Nucleic Acids Research Advance Access originally published online on February 25, 2009
Nucleic Acids Research 2009 37(7):2336-2345; doi:10.1093/nar/gkp096
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Nucleic Acids Research, 2009, Vol. 37, No. 7 2336-2345
© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Nucleic Acid Enzymes |
Functional complementation of UvsX and UvsY mutations in the mediation of T4 homologous recombination
Department of Biochemistry, University of Vermont College of Medicine, Burlington, VT 05405, USA
*To whom correspondence should be addressed. Tel: 802 656 8260; Fax: 802 656 8220; Email: smorrica{at}uvm.edu.
Received November 8, 2008. Revised February 4, 2009. Accepted February 5, 2009.
Bacteriophage T4 homologous recombination events are promoted by presynaptic filaments of UvsX recombinase bound to single-stranded DNA (ssDNA). UvsY, the phage recombination mediator protein, promotes filament assembly in a concentration-dependent manner, stimulating UvsX at stoichiometric concentrations but inhibiting at higher concentrations. Recent work demonstrated that UvsX-H195Q/A mutants exhibit decreased ssDNA-binding affinity and altered enzymatic properties. Here, we show that unlike wild-type UvsX, the ssDNA-dependent ATPase activities of UvsX-H195Q/A are strongly inhibited by both low and high concentrations of UvsY protein. This inhibition is partially relieved by UvsY mutants with decreased ssDNA-binding affinity. The UvsX-H195Q mutant retains weak DNA strand exchange activity that is inhibited by wild-type UvsY, but stimulated by ssDNA-binding compromised UvsY mutants. These and other results support a mechanism in which the formation of competent presynaptic filaments requires a hand-off of ssDNA from UvsY to UvsX, with the efficiency of the hand-off controlled by the relative ssDNA-binding affinities of the two proteins. Other results suggest that UvsY acts as a nucleotide exchange factor for UvsX, enhancing filament stability by increasing the lifetime of the high-affinity, ATP-bound form of the enzyme. Our findings reveal new details of the UvsX/UvsY relationship in T4 recombination, which may have parallels in other recombinase/mediator systems.