Activating mutations of Tn3 resolvase marking interfaces important in recombination catalysis and its regulation

Abstract

SummaryCatalysis of DNA recombination by Tn 3 resolvase is conditional on prior formation of a synapse, comprising 12 resolvase subunits and two recombination sites ( res ). Each res binds a resolvase dimer at site I, where strand exchange takes place, and additional dimers at two adjacent 'accessory' binding sites II and III. 'Hyperactive' resolvase mutants, that catalyse strand exchange at site I without accessory sites, were selected in E. coli . Some single mutants can resolve a res ¥ ¥ ¥ ¥ site I plasmid (that is, with one res and one site I), but two or more activating mutations are necessary for efficient resolution of a site I ¥ ¥ ¥ ¥ site I plasmid. Site I ¥ ¥ ¥ ¥ site I resolution by hyperactive mutants can be further stimulated by mutations at the crystallographic 2-3 ¢ ¢ ¢ ¢ interface that abolish activity of wild-type resolvase. Activating mutations may allow regulatory mechanisms of the wild-type system to be bypassed, by stabilizing or destabilizing interfaces within and between subunits in the synapse. The positions and characteristics of the mutations support a mechanism for strand exchange by serine recombinases in which the DNA is on the outside of a recombinase tetramer, and the tertiary/quaternary structure of the tetramer is reconfigured.

Characterization of DNA Sequences Required for the CcrAB-Mediated Integration of Staphylococcal Cassette Chromosome mec , a Staphylococcus aureus Genomic Island

“…SCCmec integrates into and excises from the chromosome in a site-specific manner mediated by a group of serine recombinases, CcrAB and CcrC, encoded within the element (14,16,17,19,31). Serine recombinases, such as phage integrases, bring together two attachment (att) sites and then catalyze DNA cleavage, strand exchange, and recombination (1,5,28,29,30). For SCCmec insertion, CcrB binds to specific sites, one on the incoming, circular SCCmec and the other on the staphylococcal chromosome (attS and attB, respectively), and mediates integration with the help of CcrA by protein-protein interaction.…”

A possible way that φC31 integrase regulates the recombination direction

“…The mutations in V129 have no effect on other responsible domains. As V129G and V129M mutants act efficiently as normal integrase in synapsis but appear to be defective in attB×attP recombination, V129 may also play a critical role in activating the post-synapsis cleavage besides synapsis , which is distinct from resolvases (Burke et al, 2004;Li et al, 2005;Keenholtz et al, 2011). Therefore, the latter explanation appears to be more reasonable.…”

“…Aside from φC31 integrase, some serine resolvases can also be activated by mutations (Burke et al, 2004;Li et al, 2005;Keenholtz et al, 2011). Their crystal structures have been well determined (Li et al, 2005;Keenholtz et al, 2011), pointing to the same activating principle that most of the mutations tend to stabilize the resolvase tetramer or destabilize its dimeric form (Li et al, 2005;Keenholtz et al, 2011).…”

“…isolated several mutated integrases that gain the ability to recombine attL and attR without affecting the attB×attP recombination. Interestingly, these mutants are all located in a putative coiled-coil region of CTD , rather than NTD like in serine resolvases (Burke et al, 2004;Li et al, 2005;Keenholtz et al, 2011). The putative coiled-coil structure contains two regions, E449-D477 and R489-A519 , which are termed C1 and C2, respectively, in this article.…”

“…This is consistent with the observation that some φC31 integrase variants have more stable dimeric forms and display a decreased recombination activity . Since the dimeric and tetrameric interfaces of serine recombinases are mainly composed of NTDs (Burke et al, 2004;Li et al, 2005;McEwan et al, 2009;Keenholtz et al, 2011), mutations in this domain can easily change the equilibrium between recombinase dimer and tetramer as in serine resolvases. Thus, it appears that the mutations in φC31 integrase CTD affect integrase synapsis in a rather intricate manner.…”