title: Site-directed mutagenesis studies of tn5 transposase residues involved in synaptic complex formation.
abstract: Transposition (the movement of discrete segments of DNA, resulting in rearrangement of genomic DNA) initiates when transposase forms a dimeric DNA-protein synaptic complex with transposon DNA end sequences. The synaptic complex is a prerequisite for catalytic reactions that occur during the transposition process. The transposase-DNA interactions involved in the synaptic complex have been of great interest. Here we undertook a study to verify the protein-DNA interactions that lead to synapsis in the Tn5 system. Specifically, we studied (i) Arg342, Glu344, and Asn348 and (ii) Ser438, Lys439, and Ser445, which, based on the previously published cocrystal structure of Tn5 transposase bound to a precleaved transposon end sequence, make cis and trans contacts with transposon end sequence DNA, respectively. By using genetic and biochemical assays, we showed that in all cases except one, each of these residues plays an important role in synaptic complex formation, as predicted by the cocrystal structure.
authors: Bay Paul Center, Marine Biological Laboratory, Woods Hole, MA 02543, USA.
authors: Soheila
authors: S
authors: Vaezeslami
authors: Rachel
authors: R
authors: Sterling
authors: William S
authors: WS
authors: Reznikoff
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D004251:DNA Transposable Elements / Q000502:physiology*; D004269:DNA, Bacterial / Q000378:metabolism; D008954:Models, Biological; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D011485:Protein Binding / Q000235:genetics*; D011487:Protein Conformation; D019895:Transposases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D004251:DNA Transposable Elements; D004269:DNA, Bacterial; C473503:Tn5 transposase; D019895:Transposases
references: J Biol Chem. 1999 Dec 24;274(52):37021-9
references: 10601258
references: Mol Microbiol. 2006 Dec;62(6):1558-68
references: 17074070
references: Science. 2000 Jul 7;289(5476):77-85
references: 10884228
references: Proc Natl Acad Sci U S A. 2000 Aug 1;97(16):8944-9
references: 10908658
references: J Mol Biol. 2000 Sep 8;302(1):49-63
references: 10964560
references: J Biol Chem. 2001 Jun 22;276(25):23135-43
references: 11283001
references: J Bacteriol. 2002 Jan;184(1):233-40
references: 11741865
references: J Biol Chem. 2002 Mar 29;277(13):11284-91
references: 11805107
references: J Biol Chem. 2002 May 17;277(20):17623-9
references: 11877443
references: J Mol Biol. 2002 Oct 4;322(5):971-82
references: 12367522
references: J Biol Chem. 2003 Jan 17;278(3):1904-9
references: 12424243
references: Mol Microbiol. 2003 Mar;47(5):1199-206
references: 12603728
references: Nat Rev Mol Cell Biol. 2003 Nov;4(11):865-77
references: 14682279
references: Anal Biochem. 1976 May 7;72:248-54
references: 942051
references: Nature. 1983 Jul 21-27;304(5923):280-2
references: 6306482
references: J Mol Biol. 1984 Aug 25;177(4):645-61
references: 6090674
references: Gene. 1988 Mar 31;63(2):277-85
references: 2838392
references: Cell. 1991 Jul 12;66(1):129-40
references: 1649006
references: Annu Rev Microbiol. 1993;47:945-63
references: 7504907
references: J Mol Biol. 1994 Aug 12;241(2):166-77
references: 8057357
references: Genes Dev. 1994 Oct 1;8(19):2363-74
references: 7958902
references: J Mol Biol. 1997 Aug 22;271(3):362-73
references: 9268665
references: J Biol Chem. 1998 Mar 27;273(13):7367-74
references: 9516433
references: J Mol Biol. 1998 Mar 13;276(5):913-25
references: 9566196
references: Proc Natl Acad Sci U S A. 1998 Sep 1;95(18):10716-21
references: 9724770
references: Biochem Soc Trans. 2006 Apr;34(Pt 2):320-3
references: 16545104
references: Nucleic Acids Res. 2006;34(9):2820-32
references: 16717287
references: Biochemistry. 2006 Dec 26;45(51):15552-62
references: 17176076
references: Biochem Biophys Res Commun. 1999 Dec 29;266(3):729-34
references: 10603311