Role of arginine-43 and arginine-69 of the Hin recombinase catalytic domain in the binding of Hin to the hix DNA recombination sites

The Hin recombinase mediates the site-specific inversion of a segment of the Salmonella chromosome between two flanking 26 bp hix DNA recombination sites. Mutations in two amino acid residues, R43 and R69 of the catalytic domain of the Hin recombinase, were identified that can compensate for loss of binding resulting from elimination of certain major and minor groove contacts within the hix recombination sites. With one exception, the R43 and R69 mutants were also able to bind a hix sequence with an additional 4 bp added to the centre of the site, unlike wild-type Hin. Purified Hin mutants R43H and R69C had both partial cleavage and inversion activities in vitro while mutants R43L, R43C, R69S, and R69P had no detectable cleavage and inversion activities. These data support a model in which the catalytic domain plays a role in DNA-binding specificity, and suggest that the arginine residues at positions 43 and 69 function to position the Hin recombinase on the DNA for a step in the recombination reaction which occurs either at and/or prior to DNA cleavage.     

Testing water-mediated DNA recognition by the Hin recombinase

The Hin recombinase specifically recognizes its DNA-binding site by means of both major and minor groove interactions. A previous X-ray structure, together with new structures of the Hin DNA-binding domain bound to a recombination half-site that were solved as part of the present study, have revealed that two ordered water molecules are present within the major groove interface. In this report, we test the importance of these waters directly by X-ray crystal structure analysis of complexes with four mutant DNA sequences. These structures, combined with their Hin-binding properties, provide strong support for the critical importance of one of the intermediate waters. A lesser but demonstrable role is ascribed to the second water molecule. The mutant structures also illustrate the prominent roles of thymine methyls both in stabilizing intermediate waters and in interfering with water or amino acid side chain interactions with DNA.     

Importance of minor-groove contacts for recognition of DNA by the binding domain of Hin recombinase

Incorporation of the DNA-cleaving moiety EDTA.Fe at discrete amino acid residues along a DNA-binding protein allows the positions of these residues relative to DNA bases, and hence the organization of the folded protein, to be mapped by high-resolution gel electrophoresis. A 52-residue protein, based on the sequence-specific DNA-binding domain of Hin recombinase (139-190), with EDTA at the NH2 terminus cleaves DNA at Hin recombination sites. The cleavage data for EDTA-Hin(139-190) reveal that the NH2 terminus of Hin(139-190) is bound in the minor groove of DNA near the symmetry axis of Hin-binding sites [Sluka, J. P., Horvath, S. J., Bruist, M. F., Simon, M. I., & Dervan, P. B. (1987) Science 238, 1129]. Six proteins, varying in length from 49 to 60 residues and corresponding to the DNA-binding domain of Hin recombinase, were synthesized by solid-phase methods: Hin(142-190), Hin(141-190), Hin(140-190), Hin(139-190), Hin(135-190), and Hin(131-190) were prepared with and without EDTA at the NH2 termini in order to test the relative importance of the residues Gly139-Arg140-Pro141-Arg142, located near the minor groove, for sequence-specific recognition at five imperfectly conserved 12-base-pair binding sites. Footprinting and affinity cleaving reveal that deletion of Gly139 results in a protein with affinity and specificity similar to those of Hin(139-190) but that deletion of Gly139-Arg140 affords a protein with altered affinities and sequence specificities for the five binding sites. It appears that Arg140 in the DNA-binding domain of Hin is important for recognition of the 5'-AAA-3' sequence in the minor groove of DNA. Our results indicate modular DNA and protein interactions with two adjacent DNA sites (major and minor grooves, respectively) bound on the same face of the helix by two separate parts of the protein.     

Identification of the DNA-binding domain of the FLP recombinase

We have subjected the FLP protein of the 2-micron plasmid to partial proteolysis by proteinase K and have found that FLP can be digested into two major proteinase K-resistant peptides of 21 and 13 kDa, respectively. The 21-kDa peptide contains a site-specific DNA-binding domain that binds to the FLP recognition target (FRT) site with an affinity similar to that observed for the native FLP protein. This peptide can induce DNA bending upon binding to a DNA fragment containing the FRT site, but the angle of the bend (approximately 24 degrees) is smaller in magnitude than that induced by the native FLP protein (60 degrees). The additional DNA bending induced by the interaction between two native FLP molecules bound to the FRT site is not observed with the 21-kDa DNA-binding peptide. Amino-terminal sequencing has been used to map this peptide to an internal region of FLP that begins at residue Leu-148. It is likely that the DNA-binding peptide includes the catalytic site of the FLP protein.     

The effects of symmetrical recombination site hixC on Hin recombinase function.

An artificial recombination site hixC composed of two identical half-sites that bind the Hin recombinase served as a better operator in vivo than the wild type site hixL (Hughes, K. T., Youderian, P., and Simon, M. I (1988) Genes & Dev. 2, 937-948). In vitro binding assays such as gel retardation assay and methylation protection assay demonstrated that Hin binds to hixC as tightly as it binds to hixL, even when the sites are located in negatively supercoiled plasmids. However, hixC served as a poor recombination site when it was subjected to the standard inversion assay in vitro. hixC showed a 16-fold slower inversion rate than the wild type. A series of biochemical assays designed to probe different stages of the Hin-mediated inversion reaction, demonstrated that Hin dimers bound to hixC have difficulty in forming paired hix site intermediates. KMnO4 and S1 nuclease assays detected an anomalous structure of the center of hixC only when the site was in negatively supercoiled plasmids. Mutational analysis in the central region of hixC and assays of paired hix site formation with topoisomers of the hixC substrate plasmid suggested that Hin is not able to pair hixC sites because of the presence of the anomalous structure in the center of the site. The structure does not behave like a DNA "cruciform" since Hin dimers still bind efficiently to the site. It is thought to consist of a short denatured "bubble" encompassing 2 base pairs. During the study of mutations in the center of hixC, it was found that Hin is not able to cleave DNA if a guanine residue is one of the two central nucleotides close to the cleavage site. Furthermore, Hin acts in a concerted fashion and cannot cleave any DNA strand if one of the four strands in the inversion intermediate is not cleavable.     

A putative DNA-binding domain in the NUCKS protein

We have studied the DNA-binding properties of a NUCKS-derived, synthetic peptide containing an extended GRP motif. This peptide binds to random-sequence DNA, but did not bind preferentially to poly(dA-dT). A synthetic peptide with the same amino acid composition but with a random sequence did not bind to DNA, suggesting that the structure of the DNA-binding domain plays a pivotal role in the interaction with DNA. NMR and graphic modeling were employed to investigate the structure of the synthetic peptide. It was shown that the DNA-binding peptide constituted an alpha helix in phosphate buffer at pH 5.5. Docking results indicated an almost perfect fit for this small, helical peptide into the major groove of DNA with the possibility of four basic residues interacting with the phosphate backbone of DNA. One consensus site for phosphorylation by Cdk1 is located in the N-terminal end of the DNA-binding peptide. Upon phosphorylation of this site, the binding to DNA was completely prohibited. Immunofluorescence experiments showed that NUCKS was located in the nuclei in proliferating cells in interphase of the cell cycle, but was distributed throughout the cytoplasm in mitotic cells.     

Hin recombinase mutants functionally disrupted in interactions with Fis

A previous genetic screen was designed to separate Hin recombinase mutants into distinct classes based on the stage in the recombination reaction at which they are blocked (O. Nanassy, Zoltan, and K. T. Hughes, Genetics 149:1649-1663, 1998). One class of DNA binding-proficient, recombination-deficient mutants was predicted by genetic classification to be defective in the step prior to invertasome formation. Based on the genetic criteria, mutants from this class were also inferred to be defective in interactions with Fis. In order to understand how the genetic classification relates to individual biochemical steps in the recombination reaction these mutants, R123Q, T124I, and A126T, were purified and characterized for DNA cleavage and recombination activities. Both the T124I and A126T mutants were partially active, whereas the R123Q mutant was inactive. The A126T mutant was not as defective for recombination as the T124I allele and could be partially rescued for recombination both in vivo and in vitro by increasing the concentration of Fis protein. Rescue of the A126T allele required the Fis protein to be DNA binding proficient. A model for a postsynaptic role for Fis in the inversion reaction is presented.     

Backbone dynamics of the c-Myb DNA-binding domain complexed with a specific DNA

The DNA-binding domain of c-Myb consists of three imperfect tandem repeats, R1, R2, and R3. Each repeat contains three helices. The minimal DNA-binding domain is an R2R3 fragment. Here, we have examined the backbone dynamics of R2R3 in its DNA-bound form by NMR. Upon binding to DNA, the N- and C-termini, and the linker between R2 and R3 become less flexible. In the free form the third helix of R2 exhibits slow conformational exchange fluctuations owing to a cavity in the hydrophobic core of R2. Upon binding to DNA, the conformational exchange contributions in R2 are reduced but remain significant in NMR relaxation measurements. Upon binding to DNA, the third helix of R3 comes to exhibit significant chemical exchange contributions. These findings suggest that the orientations of the third helices of both R2 and R3 as to DNA are being chemically exchanged. In the DNA-bound form both R2 and R3 exhibit similar dynamical characters, except for amino acids Trp 95, Thr 96, and Val 103 of R2, which are located around the cavity of the unbound form. Upon binding to DNA, since Trp 95 moves into the cavity to fill it up, the local conformational exchange contributions seem to be still observable around the filled cavity.     

Determinants for DNA-binding site recognition by the glucocorticoid receptor.

The glucocorticoid receptor binds with high specificity to glucocorticoid response elements, discriminating them from other closely related binding sites. Three amino acids in the recognition alpha-helix of the DNA-binding domain of the receptor are primarily responsible for this specific DNA binding activity. In this study we analyze in detail how these residues determine the specific DNA binding by studying a series of mutant glucocorticoid receptor DNA-binding domains containing all combinations of glucocorticoid and estrogen receptor-specific residues at these positions. Statistical analysis of the results enables us to create models describing the association between amino acids and base pairs. Several strategies appear to be used in accomplishing discrimination between the glucocorticoid and estrogen response elements. Single residues (i.e., Val-443 in the glucocorticoid receptor and Glu-439 in the estrogen receptor) appear to form both positive contacts with specific base pairs in the cognate binding site and negative contacts in the non-cognate site. In the glucocorticoid receptor Ser-440 is pleiotropically negative for all sites tested but the negative effect is stronger for the estrogen response element thus contributing to binding site discrimination. Furthermore, combinations of amino acids appear to act synergistically, most often causing a reduction in binding to non-cognate sites.     

Effects of dimer interface mutations in Hin recombinase on DNA binding and recombination

Previous biochemical assays and a structural model of the protein have indicated that the dimer interface of the Hin recombinase is composed of two alpha-helices. To elucidate the structure and function of the helix, amino acids at the N-terminal end of the helix, where the two helices make their most extensive contact, were randomized, and inversion-incompetent mutants were selected. To investigate why the mutants lost their inversion activities, the DNA binding, hix pairing, invertasome formation, and DNA cleavage activities were assayed using in vivo and in vitro methodologies. The results indicated that the mutants could be divided into four classes based on their DNA binding activity. We propose that the alpha-helices might serve to place a DNA binding motif of Hin in the correct spatial relationship to the minor groove of the recombination site. All the mutants except those that failed to bind DNA were able to perform hix pairing and invertasome formation, suggesting that the dimer interface is not involved in either of these processes. The inversion-incompetent phenotype of the binders was caused by the inability of mutants to perform DNA cleavage. The mutants that showed less binding ability than the wild type nevertheless exhibited a wild-type level of hix pairing activity, because the hix pairing activity overcomes the defect in DNA binding. This phenotype of the mutants that are impaired in DNA binding suggests that the binding domains of Hin may mediate Hin-Hin interaction during hix pairing.     

Participation of water in Hin recombinase--DNA recognition.

The participation of water molecules in the interaction between the Hin recombinase and its operator DNA has been detected by analysis of the dissociation constant in the presence of varying concentrations of neutral solutes and cosolvents. The dissociation constant as measured by gel mobility shift assays increased as the concentration of dimethyl sulfoxide, glycerol, sucrose, or polyethylene glycol was increased. Osmotic pressure is the only property that correlates with the change in the dissociation constant for all compounds. This data indicates that binding of a small population of water molecules accompanies formation of the Hin-DNA complex, and points to a novel role for solvent molecules in assisting site specific interaction between DNA-binding proteins and their cognate DNA sequence.     

基于Cre重组酶体系的鸡卵清蛋白基因打靶载体的构建

利用胚胎干细胞基因打靶技术制备转基因鸡是研制鸡输卵管反应器的最佳技术路线。为建立基于Cre/loxp系统的鸡卵清蛋白基因(Ovalbumingene,OV)位点的双交换打靶载体系统,本研究克隆了鸡的OV基因7.8kb片段,并与克隆的内部核糖体进入位点(IRES)、人工合成的含有Cre重组酶识别位点变异体交换盒m2/loxp71EGFPloxp66,一起构建了含有Hsvtk负筛选标记的针对鸡卵清蛋白基因位点的敲入型共表达基因打靶载体pSSCm2/71EGFP66IRESOV7.8;以猪β干扰素基因(βInterferon,IFNβ)为目的基因构建了穿梭载体pMDm2/66MCSIFNMCSLoxp71,经过限制酶酶切及部分测序鉴定,所构建载体结构正确。进一步将它们共转化组成性表达Cre的细菌BM25.8,验证了loxp突变位点对重组反应的有效性     

Mapping the active site polarity in structures of endothelial nitric oxide synthase heme domain complexed with isothioureas

Analyzing the active site topology and plasticity of nitric oxide synthase (NOS) and understanding enzyme-drug interactions are crucial for the development of potent, isoform-selective NOS inhibitors. A small hydrophobic pocket in the active site is identified in the bovine eNOS heme domain structures complexed with potent isothiourea inhibitors: seleno analogue of S-ethyl-isothiourea, S-isopropyl-isothiourea, and 2-aminothiazoline, respectively. These structures reveal the importance of nonpolar van der Waals contacts in addition to the well-known hydrogen bonding interactions between inhibitor and enzyme. The scaffold of a potent NOS inhibitor should be capable of donating hydrogen bonds to as well as making nonpolar contacts with amino acids in the NOS active site.     

Sequence-specific interaction of the Salmonella Hin recombinase in both major and minor grooves of DNA.

The Hin recombinase of Salmonella catalyzes a site-specific recombination event which leads to flagellar phase variation. Starting with a fully symmetrical recombination site, hixC, a set of 40 recombination sites which vary by pairs of single base substitutions was constructed. This set was incorporated into the Salmonella-specific bacteriophage P22 based challenge phage selection and used to define the DNA sequence determinants for the binding of Hin to DNA in vivo. The critical sequence-specific contacts between a Hin monomer and a 13 bp hix half-site are at two T:A base pairs in the major groove of the DNA which are separated by one base pair, and two consecutive A:T contacts in the minor groove. The base substitutions in the major groove recognition portion which were defective in binding Hin still retained residual binding capability in vivo, while the base pair substitutions affecting the minor groove recognition region lost all in vivo binding. Using in vitro binding assays, Hin was found to bind to hix symmetrical sites with A:T base pairs or I:C base pairs in the minor groove recognition sequences, but not to G:C base pairs. In separate in vitro binding assays, Hin was equally defective in binding to either a G:C or a I:C contact in a major groove recognition sequence. Results from in vitro binding assays to hix sites in which 3-deazaadenine was substituted for adenine are consistent with Hin making a specific contact to either the N3 of adenine or O2 of thymine in the minor groove within the hix recombination site on each symmetric half-site. These results taken with the results of previous studies on the DNA binding domain of Hin suggest a sequence-specific minor groove DNA binding motif.