Kinetic and structural probing of the precleavage synaptic complex (type 0) formed during phage Mu transposition. Action of metal ions and reagents specific to single-stranded DNA.
In an earlier kinetic study (Wang, Z., and Harshey, R. M. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 699-703), we showed that supercoiling free energy was utilized during Mu transposition to lower the activation barrier of some rate-limiting step in the formation of the cleaved Mu end synaptic complex (type I complex). We report here results from kinetic studies on the assembled but uncleaved synaptic complex (type 0). Based on the estimated rate constants for the formation of type 0 and type I complexes, as well as their temperature and superhelicity dependence, we infer that the type 0 complex is an authentic intermediate in the pathway to Mu end cleavage. Our results are consistent with type 0 production being the rate-limiting step in the overall type I reaction. The conversion of type 0 to type I complex is a fast reaction, does not show strong temperature dependence, and is apparently independent of substrate superhelicity. We have explored the DNA structure within the type 0 complex using chemical and enzymatic probes. The observed susceptibility of DNA outside the Mu ends to single-strand-specific reagents suggests that a helix opening event is associated with type 0 formation. This structural perturbation could account, at least partly, for the high activation barrier to the reaction. There is a close correlation between the appearance of single strandedness near the Mu ends and the superhelicity of the DNA substrate. It is possible that supercoiling energy is utilized in effecting specific conformational transitions within DNA. We have found that Zn2+ and Co2+ ions, like Mg2+ and Mn2+ ions, can efficiently cleave the type 0 complex. However, unlike Mg2+ and Mn2+ ions, Zn2+ and Co2+ ions cannot support assembly of type 0. We discuss the implications of our findings for the mechanism of Mu transposition.
Wang
Z
Z
Department of Microbiology, University of Texas, Austin, Texas 78712, USA.
Namgoong
S Y
SY
Zhang
X
X
Harshey
R M
RM
D010583:Bacteriophage mu / Q000235:genetics*; D001483:Base Sequence; D002413:Cations, Divalent / Q000494:pharmacology*; D003035:Cobalt / Q000494:pharmacology; D004278:DNA, Superhelical / Q000737:chemistry* / Q000187:drug effects / Q000302:isolation & purification; D004279:DNA, Viral / Q000737:chemistry* / Q000187:drug effects / Q000302:isolation & purification; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008670:Metals / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation / Q000187:drug effects*; D010957:Plasmids; D015032:Zinc / Q000494:pharmacology
D002413:Cations, Divalent; D004278:DNA, Superhelical; D004279:DNA, Viral; D008670:Metals; D003035:Cobalt; D008345:Manganese; D008274:Magnesium; D015032:Zinc