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

Author:
Zaphiropoulos, Peter G.
Source:
Nucleic acids research 1998 v.26 no.12 pp. 2843-2848
ISSN:
0305-1048
Subject:
DNA damage, DNA replication, DNA-directed DNA polymerase, Thermus aquaticus, chemical elements, complementary DNA, exons, liver, models, nucleotides, plasmids, polymerization, rats, reverse transcriptase polymerase chain reaction
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.
Agid:
2271445