Ty1 in vitro integration: effects of mutations in cis and in trans.
Mutations within the TYB gene of Ty1 encoding integrase (IN) as well as alterations in its substrate, a linear DNA molecule, were examined for their effects on in vitro IN activity, using a recently developed physical assay. Five different codon-insertion mutations, two frameshift mutations, and one missense mutation, previously identified as transposition-deficient mutations, were tested. Virus-like particles, the source of IN, from two different protease mutants and a reverse transcriptase mutant exhibited near-normal to normal IN activity. Two frameshift mutations mapping within the phylogenetically variable C-terminal domain of IN resulted in significant in vitro IN activity. In contrast, three mutations within the amino-terminal conserved domain of IN completely abolished IN activity. When the substrate termini were mutated, we found that substrates with as few as 4 bp of Ty1 termini were capable of efficiently generating integration products. Surprisingly, certain substrates that lacked obvious similarity to Ty1 termini were also readily integrated into both linear and circular targets, whereas others were not used as substrates at all. Termini rich in adenosine residues were among the more active substrates; however, certain substrates lacking terminal adenosine residues can form small quantities of integration products, including complete integration reactions.
Braiterman
L T
LT
Department of Molecular Biology & Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Boeke
J D
JD
D000595:Amino Acid Sequence; D001483:Base Sequence; D004252:DNA Mutational Analysis; D004254:DNA Nucleotidyltransferases / Q000378:metabolism; D004251:DNA Transposable Elements; D004271:DNA, Fungal / Q000235:genetics; D019426:Integrases; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D012331:RNA, Fungal / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D011995:Recombination, Genetic; D012091:Repetitive Sequences, Nucleic Acid; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013379:Substrate Specificity
D004251:DNA Transposable Elements; D004271:DNA, Fungal; D009838:Oligodeoxyribonucleotides; D012331:RNA, Fungal; D004254:DNA Nucleotidyltransferases; D019426:Integrases; D012194:RNA-Directed DNA Polymerase