Efficient homologous recombination of Ty1 element cDNA when integration is blocked.
Integration of the yeast retrotransposon Ty1 into the genome requires the self-encoded integrase (IN) protein and specific terminal nucleotides present on full-length Ty1 cDNA. Ty1 mutants with defects in IN, the conserved termini of Ty1 cDNA, or priming plus-strand DNA synthesis, however, were still able to efficiently insert into the genome when the elements were expressed from the GAL1 promoter present on a multicopy plasmid. As with normal transposition, formation of the exceptional insertions required an RNA intermediate, Ty1 reverse transcriptase, and Ty1 protease. In contrast to Ty1 transposition, at least 70% of the chromosomal insertions consisted of complex multimeric Ty1 elements. Ty1 cDNA was transferred to the inducing plasmid as well as to the genome, and transfer required the recombination and repair gene RAD52. Furthermore, multimeric insertions occurred without altering the levels of total Ty1 RNA, virus-like particle-associated RNA or cDNA, Ty1 capsid proteins, or IN. These results suggest that Ty1 cDNA is utilized much more efficiently for homologous recombination when IN-mediated integration is blocked.
Sharon
G
G
Laboratory of Eukaryotic Gene Expression, NCI-Frederick Cancer Research and Development Center, ABL-Basic Research Program, Maryland 21702-1201.
Burkett
T J
TJ
Garfinkel
D J
DJ
D001483:Base Sequence; D004254:DNA Nucleotidyltransferases / Q000235:genetics; D004251:DNA Transposable Elements / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics; D010450:Endopeptidases / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics; D015321:Gene Rearrangement; D008040:Genetic Linkage; D006639:Histidine / Q000096:biosynthesis / Q000235:genetics; D019426:Integrases; D008969:Molecular Sequence Data; D016296:Mutagenesis; D012194:RNA-Directed DNA Polymerase / Q000235:genetics; D051721:Rad52 DNA Repair and Recombination Protein; D011995:Recombination, Genetic; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins
D004251:DNA Transposable Elements; D018076:DNA, Complementary; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C497197:RAD52 protein, S cerevisiae; D051721:Rad52 DNA Repair and Recombination Protein; D029701:Saccharomyces cerevisiae Proteins; D006639:Histidine; D004254:DNA Nucleotidyltransferases; D019426:Integrases; D012194:RNA-Directed DNA Polymerase; D010450:Endopeptidases