Inversion of the phosphate chirality at the target site of Mu DNA strand transfer: evidence for a one-step transesterification mechanism.
Central to transposition of phage Mu are two reactions mediated by the MuA protein. First, MuA introduces single-stranded cuts at the ends of the Mu DNA to generate 3' OH termini. In the subsequent strand-transfer step, the MuA-Mu DNA end complex cuts a target DNA and joins the Mu 3' ends to the 5' ends of the target. DNA containing chiral phosphorothioates was used to demonstrate inversion of the chirality during the course of strand transfer. This result strongly supports a one-step transesterification mechanism in which the 3' OH of the cleaved donor DNA is the attacking nucleophile. Furthermore, this donor 3' OH group was essential for target DNA cleavage. In contrast, during lambda integration the phosphate chirality was retained, as expected for a two-step transesterification involving a covalent protein-DNA intermediate.
Mizuuchi
K
K
Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health Bethesda, Maryland 20892.
Adzuma
K
K
D010582:Bacteriophage lambda / Q000235:genetics; D010583:Bacteriophage mu / Q000235:genetics*; D001483:Base Sequence; D004279:DNA, Viral / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009713:Nucleotidyltransferases / Q000378:metabolism*; D015345:Oligonucleotide Probes; D009946:Organothiophosphorus Compounds / Q000494:pharmacology; D010710:Phosphates; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D015183:Restriction Mapping; D013237:Stereoisomerism; D019895:Transposases
D004279:DNA, Viral; D015345:Oligonucleotide Probes; D009946:Organothiophosphorus Compounds; D010710:Phosphates; D009713:Nucleotidyltransferases; D019895:Transposases