Two structural features of λ integrase that are critical for DNA cleavage by multimers but not by monomers

Abstract

Despite many years of genetic and biochemical studies on the integrase (Int) recombination system, it is still not known whether the Int protein is competent for DNA cleavage as a monomer. We have addressed this question, as part of a larger study of Int functions critical for the formation of higher-order complexes, by isolating ''multimer-specific'' mutants. We identify a pair of oppositely charged residues, E153 and R169, that comprise an intermolecular salt bridge within a functional Int multimer. Mutation of either of these residues significantly reduces both the cleavage of full-att sites and the resolution of Holliday junctions without compromising the cleavage of half-att site substrates. Allelespecific suppressor mutations were generated at these residues. Their interaction with wild-type Int on preformed Holliday junctions indicates that the mutated residues comprise an intermolecular salt bridge. We have also shown that the most C-terminal seven residues of Int, which comprise another previously identified subunit interface, inhibit DNA cleavage by monomeric but not multimeric Int. Taken together, our results lead us to conclude that Int can cleave DNA as a monomer. We also identify and discuss unique structural features of Int that act negatively to reduce its activity as a monomer and other features that act positively to enhance its activity as a multimer. T he integrase protein (Int) of Escherichia coli phage (1), which mediates the integration and excision of the viral genome in to and out of its host chromosome by recombination at specific loci (att sites) (2), belongs to the large Int family of recombinases (for review see refs. 3 and 4). They use transient covalent phospho-tyrosine intermediates to first generate and then resolve Holliday junction (HJ) recombination intermediates by means of two sequential pairs of strand exchanges. The locus of these reactions on each partner DNA duplex is a pair of 9-to 13-bp inverted binding sites for the recombinase (core-type sites) separated by a 6-to 8-bp overlap region (7 bp in the case of ) whose boundaries are defined by the staggered and precisely positioned DNA cleavage sites. The att sites of the pathway, and other ''heterobivalent'' recombinases have the additional complexity of nearby binding sites for accessory DNA bending proteins and binding sites for a second, N-terminal, recombinase domain that binds distant ''arm-type'' DNA sites.Mechanistic studies of the Int family have benefited enormously from the availability of crystal structures of several family members (reviewed in ref. 5), the most relevant to this work being the structures for the catalytic domain of Int (6), the covalent complex with DNA of the Int C75 domain (which is analogous to the monovalent recombinases Cre and Flp) (7), and the Cre recombinase complexed with a HJ and a recombination synapse (8, 9). The common motif that emerges from all of the -family structures is a C-terminal catalytic domain (containing the tyrosine nucleophile and active site pocket) appropriately po...

Receipt of the C-terminal Tail from a Neighboring λ Int Protomer Allosterically Stimulates Holliday Junction Resolution

“…We believe that these aberrant hairpin products arise from uncoordinated DNA cleavages at "non-partner" Int binding sites, either during or after resolution. 14,16 These findings extend previous results 2, 3,5,6 indicating that while the C-terminal tail is not required for DNA cleavage by monomeric Int, it plays a critical role in the function and proper coordination of multimeric Int.…”

“…Consistent with the predictions of this structure, deletion of the Cterminal tail alleviates this repression and enhances the monomeric activity of Int. 2,5,6 In contrast, in an Int tetramer, which likely comprises the functional core of the recombinogenic complex, the C-terminal tail contacts the β5 strand of a neighboring Int to form a cyclic arrangement of linkages. 13 As a result of these intermolecular contacts (Figure 1(c)), the Tyr342 nucleophile is positioned near the active site (Figure 1(b)).…”

Non-equivalent Interactions between Amino-terminal Domains of Neighboring λ Integrase Protomers Direct Holliday Junction Resolution

“…19 Some of the asymmetry that delineates active from inactive partner pairs during recombination derives from the carboxy-terminal tail, which in all of the structurally characterized Int family members makes cyclic intermolecular connections within the HJ tetramer. [14][15][16][17]27 Here, we present evidence that an additional candidate for mediating and/or sensing the asymmetry necessary for orderly resolution of an HJ recombination intermediate is the amino-terminal DNA-binding domain, which is unique to the heterobivalent recombinases and plays a central role in regulation and directionality.…”

“…The two pairs of DNA strand cleavage/ligation reactions are separated physically by a 7 bp overlap region that is exchanged between recombining partners and they are temporally separated by conformational rearrangements that ensure only one pair of partner Ints is active at a time. A 2-fold symmetry delineates the "active" from the "inactive" partners within tetrameric complexes of tyrosine recombinases, [10][11][12][13][14][15][16][17] and was therefore not expected to extend to the small amino-terminal domain (residues 1-74), which is unique to the heterobivalent family members and considered to function primarily in the regulation and control of directionality.…”

Trans Cooperativity by a Split DNA Recombinase: The Central and Catalytic Domains of Bacteriophage Lambda Integrase Cooperate in Cleaving DNA Substrates When the Two Domains Are not Covalently Linked

“…2 DNA cleavage and recombination by λ-Int at its cognate "core" sites proceeds upon assembly of four protomers of the enzyme at a pair of T-rich semisymmetrical inverted sequences on the phage and bacterial genomic DNA. 3 Although the enzyme functions as a tetramer during recombination, the enzyme can also bind and cleave DNA without tetramer assembly, 4 as experiments in vitro show that it exhibits topoisomerase activity 5 and is capable of cleaving small DNA substrates consisting of core half-sites. 6 The 356-residue λ-Int enzyme consists of three structurally independent domains connected by extended peptide linkers: the amino-terminal "arm binding" domain (residues 1-64), the central "corebinding" domain (residues 65-169), and the C-terminal catalytic domain (residues 170-356).…”