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Nucleic Acids Research, 2000, Vol. 28, No. 16 3117-3124
© 2000 Oxford University Press

In vitro expansion of mammalian telomere repeats by DNA polymerase {alpha}-primase

Katsura Nozawa, Motoshi Suzuki, Masaharu Takemura and Shonen Yoshida*

Laboratory of Cancer Cell Biology, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine, Tsurumai-cho 65, Showa-ku, Nagoya 466-8550, Japan

Among the polymerases, DNA polymerase {alpha}-primase is involved in lagging strand DNA synthesis. A previous report indicated that DNA polymerase {alpha}-primase initiates primer RNA synthesis with purine bases on a single-stranded G-rich telomere repeat. In this study, we found that DNA polymerase {alpha}-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 {alpha}-primase synthesized the nascent DNA fragments by extending the primer. It was remarkable that DNA polymerase {alpha}-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 {alpha} 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.

* To whom correspondence should be addressed. Tel: +81 52 744 2453; Fax: +81 52 744 2457; Email: syoshida@tsuru.med.nagoya-u.ac.jp


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