Mutational Analysis of the Mu Transposase

Abstract

Mu transposase is a member of a protein family that includes many transposases and the retroviral integrases. These recombinases catalyze the DNA cleavage and joining reactions essential for transpositional recombination. Here we demonstrate that, consistent with structural predictions, aspartate 336 of Mu transposase is required for catalysis of both DNA cleavage and DNA joining. This residue, although located 55 rather than 35 residues NH 2 -terminal of the essential glutamate, is undoubtedly the analog of the second aspartate of the AspAsp-35-Glu motif found in other family members. The core domain of Mu transposase consists of two subdomains: the NH 2 -terminal subdomain (IIA) contains the conserved Asp-Asp-Glu motif residues, whereas the smaller COOH-terminal subdomain (IIB) contains a large positively charged region exposed on its surface. To probe the function of domain IIB, we constructed mutant proteins carrying deletion or substitution mutations within this region. The activity of the deletion proteins revealed that domains IIA and IIB can be provided by different subunits in the transposase tetramer. Substitution mutations at two pairs of exposed lysine residues within the positively charged surface of domain IIB render transposase defective in transposition at a reaction step after DNA cleavage but prior to DNA joining. The severity of this defect depends on the structure of the DNA flanking the cleavage site. Thus, these data suggest that domain IIB is involved in manipulating the DNA near the cleavage site and that this function is important during the transition between the DNA cleavage and the DNA joining steps of recombination.Transposons are genetic elements that move from one site on DNA to another by transpositional recombination. Most transposons encode a single transposase protein responsible for the DNA cleavage and DNA joining reactions required during the initial recombination steps (1, 2). Retroviral integration follows a very similar recombination pathway as transposition (3, 4), and recent studies clearly establish that the transposases and retroviral integrases are a protein family whose members contain regions of similar amino acid sequence, protein structure, and function (5, 6).The transposase of bacteriophage Mu (MuA) is one of the best characterized members of the transposase/integrase family. MuA promotes two chemical steps during recombination: 1) donor DNA cleavage, which is the endonucleolytic cleavage of the phosphodiester bond between the element and the flanking host DNA, and 2) DNA strand transfer, the coupled DNA cleavage and joining reaction that covalently links the 3Ј ends of the element DNA to the DNA at the target site (5) (reviewed in Refs.

Reorganization of the Mu Transpososome Active Sites during a Cooperative Transition between DNA Cleavage and Joining

“…Transposase subunits anchor both ends of the Mu genome within the transpososome (6, 9, 10) by specifically binding repeat sequences near the Mu DNA ends through an N-terminal domain (11,12). Additional protein regions, within and adjacent to the catalytic core, have been implicated in nonspecific DNA binding (12)(13)(14) and are proposed to contribute amino acids to the active sites (8,(15)(16)(17). The catalytic core (14) contains the conserved DDE residues characteristic of the larger transposase/integrase family.…”

“…The catalytic core (14) contains the conserved DDE residues characteristic of the larger transposase/integrase family. These active site DDE residues are required specifically for the chemical steps of transposition (15,18,19) and coordinate the necessary catalytic divalent metal ions (20 -22). The transposase catalytic core and described DNA-binding domains are the minimal regions required for transposition in vitro when the Mu DNA ends are contained within short DNA fragments (23).…”

Effect of Mutations in the C-terminal Domain of Mu B on DNA Binding and Interactions with Mu A Transposase

“…Domain I is responsible for DNA binding of the enhancer element and the Mu ends (20,21). Domain II can be divided into two functionally distinct and complementary subdomains: domain II␣, which contains the conserved DDE motif believed to coordinate the divalent metal ion (22,23), and domain II␤, which is involved with transpososome assembly and may bind DNA (24,25). Domain III also contains two subdomains, domain III␣ and domain III␤.…”

Mutational analysis of RAG1 and RAG2 identifies three catalytic amino acids in RAG1 critical for both cleavage steps of V(D)J recombination

“…Each of these proteins contains a catalytic triad of two aspartic acids (D) and a glutamic acid (E), commonly referred to as the DDE motif (Kulkosky et al 1992;Grindley and Leschziner 1995). The DDE motif is required for DNA cleavage: Mutation of any one of the three residues results in a severe (∼100-fold) decrease in activity (Engelman and Craigie 1992; Kulkosky et al 1992;Baker and Luo 1994;Kim et al 1995;Bolland and Kleckner 1996;Sarnovsky et al 1996;Krementsova et al 1998). Whereas the precise role of the DDE motif in phosphodiester bond cleavage has not been fully elucidated, crystallographic analysis of several superfamily members indicates that the two aspartic acids coordinate divalent metal ion(s) that are necessary for catalysis (Grindley and Leschziner 1995).…”