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.
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- Bailleul B. During in vivo maturation of eukaryotic nuclear mRNA, splicing yields excised exon circles. Nucleic Acids Res. 1996 Mar 15;24(6):1015–1019. doi: 10.1093/nar/24.6.1015. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Braun S., Domdey H., Wiebauer K. Inverse splicing of a discontinuous pre-mRNA intron generates a circular exon in a HeLa cell nuclear extract. Nucleic Acids Res. 1996 Nov 1;24(21):4152–4157. doi: 10.1093/nar/24.21.4152. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Canceill D., Ehrlich S. D. Copy-choice recombination mediated by DNA polymerase III holoenzyme from Escherichia coli. Proc Natl Acad Sci U S A. 1996 Jun 25;93(13):6647–6652. doi: 10.1073/pnas.93.13.6647. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Capel B., Swain A., Nicolis S., Hacker A., Walter M., Koopman P., Goodfellow P., Lovell-Badge R. Circular transcripts of the testis-determining gene Sry in adult mouse testis. Cell. 1993 Jun 4;73(5):1019–1030. doi: 10.1016/0092-8674(93)90279-y. [DOI] [PubMed] [Google Scholar]
- Chetverin A. B., Chetverina H. V., Demidenko A. A., Ugarov V. I. Nonhomologous RNA recombination in a cell-free system: evidence for a transesterification mechanism guided by secondary structure. Cell. 1997 Feb 21;88(4):503–513. doi: 10.1016/S0092-8674(00)81890-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
- Cocquerelle C., Daubersies P., Majérus M. A., Kerckaert J. P., Bailleul B. Splicing with inverted order of exons occurs proximal to large introns. EMBO J. 1992 Mar;11(3):1095–1098. doi: 10.1002/j.1460-2075.1992.tb05148.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cocquerelle C., Mascrez B., Hétuin D., Bailleul B. Mis-splicing yields circular RNA molecules. FASEB J. 1993 Jan;7(1):155–160. doi: 10.1096/fasebj.7.1.7678559. [DOI] [PubMed] [Google Scholar]
- Deirdre A., Scadden J., Smith C. W. Interactions between the terminal bases of mammalian introns are retained in inosine-containing pre-mRNAs. EMBO J. 1995 Jul 3;14(13):3236–3246. doi: 10.1002/j.1460-2075.1995.tb07326.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dietrich R. C., Incorvaia R., Padgett R. A. Terminal intron dinucleotide sequences do not distinguish between U2- and U12-dependent introns. Mol Cell. 1997 Dec;1(1):151–160. doi: 10.1016/s1097-2765(00)80016-7. [DOI] [PubMed] [Google Scholar]
- Gordenin D. A., Malkova A. L., Peterzen A., Kulikov V. N., Pavlov Y. I., Perkins E., Resnick M. A. Transposon Tn5 excision in yeast: influence of DNA polymerases alpha, delta, and epsilon and repair genes. Proc Natl Acad Sci U S A. 1992 May 1;89(9):3785–3789. doi: 10.1073/pnas.89.9.3785. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hall S. L., Padgett R. A. Requirement of U12 snRNA for in vivo splicing of a minor class of eukaryotic nuclear pre-mRNA introns. Science. 1996 Mar 22;271(5256):1716–1718. doi: 10.1126/science.271.5256.1716. [DOI] [PubMed] [Google Scholar]
- Jansen R., Ledley F. D. Disruption of phase during PCR amplification and cloning of heterozygous target sequences. Nucleic Acids Res. 1990 Sep 11;18(17):5153–5156. doi: 10.1093/nar/18.17.5153. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lai M. M. RNA recombination in animal and plant viruses. Microbiol Rev. 1992 Mar;56(1):61–79. doi: 10.1128/mr.56.1.61-79.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- MESELSON M., WEIGLE J. J. Chromosome brekage accompanying genetic recombination in bacteriophage. Proc Natl Acad Sci U S A. 1961 Jun 15;47:857–868. doi: 10.1073/pnas.47.6.857. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Madsen C. S., Ghivizzani S. C., Hauswirth W. W. In vivo and in vitro evidence for slipped mispairing in mammalian mitochondria. Proc Natl Acad Sci U S A. 1993 Aug 15;90(16):7671–7675. doi: 10.1073/pnas.90.16.7671. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Marton A., Delbecchi L., Bourgaux P. DNA nicking favors PCR recombination. Nucleic Acids Res. 1991 May 11;19(9):2423–2426. doi: 10.1093/nar/19.9.2423. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Meyerhans A., Vartanian J. P., Wain-Hobson S. DNA recombination during PCR. Nucleic Acids Res. 1990 Apr 11;18(7):1687–1691. doi: 10.1093/nar/18.7.1687. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mikheeva S., Hakim-Zargar M., Carlson D., Jarrell K. Use of an engineered ribozyme to produce a circular human exon. Nucleic Acids Res. 1997 Dec 15;25(24):5085–5094. doi: 10.1093/nar/25.24.5085. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Negroni M., Ricchetti M., Nouvel P., Buc H. Homologous recombination promoted by reverse transcriptase during copying of two distinct RNA templates. Proc Natl Acad Sci U S A. 1995 Jul 18;92(15):6971–6975. doi: 10.1073/pnas.92.15.6971. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nigro J. M., Cho K. R., Fearon E. R., Kern S. E., Ruppert J. M., Oliner J. D., Kinzler K. W., Vogelstein B. Scrambled exons. Cell. 1991 Feb 8;64(3):607–613. doi: 10.1016/0092-8674(91)90244-s. [DOI] [PubMed] [Google Scholar]
- Odelberg S. J., Weiss R. B., Hata A., White R. Template-switching during DNA synthesis by Thermus aquaticus DNA polymerase I. Nucleic Acids Res. 1995 Jun 11;23(11):2049–2057. doi: 10.1093/nar/23.11.2049. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Papanicolaou C., Ripley L. S. Polymerase-specific differences in the DNA intermediates of frameshift mutagenesis. In vitro synthesis errors of Escherichia coli DNA polymerase I and its large fragment derivative. J Mol Biol. 1989 May 20;207(2):335–353. doi: 10.1016/0022-2836(89)90258-1. [DOI] [PubMed] [Google Scholar]
- Parker R., Siliciano P. G. Evidence for an essential non-Watson-Crick interaction between the first and last nucleotides of a nuclear pre-mRNA intron. Nature. 1993 Feb 18;361(6413):660–662. doi: 10.1038/361660a0. [DOI] [PubMed] [Google Scholar]
- Pasman Z., Been M. D., Garcia-Blanco M. A. Exon circularization in mammalian nuclear extracts. RNA. 1996 Jun;2(6):603–610. [PMC free article] [PubMed] [Google Scholar]
- Päbo S., Irwin D. M., Wilson A. C. DNA damage promotes jumping between templates during enzymatic amplification. J Biol Chem. 1990 Mar 15;265(8):4718–4721. [PubMed] [Google Scholar]
- Tarn W. Y., Steitz J. A. A novel spliceosome containing U11, U12, and U5 snRNPs excises a minor class (AT-AC) intron in vitro. Cell. 1996 Mar 8;84(5):801–811. doi: 10.1016/s0092-8674(00)81057-0. [DOI] [PubMed] [Google Scholar]
- Tease C. Cytological detection of crossing-over in BUdR substituted meiotic chromosomes using the fluorescent plus Giemsa technique. Nature. 1978 Apr 27;272(5656):823–824. doi: 10.1038/272823a0. [DOI] [PubMed] [Google Scholar]
- Zaphiropoulos P. G. Circular RNAs from transcripts of the rat cytochrome P450 2C24 gene: correlation with exon skipping. Proc Natl Acad Sci U S A. 1996 Jun 25;93(13):6536–6541. doi: 10.1073/pnas.93.13.6536. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zaphiropoulos P. G. Exon skipping and circular RNA formation in transcripts of the human cytochrome P-450 2C18 gene in epidermis and of the rat androgen binding protein gene in testis. Mol Cell Biol. 1997 Jun;17(6):2985–2993. doi: 10.1128/mcb.17.6.2985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- d'Alençon E., Petranovic M., Michel B., Noirot P., Aucouturier A., Uzest M., Ehrlich S. D. Copy-choice illegitimate DNA recombination revisited. EMBO J. 1994 Jun 1;13(11):2725–2734. doi: 10.1002/j.1460-2075.1994.tb06563.x. [DOI] [PMC free article] [PubMed] [Google Scholar]