In vitro expansion of mammalian telomere repeats by DNA polymerase alpha-primase.
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)(5)T-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 approximately 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.
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.
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K
Nozawa
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M
Suzuki
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M
Takemura
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S
Yoshida
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D004256:DNA Polymerase I / Q000378:metabolism*; D019915:DNA Primase / Q000378:metabolism*; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D006147:Guanine; D010957:Plasmids; D012091:Repetitive Sequences, Nucleic Acid; D017384:Sequence Deletion; D013379:Substrate Specificity; D016615:Telomere / Q000378:metabolism*; D013698:Templates, Genetic; D013950:Thymus Gland / Q000201:enzymology
D017931:DNA Primers; D006147:Guanine; D019915:DNA Primase; C116932:DNA polymerase alpha-primase; D004256:DNA Polymerase I