Abstract
BackgroundA large subfamily of serine recombinases contains long polypeptide segments appended to the C-terminal end of the conserved catalytic domain. Members of this subfamily often function as phage integrases but also mediate transposition and regulate terminal differentiation processes in eubacteria. Although a few members of this subfamily have been studied in purified in vitro systems, key mechanistic aspects of reactions promoted by these recombinases remain to be determined, particularly with respect to the functions of the large C-terminal domain.ResultsWe have developed and characterized a robust in vitro recombination reaction by the Listeria phage A118 integrase, a member of the subfamily of serine recombinases containing a large C-terminal domain. The reaction occurs in a simple buffered salt solution and exhibits a modest stimulation by divalent cations or spermidine and DNA supercoiling. Recombination with purified A118 integrase is unidirectional, being efficient only between attP and attB DNA sites to either join separate DNA molecules (intermolecular recombination) or to generate deletions or inversions depending on the relative orientation of att sites in cis (intramolecular recombination). The minimal attP site is 50 bp but requires only 44 bp of base sequence information, whereas the minimal attB site is 42 bp and requires 38 bp of base sequence information. DNA exchange occurs between the central 2 bp of attP and attB. Identity between these two base pairs is required for recombination, and they solely determine the orientation of recombination sites. The integrase dimer binds efficiently to full att sites, including the attL and attR integration products, but poorly and differentially to each half-site. The large C-terminal domain can be separated from the N-terminal catalytic by partial proteolysis and mediates non-cooperative DNA binding to att sites.ConclusionsThe basic properties of the phage A118 integrase reaction and its substrate requirements have been elucidated. A118 integrase thus joins the handful of biochemically characterized serine integrases that are serving as models for mechanistic studies on this important class of recombinases. Information reported here will also be useful in exploiting this recombinase for genetic engineering.
Highlights
A large subfamily of serine recombinases contains long polypeptide segments appended to the C-terminal end of the conserved catalytic domain
Partial proteolysis of the A118 integrase by chymotrypsin or proteinase K demonstrated that the full-length enzyme consists of two distinct folded domains, an Nterminal domain corresponding to the catalytic core that is common to all serine recombinases linked to a large domain that constitutes over two-thirds of the protein (Figure 2E)
There are a number of fundamental questions regarding the mechanism of recombination reactions catalyzed by serine integrases, with respect to the roles of their C-terminal domains, whose sizes dwarf the much smaller catalytic domains
Summary
A large subfamily of serine recombinases contains long polypeptide segments appended to the C-terminal end of the conserved catalytic domain. Members of this subfamily often function as phage integrases and mediate transposition and regulate terminal differentiation processes in eubacteria. A few members of this subfamily have been studied in purified in vitro systems, key mechanistic aspects of reactions promoted by these recombinases remain to be determined, with respect to the functions of the large C-terminal domain. Members of the tyrosine recombinase family generate single-strand DNA breaks through the nucleophilic attack of a tyrosine hydroxyl. Members of the serine recombinase family generate double strand breaks in DNA through the concerted action of a pair of active site serine residues within the dimeric enzyme. DNA exchange occurs by translocation of subunits that are covalently linked to the cleaved DNA ends within the recombination complex in a reaction known as subunit rotation [10,11,12,13,14,15]
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