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Journal of Bacteriology, May 2005, p. 3431-3437, Vol. 187, No. 10
0021-9193/05/$08.00+0     doi:10.1128/JB.187.10.3431-3437.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Piv Site-Specific Invertase Requires a DEDD Motif Analogous to the Catalytic Center of the RuvC Holliday Junction Resolvases

John M. Buchner, Anne E. Robertson, David J. Poynter,{dagger} Shelby S. Denniston,{ddagger} and Anna C. Karls*

Department of Microbiology, University of Georgia, Athens, Georgia 30602-2605

Received 27 December 2004/ Accepted 7 February 2005

Piv, a unique prokaryotic site-specific DNA invertase, is related to transposases of the insertion elements from the IS110/IS492 family and shows no similarity to the site-specific recombinases of the tyrosine- or serine-recombinase families. Piv tertiary structure is predicted to include the RNase H-like fold that typically encompasses the catalytic site of the recombinases or nucleases of the retroviral integrase superfamily, including transposases and RuvC-like Holliday junction resolvases. Analogous to the DDE and DEDD catalytic motifs of transposases and RuvC, respectively, four Piv acidic residues D9, E59, D101, and D104 appear to be positioned appropriately within the RNase H fold to coordinate two divalent metal cations. This suggests mechanistic similarity between site-specific inversion mediated by Piv and transposition or endonucleolytic reactions catalyzed by enzymes of the retroviral integrase superfamily. The role of the DEDD motif in Piv catalytic activity was addressed using Piv variants that are substituted individually or multiply at these acidic residues and assaying for in vivo inversion, intermolecular recombination, and DNA binding activities. The results indicate that all four residues of the DEDD motif are required for Piv catalytic activity. The DEDD residues are not essential for inv recombination site recognition and binding, but this acidic tetrad does appear to contribute to the stability of Piv-inv interactions. On the basis of these results, a working model for Piv-mediated inversion that includes resolution of a Holliday junction is presented.

* Corresponding author. Mailing address: Department of Microbiology, University of Georgia, Biological Sciences Building, Cedar St., Athens, GA 30602-2605. Phone: (706) 583-0822. Fax: (706) 542-2674. E-mail: akarls{at}uga.edu.

{dagger} Present address: Wake Forest University School of Medicine, Winston-Salem, NC.

{ddagger} Present address: University of Tennessee Health Science Center, College of Pharmacy, Memphis, TN.

Journal of Bacteriology, May 2005, p. 3431-3437, Vol. 187, No. 10
0021-9193/05/$08.00+0     doi:10.1128/JB.187.10.3431-3437.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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