The integrase of the conjugative transposon Tn916 directs strand- and sequence-specific cleavage of the origin of conjugal transfer, oriT, by the endonuclease Orf20.

ABSTRACT: Orf20 of the conjugative transposon Tn916 was purified as a chimeric protein fused to maltose binding protein (MBP-Orf20). The chimeric protein possessed endonucleolytic activity, cleaving both strands of the Tn916 origin of conjugal transfer (oriT) at several distinct sites and favoring GT dinucleotides. Incubation of the oriT DNA with purified Tn916 integrase (Int) and MBP-Orf20 resulted in strand- and sequence-specific cleavage of oriT at a TGGT motif in the transferred strand. This motif lies immediately adjacent to a sequence in oriT previously shown to be protected from DNase I cleavage by Int. The endonucleolytic cleavages produced by Orf20 generated a 3' OH group that could be radiolabeled by dideoxy ATP and terminal transferase. The production of a 3' OH group distinguished these

SUBMITTER: Rocco JM 

PROVIDER: S-EPMC1428151 | biostudies-literature | 2006 Mar

REPOSITORIES: biostudies-literature