Tangle analysis of site-specific recombination: Gin and Xer systems.

摘要

The topological approach to enzymology incubates circular DNA substrate molecules in an enzyme solution, and uses the enzyme-mediated changes in the geometry and topology of the substrate to infer enzyme binding and mechanism. I apply the tangle model [Summers et al., Q. J. Biophys. 289(1995), 253] to analyze knotted and linked products of site-specific recombination mediated by Gin, mutant Gin and Xer. Based on the experimental results of Kanaar et al. [Cell 62(1990), 353], I find that, under biologically reasonable assumptions, there is a unique mechanism of action that accounts for the products of processive recombination mediated by Gin. Crisona et al. [Journal of Molecular Biology 243(1994), 437] studied the topological changes induced in unknotted DNA substrates by a mutant of Gin. With the tangle model I show that this mutant is not specific for the same synaptic complex as the wild type, and that it uses at least two different recombinational schemes.; The second part of my work deals with Xer recombination system from Escherichia coli. Colloms et al. [Cell 88(1997), 855] found that Xer recombination on unknotted DNA substrates with directly repeated sites produces a right-handed 4-crossing torus link with anti-parallel sites. In Bath et al. [Journal of Molecular Biology 289(1999), 873], 6-crossing right-handed torus DNA links with anti-parallel Xer sites were used as substrates for Xer, producing DNA knots whose gel velocities indicate 7 crossings. Under certain assumptions, I am able to elucidate all possible Xer mechanisms on both the unknotted and the linked substrates. My results find very few other possible mechanisms in addition to the ones proposed by Colloms and Bath. Some of the mechanisms I found can be rejected on biological grounds. The results support a general model for Xer action that can be confirmed experimentally. I propose an experiment which will uniquely characterize Xer binding and mechanism.