Non-homologous recombination mediated by Thermus aquaticus DNA polymerase I. Evidence supporting a copy choice mechanism

Nucleic Acids Res. 1998 Jun 15;26(12):2843-8. doi: 10.1093/nar/26.12.2843.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • Cytochrome P-450 Enzyme System / genetics
  • Cytochrome P450 Family 2
  • DNA, Complementary
  • Exons / genetics
  • Liver
  • Models, Genetic
  • Molecular Sequence Data
  • Polymerase Chain Reaction* / methods
  • RNA Splicing / genetics
  • Rats
  • Recombination, Genetic / physiology*
  • Repetitive Sequences, Nucleic Acid / genetics
  • Steroid 21-Hydroxylase / genetics
  • Taq Polymerase / metabolism*
  • Templates, Genetic

Substances

  • DNA, Complementary
  • Cytochrome P-450 Enzyme System
  • Cyp2c6v1 protein, rat
  • Cytochrome P450 Family 2
  • Steroid 21-Hydroxylase
  • Taq Polymerase