M, Takemura, M & Yoshida, S 2000, 'In vitro expansion of mammalian telomere repeats by DNA polymerase α-primase', Nucleic Acids Research, vol. 28, no. 16, pp. 3117-3124.

In vitro expansion of mammalian telomere repeats by DNA polymerase α-primase. / Nozawa, K.; Suzuki, Motoshi; Takemura, M.; Yoshida, S.

In: Nucleic Acids Research, Vol. 28, No. 16, 15.08.2000, p. 3117-3124.

Research output: Contribution to journalArticle

TY - JOUR

T1 - In vitro expansion of mammalian telomere repeats by DNA polymerase α-primase

AU - Nozawa, K.

AU - Suzuki, Motoshi

AU - Takemura, M.

AU - Yoshida, S.

PY - 2000/8/15

Y1 - 2000/8/15

N2 - Among the polymerases, DNA polymerase α-primase is involved in lagging strand DNA synthesis. A previous report indicated that DNA polymerase α-primase initiates primer RNA synthesis with purine bases on a single-stranded G-rich telomere repeat. In this study, we found that DNA polymerase α-primase precisely initiated with adenosine opposite the 3'-side thymidine in the G-rich telomere repeat 5'-(TTAGGG)(n)-3' under rATP-rich conditions. Then, DNA polymerase α-primase synthesized the nascent DNA fragments by extending the primer. It was remarkable that DNA polymerase α-primase further expanded the product DNA far beyond the length of the template DNA, as ladders of multiple hexanucleotides on polyacrylamide gel electrophoresis. Using an oligomer duplex 5'-A(GGGTTA)5-3'/5'-(TAACCC)5T-3' as a template-primer, we show that both the Klenow fragment of Escherichia coli DNA polymerase I and HIV reverse transcriptase could expand telomere DNA sequences as well, giving products greater than the size of the template DNA. The maximum product lengths with these polymerases were ~40-90 nt longer than the template length. Our data imply that DNA polymerases have an intrinsic activity to expand the hexanucleotide repeats of the telomere sequence by a slippage mechanism and that DNA polymerase α uses both the repeat DNA primers and the de novo RNA primers for expansion. On the other hand, a plasmid harboring a eukaryotic telomere repeat showed remarkable genetic instability in E.coli. The telomere repeats exhibited either expansions or deletions by multiple hexanucleotide repeats during culture for a number of generations, suggesting involvement of the slippage mechanism in the instability of telomeric DNA in vivo.

AB - Among the polymerases, DNA polymerase α-primase is involved in lagging strand DNA synthesis. A previous report indicated that DNA polymerase α-primase initiates primer RNA synthesis with purine bases on a single-stranded G-rich telomere repeat. In this study, we found that DNA polymerase α-primase precisely initiated with adenosine opposite the 3'-side thymidine in the G-rich telomere repeat 5'-(TTAGGG)(n)-3' under rATP-rich conditions. Then, DNA polymerase α-primase synthesized the nascent DNA fragments by extending the primer. It was remarkable that DNA polymerase α-primase further expanded the product DNA far beyond the length of the template DNA, as ladders of multiple hexanucleotides on polyacrylamide gel electrophoresis. Using an oligomer duplex 5'-A(GGGTTA)5-3'/5'-(TAACCC)5T-3' as a template-primer, we show that both the Klenow fragment of Escherichia coli DNA polymerase I and HIV reverse transcriptase could expand telomere DNA sequences as well, giving products greater than the size of the template DNA. The maximum product lengths with these polymerases were ~40-90 nt longer than the template length. Our data imply that DNA polymerases have an intrinsic activity to expand the hexanucleotide repeats of the telomere sequence by a slippage mechanism and that DNA polymerase α uses both the repeat DNA primers and the de novo RNA primers for expansion. On the other hand, a plasmid harboring a eukaryotic telomere repeat showed remarkable genetic instability in E.coli. The telomere repeats exhibited either expansions or deletions by multiple hexanucleotide repeats during culture for a number of generations, suggesting involvement of the slippage mechanism in the instability of telomeric DNA in vivo.

UR - http://www.scopus.com/inward/record.url?scp=0034663532&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0034663532&partnerID=8YFLogxK

M3 - Article

C2 - 10931927

AN - SCOPUS:0034663532

VL - 28

SP - 3117

EP - 3124

JO - Nucleic Acids Research

JF - Nucleic Acids Research

SN - 0305-1048

IS - 16

ER -