Trapping DNA polymerases using triplex-forming oligodeoxyribonucleotides.
Triplexes (triple helices) formed within DNA templates prior to or during DNA synthesis cause DNA polymerase to terminate [Samadashwily et al., EMBO J. 13 (1993) 4975-4983]. Here, we show that triplex-forming oligodeoxyribonucleotides (oligos) efficiently trap DNA polymerases at target DNA sequences within single-stranded (ss) templates. This was observed for all studied DNA polymerases, including Sequenase and the thermophilic Taq and Vent polymerases. The termination rate depends on the fine structure of a triplex, as well as on ambient conditions such as temperature and the concentration of magnesium ions. Inhibition of DNA synthesis was observed not only when triplexes blocked the path of DNA polymerase, but also when a polymerization primer was involved in triplex formation. Escherichia coli ss-binding (SSB) protein helps DNA polymerase overcome the triplex barrier, but with an efficiency dramatically dependent on the triplex configuration. These results describe a novel method for blocking DNA replication at target homopurine-homopyrimidine sequences by means of triplex-forming oligos in direct analogy with similar results during transcription.
Department of Genetics, University of Illinois at Chicago 60612.
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Samadashwily
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Mirkin
D001483:Base Sequence; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism*; D013696:Temperature; D013698:Templates, Genetic
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D004259:DNA-Directed DNA Polymerase