Document 0693
 DOCN  M9590693
 TI    The localization of catalytic activities in Tn7 transposition
 DT    9509
 AU    May E; Sarnovsky R; Craig N; Department of MB & G, Johns Hopkins School
       of Medicine
 SO    NIH Conf Retroviral Integrase. 1995 Jan 19-20;:(Participants' abstracts
       and posters, abstract no. 6). Unique Identifier : AIDSLINE AIDS/95920026
 AB    We study the bacterial transposable element Tn7. Tn7 moves using a cut
       and paste mechanism which on the molecular level is similar to the
       movement of retroviruses. The Tn7 transposition machinery generates a
       pair of staggered breaks at the ends of the element, creating 3'
       recessed ends. These 3' ends, which terminate in CA, are subsequently
       jointed to the target DNA at displaced positions to generate an
       insertion product flanked by small direct repeats. Our research has
       focused on the determination of which of the transposon-encoded proteins
       is/are responsible for the initial excision events from the donor DNA
       molecule. Previous experiments have shown that all catalytic activities
       lie within the TnsA and TnsB proteins. These two proteins share a
       limited homology with each other and a large family of proteins,
       including retroviral integrases: the DD-35-E motif. Mutation at any of
       the three conserved acidic residues in TnsB yields protein which blocks
       3' strand cleavage and joining activities in a full TnsABC+D in vitro
       reaction. However 5' strand cleavage proceeds normally. Conversely, the
       use of TnsA mutant at the center acidic residue in its putative DD-35-E
       motif yields products where the 3' strand cleavage and joining
       activities are intact, but the 5' strand cleavage activity is dead. The
       simplest interpretation of these results is that TnsA cleaves the
       strands 5' of the transposon, while TnsB cleaves the strands 3' of the
       transposon and subsequently transfers them to the target DNA. When the
       5' strand cleavage activity is blocked in vitro, the resultant product
       is a fusion of the donor and target molecules, similar to the product of
       Bacteriophage Mu transposition. A surprising result was found when TnsA
       mutant in this activity was tested in vivo. When we tested mini-Tn7
       elements carrying a mutant TnsA gene (but wild type versions of the
       other Tns genes) in a mating-out assay, mobilization of a marker outside
       the transposon was observed. So it appears that the fusion product is
       generated in vivo and can be replicatively processed to form cointegrate
       molecules, which had as yet been unobserved as a Tn7
       transposition-mediated product.
 DE    DNA Repair  DNA, Complementary/GENETICS  DNA, Viral/GENETICS
       Electrophoresis, Gel, Two-Dimensional  HIV-1/*GENETICS  *Transcription,
       Genetic  Virus Integration/*GENETICS  MEETING ABSTRACT

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).