Effect of Mutations in the C-terminal Domain of Mu B on DNA Binding and Interactions with Mu A Transposase
2003; Elsevier BV; Volume: 278; Issue: 33 Linguagem: Inglês
10.1074/jbc.m303693200
ISSN1083-351X
AutoresColin J. Coros, Yukiko Sekino, Tania A. Baker, George Chaconas,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoBacteriophage Mu transposition requires two phage-encoded proteins, the transposase, Mu A, and an accessory protein, Mu B. Mu B is an ATP-dependent DNA-binding protein that is required for target capture and target immunity and is an allosteric activator of transpososome function. The recent NMR structure of the C-terminal domain of Mu B (Mu B223–312) revealed that there is a patch of positively charged residues on the solvent-exposed surface. This patch may be responsible for the nonspecific DNA binding activity displayed by the purified Mu B223–312 peptide. We show that mutations of three lysine residues within this patch completely abolish nonspecific DNA binding of the C-terminal peptide (Mu B223–312). To determine how this DNA binding activity affects transposition we mutated these lysine residues in the full-length protein. The full-length protein carrying all three mutations was deficient in both strand transfer and allosteric activation of transpososome function but retained ATPase activity. Peptide binding studies also revealed that this patch of basic residues within the C-terminal domain of Mu B is within a region of the protein that interacts directly with Mu A. Thus, we conclude that this protein segment contributes to both DNA binding and protein-protein contacts with the Mu transposase. Bacteriophage Mu transposition requires two phage-encoded proteins, the transposase, Mu A, and an accessory protein, Mu B. Mu B is an ATP-dependent DNA-binding protein that is required for target capture and target immunity and is an allosteric activator of transpososome function. The recent NMR structure of the C-terminal domain of Mu B (Mu B223–312) revealed that there is a patch of positively charged residues on the solvent-exposed surface. This patch may be responsible for the nonspecific DNA binding activity displayed by the purified Mu B223–312 peptide. We show that mutations of three lysine residues within this patch completely abolish nonspecific DNA binding of the C-terminal peptide (Mu B223–312). To determine how this DNA binding activity affects transposition we mutated these lysine residues in the full-length protein. The full-length protein carrying all three mutations was deficient in both strand transfer and allosteric activation of transpososome function but retained ATPase activity. Peptide binding studies also revealed that this patch of basic residues within the C-terminal domain of Mu B is within a region of the protein that interacts directly with Mu A. Thus, we conclude that this protein segment contributes to both DNA binding and protein-protein contacts with the Mu transposase. Bacteriophage Mu is a temperate phage that undergoes two forms of transposition during its life cycle. The first form of transposition occurs when Mu DNA integrates into its host genome through a nonreplicative transposition event called conservative integration (1Liebart J.C. Ghelardini P. Paolozzi L. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 4362-4366Crossref PubMed Scopus (35) Google Scholar, 2Chaconas G. Kennedy D.L. Evans D. Virology. 1983; 128: 48-59Crossref PubMed Scopus (20) Google Scholar, 3Harshey R.M. Cold Spring Harbor Symp. Quant. Biol. 1984; 49: 273-278Crossref PubMed Google Scholar). The second form occurs after integration, when the Mu DNA amplifies itself through a process known as replicative transposition (4Chaconas G. Harshey R.M. Sarvetnick N. Bukhari A.I. J. Mol. Biol. 1981; 150: 341-359Crossref PubMed Scopus (40) Google Scholar). The conservative integration reaction has never been reproduced in vitro, but the replicative transposition pathway has been characterized extensively using purified substrate and both host-encoded and phage-encoded proteins (5Chaconas G. Harshey R.M. Craig N.L. Lambowitz A. Gellert M. Craigie R. Mobile DNA II. ASM Press, Washington D. C.2002: 384-402Crossref Google Scholar).Mu replicative transposition can be performed in vitro using a supercoiled mini-Mu substrate, which contains the left end binding region (attL), the right end binding region (attR), and the enhancer element within a donor DNA molecule. The reaction occurs through a series of higher order nucleoprotein complexes called transpososomes. Transpososome formation up to and including strand transfer occurs in the presence of two phage-encoded proteins, Mu A and Mu B, and two host encoded proteins, HU and IHF (Fig. 1). In the presence of the mini-Mu substrate, Mu A, HU, IHF, and an appropriate divalent metal ion, the initial LER complex is formed. The LER complex is a transitory three-site complex that through numerous protein-protein and protein-DNA interactions brings together the left end, the right end, and the enhancer elements (6Watson M.A. Chaconas G. Cell. 1996; 85: 435-445Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). The first stable complex formed is the Type 0 (stable synaptic complex) (7Mizuuchi M. Baker T.A. Mizuuchi K. Cell. 1992; 70: 303-311Abstract Full Text PDF PubMed Scopus (129) Google Scholar, 8Wang Z. Namgoong S.-Y. Zhang X. Harshey R.M. J. Biol. Chem. 1996; 271: 9619-9626Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The Type 0 complex is characterized by the engagement of the Mu ends by the active site and accumulates in the presence of calcium, which does not support strand cleavage. In the presence of magnesium, the 3′-ends of the Mu DNA are nicked, and the Type 0 complex is quickly converted to Type 1 complex (cleaved donor complex) (9Craigie R. Mizuuchi K. Cell. 1987; 51: 493-501Abstract Full Text PDF PubMed Scopus (121) Google Scholar, 10Surette M.G. Buch S.J. Chaconas G. Cell. 1987; 49: 253-262Abstract Full Text PDF PubMed Scopus (157) Google Scholar). The addition of Mu B, ATP, and target DNA results in the formation of the Type 2 complex (strand transfer complex) in which the 3′-ends of the Mu DNA are transferred to a target DNA molecule (9Craigie R. Mizuuchi K. Cell. 1987; 51: 493-501Abstract Full Text PDF PubMed Scopus (121) Google Scholar, 10Surette M.G. Buch S.J. Chaconas G. Cell. 1987; 49: 253-262Abstract Full Text PDF PubMed Scopus (157) Google Scholar). The Type 2 complex is the most stable transpososome and must be destabilized by the host-encoded ClpX ATPase before replication can occur (11Nakai H. Kruklitis R. J. Biol. Chem. 1995; 270: 19591-19598Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 12Levchenko I. Luo L. Baker T.A. Genes Dev. 1995; 9: 2399-2408Crossref PubMed Scopus (245) Google Scholar, 13Kruklitis R. Welty D.J. Nakai H. EMBO J. 1996; 15: 935-944Crossref PubMed Scopus (104) Google Scholar, 14Jones J.M. Welty D.J. Nakai H. J. Biol. Chem. 1998; 273: 459-465Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Target capture can also occur at the LER or Type 0 stage of the reaction (15Naigamwalla D.Z. Chaconas G. EMBO J. 1997; 16: 5227-5234Crossref PubMed Scopus (40) Google Scholar). In the absence of target DNA, Mu B and ATP stimulate intramolecular strand transfer, whereby the 3′-ends of the Mu DNA are transferred into a new DNA site within the donor molecule (16Maxwell A. Craigie R. Mizuuchi K. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 699-703Crossref PubMed Scopus (91) Google Scholar, 17Baker T.A. Mizuuchi M. Mizuuchi K. Cell. 1991; 65: 1003-1013Abstract Full Text PDF PubMed Scopus (83) Google Scholar).The Mu A protein (transposase) is 663 amino acids in length (18Harshey R.M. Getzoff E.D. Baldwin D.L. Miller J.L. Chaconas G. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 7676-7680Crossref PubMed Scopus (57) Google Scholar) and is able to perform all of the chemical steps in the transposition reaction. Mu A can be divided into three distinct globular domains (19Nakayama C. Teplow D.B. Harshey R.M. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1809-1813Crossref PubMed Scopus (69) Google Scholar). Domain I is responsible for DNA binding of the enhancer element and the Mu ends (20Mizuuchi M. Mizuuchi K. Cell. 1989; 58: 399-408Abstract Full Text PDF PubMed Scopus (119) Google Scholar, 21Leung P.C. Teplow D.B. Harshey R.M. Nature. 1989; 338: 656-658Crossref PubMed Scopus (112) Google Scholar). Domain II can be divided into two functionally distinct and complementary subdomains: domain IIα, which contains the conserved DDE motif believed to coordinate the divalent metal ion (22Baker T.A. Luo L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6654-6658Crossref PubMed Scopus (121) Google Scholar, 23Kim K. Namgoong S.Y. Jayaram M. Harshey R.M. J. Biol. Chem. 1995; 270: 1472-1479Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), and domain IIβ, which is involved with transpososome assembly and may bind DNA (24Kremenstova E. Giffen M.J. Pincus D. Baker T.A. J. Biol. Chem. 1998; 273: 31358-31365Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 25Namgoong S.Y. Kim K. Saxena P. Yang J.Y. Jayaram M. Giedroc D.P. Harshey R.M. J. Mol. Biol. 1998; 275: 221-232Crossref PubMed Scopus (19) Google Scholar). Domain III also contains two subdomains, domain IIIα and domain IIIβ. Domain IIIα has functional similarities to domain IIβ, and they may both constitute a single functional domain (24Kremenstova E. Giffen M.J. Pincus D. Baker T.A. J. Biol. Chem. 1998; 273: 31358-31365Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 25Namgoong S.Y. Kim K. Saxena P. Yang J.Y. Jayaram M. Giedroc D.P. Harshey R.M. J. Mol. Biol. 1998; 275: 221-232Crossref PubMed Scopus (19) Google Scholar, 26Naigamwalla D.Z. Coros C.J. Wu Z. Chaconas G. J. Mol. Biol. 1998; 282: 265-274Crossref PubMed Scopus (12) Google Scholar). Domain IIIβ is a protein-protein interaction region that contains sequences bound by Mu B and ClpX (12Levchenko I. Luo L. Baker T.A. Genes Dev. 1995; 9: 2399-2408Crossref PubMed Scopus (245) Google Scholar, 17Baker T.A. Mizuuchi M. Mizuuchi K. Cell. 1991; 65: 1003-1013Abstract Full Text PDF PubMed Scopus (83) Google Scholar, 27Leung P.C. Harshey R.M. J. Mol. Biol. 1991; 219: 189-199Crossref PubMed Scopus (32) Google Scholar, 28Wu Z. Chaconas G. J. Biol. Chem. 1994; 269: 28829-28833Abstract Full Text PDF PubMed Google Scholar).The 312-residue phage-encoded protein Mu B is an ATP-dependent DNA-binding protein required for efficient Mu DNA transposition (16Maxwell A. Craigie R. Mizuuchi K. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 699-703Crossref PubMed Scopus (91) Google Scholar, 29Chaconas G. Gloor G. Miller J.L. J. Biol. Chem. 1985; 260: 2662-2669Abstract Full Text PDF PubMed Google Scholar). Mu B is able to capture target DNA and interact with all of the transpososome complexes (15Naigamwalla D.Z. Chaconas G. EMBO J. 1997; 16: 5227-5234Crossref PubMed Scopus (40) Google Scholar). Mu B is also an allosteric activator of the transposase and can stimulate transpososome formation on both mutant donor DNA substrates and with partially functional mutant forms of Mu A (17Baker T.A. Mizuuchi M. Mizuuchi K. Cell. 1991; 65: 1003-1013Abstract Full Text PDF PubMed Scopus (83) Google Scholar, 26Naigamwalla D.Z. Coros C.J. Wu Z. Chaconas G. J. Mol. Biol. 1998; 282: 265-274Crossref PubMed Scopus (12) Google Scholar, 30Wu Z. Chaconas G. EMBO J. 1995; 14: 3835-3843Crossref PubMed Scopus (45) Google Scholar, 31Coros C.J. Chaconas G. J. Mol. Biol. 2001; 310: 299-309Crossref PubMed Scopus (16) Google Scholar, 32Surette M.G. Chaconas G. J. Biol. Chem. 1991; 266: 17306-17313Abstract Full Text PDF PubMed Google Scholar). Along with target capture and transpososome formation, Mu B is also responsible for preventing Mu from transposing into itself, a process known as target immunity. During target immunity, Mu A stimulates the release of Mu B bound to DNA through the hydrolysis of ATP (33Adzuma K. Mizuuchi K. Cell. 1988; 53: 257-266Abstract Full Text PDF PubMed Scopus (94) Google Scholar, 34Adzuma K. Mizuuchi K. Cell. 1989; 57: 41-47Abstract Full Text PDF PubMed Scopus (71) Google Scholar, 35Adzuma K. Mizuuchi K. J. Biol. Chem. 1991; 266: 6159-6167Abstract Full Text PDF PubMed Google Scholar).The Mu B protein has two globular domains (36Teplow D.B. Nakayama C. Leung P.C. Harshey R.M. J. Biol. Chem. 1988; 263: 10851-10857Abstract Full Text PDF PubMed Google Scholar). The 25-kDa N-terminal domain contains nonspecific DNA binding activity and has an ATPase motif (29Chaconas G. Gloor G. Miller J.L. J. Biol. Chem. 1985; 260: 2662-2669Abstract Full Text PDF PubMed Google Scholar, 36Teplow D.B. Nakayama C. Leung P.C. Harshey R.M. J. Biol. Chem. 1988; 263: 10851-10857Abstract Full Text PDF PubMed Google Scholar). The 11-kDa C-terminal domain is also able to bind nonspecifically to DNA (40Hung L.H. Chaconas G. Shaw G.S. EMBO J. 2000; 19: 5625-5634Crossref PubMed Scopus (9) Google Scholar). Site-directed mutagenesis of the N-terminal bipartite nucleotide binding motif (Walker A and Walker B boxes) results in the loss of ATPase activity (37Yamauchi M. Baker T.A. EMBO J. 1998; 17: 5509-5518Crossref PubMed Scopus (40) Google Scholar). However, the isolated N-terminal domain by itself is unable to hydrolyze ATP (36Teplow D.B. Nakayama C. Leung P.C. Harshey R.M. J. Biol. Chem. 1988; 263: 10851-10857Abstract Full Text PDF PubMed Google Scholar). Mutations in the N-terminal domain of Mu B have been shown to affect target capture, but these mutants retain their ability to interact with the transpososome complex (37Yamauchi M. Baker T.A. EMBO J. 1998; 17: 5509-5518Crossref PubMed Scopus (40) Google Scholar, 38Millner A. Chaconas G. J. Mol. Biol. 1998; 275: 233-243Crossref PubMed Scopus (7) Google Scholar). A small truncation of the C-terminal domain of Mu B blocks replicative transposition but not integration, whereas longer truncations affect both replicative and integrative transposition in vivo (39Chaconas G. Giddens E.B. Miller J.L. Gloor G. Cell. 1985; 41: 857-865Abstract Full Text PDF PubMed Scopus (29) Google Scholar).The C-terminal domain of Mu B (Mu B223–312)is a four-helix bundle (Fig. 2) and has a similar fold to that of the N-terminal region of DnaB (see Ref. 40Hung L.H. Chaconas G. Shaw G.S. EMBO J. 2000; 19: 5625-5634Crossref PubMed Scopus (9) Google Scholar). Examination of the charged residues within the peptide revealed that there is a positively charged patch of amino acids on the surface of the structure. It was proposed that this patch may be involved in the DNA binding activity that has been associated with the C-terminal domain. In this study, we first confirmed that these residues are responsible for binding DNA, and then we investigated how these positively charged residues contribute to the in vitro transposition reaction. We found that by changing three lysine residues in the C-terminal domain of Mu B to alanine residues, we generated a mutant protein that could no longer capture target or interact with the Mu transpososome. Peptide binding experiments further reveal that this region of Mu B can participate directly in Mu A-Mu B protein-protein contacts.Fig. 2Ribbon diagram of the ensemble average structure of Mu B 223–312. The ribbon diagram, generated with the Swiss Pdb Viewer, represents the average of the 20 defined solution structures previously determined from NMR data with the initial 8 residues removed (40Hung L.H. Chaconas G. Shaw G.S. EMBO J. 2000; 19: 5625-5634Crossref PubMed Scopus (9) Google Scholar). The four helixes have been colored in green, blue, yellow, and purple and labeled as helix 1, 2, 3, and 4 respectively. Also shown in red are the three lysine side chains that that were changed to alanine residues.View Large Image Figure ViewerDownload Hi-res image Download (PPT)EXPERIMENTAL PROCEDURESDNA, Reagents, and Enzymes—The 6.5-kb mini-Mu plasmid pBL08 has been previously described (6Watson M.A. Chaconas G. Cell. 1996; 85: 435-445Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). The 7.2-kb mutant mini-Mu plasmid pMS9A1 has an A to G transition at the Mu left end terminal base pair (32Surette M.G. Chaconas G. J. Biol. Chem. 1991; 266: 17306-17313Abstract Full Text PDF PubMed Google Scholar). The target DNA used was the 5.2-kb plasmid pSD7 (32Surette M.G. Chaconas G. J. Biol. Chem. 1991; 266: 17306-17313Abstract Full Text PDF PubMed Google Scholar). All plasmids were purified using a cesium chloride gradient and dialyzed extensively.Mutagenesis and Protein Purification—The HU (41Lavoie B.D. Chaconas G. Genes Dev. 1993; 7: 2510-2519Crossref PubMed Scopus (82) Google Scholar) and IHF (42Surette M.G. Chaconas G. J. Biol. Chem. 1989; 264: 3028-3034Abstract Full Text PDF PubMed Google Scholar) proteins were purified as previously described. Mu A proteins were purified as described previously (43Baker T.A. Mizuuchi M. Savilahti H. Mizuuchi K. Cell. 1993; 74: 723-733Abstract Full Text PDF PubMed Scopus (94) Google Scholar) through the P-11 step. No dithiothreitol was added to the wash step or the elution step of the P-11 column, and the protein was induced in the presence of 1% (w/v) glucose, which improved the induction and purity after the P-11 column.To introduce mutations into Mu B223–312 and the full-length Mu B genes, site-directed mutagenesis was performed using the QuikChange site-directed mutagenesis kit purchased from Stratagene, with a modified protocol. Two complementary oligonucleotides containing the desired mutation sequence were purchased (Sigma). Two separate 25-μl PCRs were set up (as per standard QuikChange protocol) containing 250 pmol of each mutant primer in separate reactions with 50 ng of wild-type Mu B expression plasmid DNA. Each separate PCR was amplified for three cycles of 95 °C for 50 s, 55 °C for 60 s, and 68 °C for 12 min with 0.5 units of Pfu Turbo DNA polymerase. The two amplification reactions were then combined to create a 50-μl PCR containing both primers and their amplification products from the first three cycles. This new reaction was supplemented with an additional 0.5 units of Pfu Turbo DNA polymerase, and the reaction was run for 18 cycles of 95 °C for 50 s, 55 °C for 50 s, and 68 °C for 12 min, followed by an additional extension for 7 min at 68 °C. The amplification product was digested with DpnI for 90 min at 37 °C and then transformed into electrocompetent DH5α cells. The mutant plasmids were then purified and sequenced.Mutagenesis and purification of the Mu B223–312 gene was performed using the Mu B223–312 gene expression construct, pHH05 from the strain GC1876, described previously (40Hung L.H. Chaconas G. Shaw G.S. EMBO J. 2000; 19: 5625-5634Crossref PubMed Scopus (9) Google Scholar). One liter of cells was grown in LB in a 4-liter Fernbach flask in the presence of 50 μg/ml ampicillin and 1% (w/v) glucose at 37 °C, with a rotation of 225 rpm, to an A 595 of 0.65. The flask was then transferred to 18 °C, with a rotation of 225 rpm and incubated for 45 min, and the cells were induced with 1 mm isopropyl-1-thio-β-d-galactopyranoside for 18 h. The cells were harvested by spinning at 8500 × g for 10 min, washed, and resuspended in 10 ml of 25 mm Tris-HCl, pH 7.5, plus 5 mm EDTA. Cells were incubated for 30 min at 4 °C with lysozyme (0.3 mg/ml), followed by the addition of 1% Triton X-100 and five 30-s bursts using a Fisher model 300 sonifier at 50-watt power. The lysed cells were then pelleted at 27,200 × g for 30 min. The supernatant was batch-absorbed at 4 °C for 45 min to 500 μl of glutathione-Sepharose 4B matrix (Amersham Biosciences) equilibrated with 25 mm Tris-HCl, pH 7.5, plus 5 mm EDTA. The matrix was then washed three times with 25 ml of 25 mm Tris-HCl, pH 7.5, plus 5 mm EDTA and three times with 25 mm Tris-HCl, pH 7.5, plus 5 mm EDTA and 1 m NaCl. The matrix was transferred into a 1 × 2.5-cm column, washed three times more with 25 mm Tris-HCl, pH 7.5, plus 5 mm EDTA, and eluted with 50 mm Tris-HCl, pH 7.5, plus 5 mm EDTA and 20 mm glutathione. The eluate was digested with 1 unit of thrombin/mg of protein for 45 min at room temperature. The digested protein was then filtered (0.22-μm filter) and loaded (1 ml/min) onto a 1-ml Resource prepacked Mono 15S column (Amersham Biosciences), equilibrated with 25 mm Tris-HCl, pH 7.5, using a high pressure liquid chromatograph (Waters). The Mu B223–312 peptide was eluted from the Mono 15S column using a 0–700 mm NaCl (Tris-HCl, pH 7.5) gradient for 40 min at a flow rate of 1 ml/min. Peak fractions were collected and dialyzed against 20 mm phosphate, pH 6.8, plus 1.5 m NaCl.Mutagenesis and purification was performed on the full-length construct using the Mu B gene expression plasmid pML02 (38Millner A. Chaconas G. J. Mol. Biol. 1998; 275: 233-243Crossref PubMed Scopus (7) Google Scholar). Mu B was purified as described previously (38Millner A. Chaconas G. J. Mol. Biol. 1998; 275: 233-243Crossref PubMed Scopus (7) Google Scholar), with the following changes: the ethanol pellet was resuspended in 25 mm Tris-HCl, pH 8.8, plus 1 mm EDTA, 10 mm MSH, and 7.0 m urea; and the DEAE-Sepharose column was equilibrated with 25 mm Tris-HCl, pH 8.8, plus 1 mm EDTA, 10 mm MSH, and 6.2 m urea and eluted with 25 mm Tris-HCl, pH 6.8, plus 1 mm EDTA, 10 mm MSH, and 6.2 m urea. The CM-Sepharose column was equilibrated with 25 mm Tris-HCl, pH 6.8, plus 1 mm EDTA, 10 mm MSH, 6.8 m urea, and 25 mm NaCl, and the protein was loaded and washed with 4 ml of equilibrium buffer before being eluted with 7 ml of 25 mm Tris-HCl, pH 7.4, plus 1 mm EDTA, 10 mm MSH, 6.8 m urea, and 100 mm NaCl. The 3K protein was purified as above using SP-Sepharose instead of CM-Sepharose. All of the proteins were greater than 95% pure with the exception of the 3K protein, which was greater than 90% pure as judged by SDS-PAGE.DNA Binding Assays—The Mu B223–312 DNA binding assays were performed using various concentrations of peptide (described in the legends of Figs. 3 and 5) in the presence of 25 mm Hepes-NaOH, pH 7.6, 150 mm NaCl, and 15 μg/ml DNA (pSD7). The reactions were incubated at 25 °C for 10 min.Fig. 3Assay of DNA binding activity of Mu B 223–312 mutant peptides. A, schematic of Mu B domain structure. The Mu B protein can be divided into two structural domains by partial proteolysis (36Teplow D.B. Nakayama C. Leung P.C. Harshey R.M. J. Biol. Chem. 1988; 263: 10851-10857Abstract Full Text PDF PubMed Google Scholar). The N-terminal domain contains an AAA+ ATPase motif (54Neuwald A.F. Aravind L. Spouge J.L. Koonin E.V. Genome Res. 1999; 9: 27-43Crossref PubMed Google Scholar) and nonspecific DNA binding activity (36Teplow D.B. Nakayama C. Leung P.C. Harshey R.M. J. Biol. Chem. 1988; 263: 10851-10857Abstract Full Text PDF PubMed Google Scholar). The C-terminal domain has a nonspecific DNA binding activity and shares structural similarity to the N-terminal domain of DnaB (40Hung L.H. Chaconas G. Shaw G.S. EMBO J. 2000; 19: 5625-5634Crossref PubMed Scopus (9) Google Scholar). B, DNA binding was assayed using gel mobility shifts by incubating 225 ng of supercoiled non-Mu plasmid (pSD7) with various amounts of purified Mu B carrying mutations in the C-terminal domain (Bc). Wild-type and mutant Mu B223–312 peptides were incubated with DNA and run on a 1% agarose gel at 80 V cm–1 for 4 h and stained with ethidium bromide. The position of the supercoiled and relaxed DNA in the absence of protein is labeled at the side of the gel. The mutant peptides assayed are noted above the gel. The 3K mutant protein is a triple alanine substitution at positions Lys233, Lys235, and Lys236. “Bc” indicates that the mutations are carried in the purified C-terminal peptide.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 5Assay of DNA binding activity of full-length mutant Mu B proteins. A, the DNA binding affinities for the wild-type and mutant full-length Mu B proteins were determined using affinity co-electrophoresis assays, essentially as described previously (44Lim W.A. Sauer R.T. Lander A.D. Methods Enzymol. 1991; 208: 196-210Crossref PubMed Scopus (58) Google Scholar). Briefly, various concentrations of wild-type and mutant proteins were embedded into the lanes of a horizontal slab gel. Then 32P-labeled 70-bp double-stranded DNA oligonucleotide was loaded into a single well spanning the width of the gel and electrophoretically run through the gel in the presence of 0.5 mm ATP. As protein-DNA complexes form during electrophoresis, migration of the radiolabeled DNA is retarded in the gel. B, quantification of affinity co-electrophoresis assay gels for wild-type, K233A, K235A, K236A, and 3K full-length Mu B proteins. DNA binding was determined as the percentage of the DNA retained at the top half of the gel (see “Experimental Procedures”). Data were fitted to the following equations, as described previously (37Yamauchi M. Baker T.A. EMBO J. 1998; 17: 5509-5518Crossref PubMed Scopus (40) Google Scholar, 44Lim W.A. Sauer R.T. Lander A.D. Methods Enzymol. 1991; 208: 196-210Crossref PubMed Scopus (58) Google Scholar): Mu B wild type, θ = θmax/(1 + (5.2 × 102/[protein]1.4)); K233A, θ = θmax/(1 + (2.9 × 104/[protein]2.2)); K235A, θ = θmax/(1 + (2.1 × 104/[protein]2.0)); K236A, θ = θmax/(1 + (1.1 × 103/[protein]1.5)); 3K, θ = θmax/(1 + (3.3 × 105/[protein]2.1)). K values represent the Mu B protein concentrations required for half-maximal DNA binding, where θ represents the percentage of DNA bound and θmax is the maximum percentage of DNA bound.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Affinity co-electrophoresis was performed essentially as described previously (44Lim W.A. Sauer R.T. Lander A.D. Methods Enzymol. 1991; 208: 196-210Crossref PubMed Scopus (58) Google Scholar). Protein was embedded into 1% LMP agarose and 32P-end-labeled 70-bp double-stranded DNA was run through the gel. The final protein concentration in the gel varied between 22.5 nm and 1.44 μm as described throughout. The gels and electrophoresis buffer contained 25 mm Tris-HCl, pH 8.0, 0.1 mg/ml bovine serum albumin, 10 mm magnesium acetate, 50 mm potassium acetate, and 0.5 mm ATP. Gels were run at 4 °C with circulating buffer at 4.2 V cm–1 for 5 h. The gels were dried and scanned using a PhosphorImager S and quantified using ImageQuant software from Amersham Biosciences. The fraction of DNA bound was taken as the percentage of radioactivity in the top half of each lane.In Vitro Reactions—The Type 2 reactions were performed under standard reaction conditions (1×): 15 μg/ml supercoiled mini-Mu plasmid, 3 μg/ml (44 nm) Mu A protein, 3.5 μg/ml (315 nm) HU, 0.2 μg/ml (18 nm) IHF, 5 μg/ml (140 nm) Mu B, 2 mm ATP, 20 μg/ml target DNA, 25 mm Hepes-NaOH, pH 7.6, plus 140 mm NaCl and 10 mm MgCl2. Reactions were incubated at 30 °C for various amounts of time as described in the legends of Figs. 4 and 6. The intramolecular strand transfer reactions were performed at 2.5× reaction conditions in the absence of target DNA at Mu B concentrations of 140 and 280 nm as indicated.Fig. 4Kinetics of Type 2 formation using purified full-length mutant Mu B proteins. Strand transfer was assayed at 30 °C under standard reaction conditions. The reactions were run on a 1% agarose gel at 2.75 V cm–1 for 12 h. The supercoiled target (ST), the supercoiled donor (SD), the relaxed target (RT), and the relaxed donor (RD) are indicated to the left of the gel. The positions of the Type 2, the Type 1, and the intramolecular products are shown to the right of the gel. The faint band observed with wild type Mu B, running between the Type 2 complex and the relaxed donor DNA, is an unstable form of the Type 2 complex where the target DNA is still supercoiled. The nomenclature for the mutant proteins is described in Fig. 3, except that all of the mutations are present in the full-length protein.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 6Assay for stimulation of donor cleavage by full-length mutant Mu B proteins. A, transposition reactions were performed using the terminal base pair mutant substrate pMS9A1 (32Surette M.G. Chaconas G. J. Biol. Chem. 1991; 266: 17306-17313Abstract Full Text PDF PubMed Google Scholar) in the absence of target DNA for 30 min at 30 °C. B, transposition reactions were performed using the Aban mutant transposase (30Wu Z. Chaconas G. EMBO J. 1995; 14: 3835-3843Crossref PubMed Scopus (45) Google Scholar) in the absence of target DNA for 30 min at 30 °C. The percentage of reaction (percent of substrate utilized) was calculated by adding the percentage of cleaved product plus the percentage of intramolecular strand transfer complex formed. The percentage of reaction was determined in the absence of Mu B protein and in the presence of 140 nm and 280 nm Mu B concentrations.View Large Image Figure ViewerDownload Hi-res image Download (PPT)ATPase Assay—The ATPase assays were performed essentially as previously described (16Maxwell A. Craigie R. Mizuuchi K. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 699-703Crossref PubMed Scopus (91) Google Scholar). Reactions contained 35 μg/ml (980 nm) Mu B in 150 mm NaCl, 10 mm MgCl2, 7.5% glycerol, 3 mm dithiothreitol, 0.3 mg/ml bovine serum albumin, 0.1 mm [γ-32P]ATP (20 nCi), and 25 mm Tris, pH 7.8, and, where indicated, 40 μg/ml (586 nm) Mu A and 50 μg/
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