Non-homologous recombination mediated by Thermus aquaticus DNA polymerase I. Evidence supporting a copy choice mechanism.
RT-PCR amplification of P450 2C6 from rat liver, using primers in opposite orientations of exon 6, resulted in PCR products containing segments of exons joined at non-consensus splice sites. Moreover, many of the PCR products identified were composed of not only a single region containing exonic segments joined at non-consensus splice sites but, instead, of several repeats of the non-canonically joined region. To investigate whether these PCR products represent pre-existing molecules or are generated during the amplification process, the liver cDNA template was replaced by a plasmid containing the P450 2C6 cDNA. Surprisingly, PCR products containing repeats of non-canonically joined exonic segments were again revealed. In some cases the position of this non-canonical joining was a sequence of one or two identical nucleotides; however, there were also a number of products lacking any nucleotide identity at the position of joining. DNA nicking and/or DNA damage is thought to favour recombination during PCR, probably by misalignment of incomplete DNA strands; however, the presence of multiple repeats of the recombined region in the PCR products identified suggests a certain repetitiveness of the underlying mechanism. It is therefore proposed that these products result from a template switching event that occurs several times during a single polymerization step, following a rolling circle model of DNA synthesis.
Zaphiropoulos
P G
PG
Department of Bioscience, Center for Nutrition and Toxicology, Karolinska Institute, Novum, 141 57 Huddinge, Sweden. peza@cnt.ki.se
D000818:Animals; D001483:Base Sequence; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics; D000072467:Cytochrome P450 Family 2; D018076:DNA, Complementary; D005091:Exons / Q000235:genetics; D008099:Liver; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D012326:RNA Splicing / Q000235:genetics; D051381:Rats; D011995:Recombination, Genetic / Q000502:physiology*; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D013255:Steroid 21-Hydroxylase / Q000235:genetics; D019914:Taq Polymerase / Q000378:metabolism*; D013698:Templates, Genetic
D018076:DNA, Complementary; D003577:Cytochrome P-450 Enzyme System; C089877:Cyp2c6v1 protein, rat; D000072467:Cytochrome P450 Family 2; D013255:Steroid 21-Hydroxylase; D019914:Taq Polymerase