NORMAL GENOMIC

• Lam WC, Thompson EH, Potapova O, Sun XC, Joyce CM, Millar DP
• 3'-5' exonuclease of Klenow fragment: role of amino acid residues withinthe single-stranded DNA binding region in exonucleolysis and duplex DNAmelting.
• Biochemistry. 2002; 41: 3943-51
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• The mechanism of the 3'-5' exonuclease activity of the Klenow fragment ofDNA polymerase I has been investigated with a combination of biochemicaland spectroscopic techniques. Site-directed mutagenesis was used to makealanine substitutions of side chains that interact with the DNA substrateon the 5' side of the scissile phosphodiester bond. Kinetic parameters for3'-5' exonuclease cleavage of single- and double-stranded DNA substrateswere determined for each mutant protein in order to probe the role of theselected side chains in the exonuclease reaction. The results indicatethat side chains that interact with the penultimate nucleotide (Q419,N420, and Y423) are important for anchoring the DNA substrate at theactive site or ensuring proper geometry of the scissile phosphate. Incontrast, side chains that interact with the third nucleotide from the DNAterminus (K422 and R455) do not participate directly in exonucleasecleavage of single-stranded DNA. Alanine substitutions of Q419, Y423, andR455 have markedly different effects on the cleavage of single- anddouble-stranded DNA, causing a much greater loss of activity in the caseof a duplex substrate. Time-resolved fluorescence anisotropy decaymeasurements with a dansyl-labeled primer/template indicate that theQ419A, Y423A, and R455A mutations disrupted the ability of the Klenowfragment to melt duplex DNA and bind the frayed terminus at theexonuclease site. In contrast, the N420A mutation stabilized binding of aduplex terminus to the exonuclease site, suggesting that the N420 sidechain facilitates the 3'-5' exonuclease reaction by introducing straininto the bound DNA substrate. Together, these results demonstrate thatprotein side chains that interact with the second or third nucleotidesfrom the terminus can participate in both the chemical step of theexonuclease reaction, by anchoring the substrate in the active site or byensuring proper geometry of the scissile phosphate, and in the prechemicalsteps of double-stranded DNA hydrolysis, by facilitating duplex melting.

• Moy FJ et al.
• High-resolution solution structure of the catalytic fragment of humancollagenase-3 (MMP-13) complexed with a hydroxamic acid inhibitor.
• J Mol Biol. 2000; 302: 671-89
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• The high-resolution solution structure of the catalytic fragment of humancollagenase-3 (MMP-13) complexed with a sulfonamide derivative of ahydroxamic acid compound (WAY-151693) has been determined bymultidimensional heteronuclear NMR. A total of 30 structures werecalculated for residues 7-164 by means of hybrid distancegeometry-simulated annealing using a total of 3280 experimental NMRrestraints. The atomic rms distribution about the mean coordinatepositions for the 30 structures is 0.43(+/-0.05) A for the backbone atoms,0.80(+/-0.09) A for all atoms, and 0.47(+/-0.04) A for all atoms excludingdisordered side-chains. The overall structure of MMP-13 is composed of abeta-sheet consisting of five beta-strands in a mixed parallel andanti-parallel arrangement and three alpha-helices where its overall foldis consistent with previously solved MMP structures. A comparison of theNMR structure of MMP-13 with the published 1.6 A resolution X-raystructure indicates that the major differences between the structures isassociated with loop dynamics and crystal-packing interactions. Theside-chains of some active-site residues for the NMR and X-ray structuresof MMP-13 adopt distinct conformations. This is attributed to the presenceof unique inhibitors in the two structures that encounter distinctinteractions with MMP-13. The major structural difference observed betweenthe MMP-13 and MMP-1 NMR structures is the relative size and shape of theS1' pocket where this pocket is significantly longer for MMP-13, nearlyreaching the surface of the protein. Additionally, MMP-1 and MMP-13exhibit different dynamic properties for the active-site loop and thestructural Zn-binding region. The inhibitor WAY-151693 is well defined inthe MMP-13 active-site based on a total of 52 distance restraints. Thebinding motif of WAY-151693 in the MMP-13 complex is consistent with ourpreviously reported MMP-1:CGS-27023A NMR structure and is similar to theMMP-13: RS-130830 X-ray structure.

• Brautigam CA, Steitz TA
• Structural principles for the inhibition of the 3'-5' exonuclease activityof Escherichia coli DNA polymerase I by phosphorothioates.
• J Mol Biol. 1998; 277: 363-77
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• A two-metal-ion catalytic mechanism has previously been proposed forseveral phosphoryl-transfer enzymes. In order to extend the structuralbasis of this mechanism, crystal structures of three single-stranded DNAsubstrates bound to the 3'-5' exonucleolytic active site of the largefragment of DNA polymerase I from Escherichia coli have been elucidated.The first is a 2.1 A resolution structure of a Michaelis complex betweenthe large fragment (or Klenow fragment, KF) and a single-stranded DNAsubstrate, stabilized by low pH and flash-freezing. The positions andidentities of the catalytic metal ions, a Zn2+ at site A and a Mg2+ atsite B, have been clearly established. The structural and kineticconsequences of sulfur substitutions in the scissile phosphate have beenexplored. A complex with the Rp isomer of phosphorothioate DNA, refined at2.2 A resolution, shows Zn2+ bound to both metal sites and amispositioning of the substrate and attacking nucleophile. The complexwith the Sp phosphorothioate at 2. 3 A resolution reveals that metal ionsdo not bind in the active site, having been displaced by a bulky sulfuratom. Steady-state kinetic experiments show that catalyzed hydrolysis ofthe Rp isomer was reduced only about 15-fold, while no enzyme activitycould be detected with the Sp phosphorothioate, consistent with thestructural observations. Furthermore, Mn2+ could not rescue the activityof the exonuclease on the Sp phosphorothioate. Taken together, thesestudies confirm and extend the proposed two-metal-ion exonucleasemechanism and provide a structural context to explain the effects ofsulfur substitutions on this and other phosphoryl-transfer enzymes. Theseexperiments also suggest that the possibility of metal-ion exclusion betaken into account when interpreting the results of Mn2+ rescueexperiments.

• Reha-Krantz LJ
• Regulation of DNA polymerase exonucleolytic proofreading activity: studiesof bacteriophage T4 "antimutator" DNA polymerases.
• Genetics. 1998; 148: 1551-7

• Cabaniss S, Deerfield DW 2nd, Monroe DM, Hiskey RG, Pedersen LG
• Is Ca(II) ion binding to prothrombin fragment 1 intrinsically cooperative,or is the cooperative binding accounted for by self-association?
• Blood Coagul Fibrinolysis. 1995; 6: 464-73
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• The recent suggestion that the apparent cooperativity seen in the bindingof Ca(II) ions to prothrombin fragment 1 is due to protein aggregation isevaluated. Since (1) we find that the Ca(II) ion binding is not dependentupon protein concentration, (2) the analytical expression for theequilibrium constant of the aggregation model is unrealistically largewhen evaluated at realistic Ca(II) ion concentrations, and (3) a verysimple allosteric cooperative binding model (Monod) can be shown to fitthe experimental data, we conclude that the aggregation explanation forthe apparent cooperativity in the Ca(II) ion binding by prothrombinfragment 1 is not correct.

• Matray TJ, Haxton KJ, Greenberg MM
• The effects of the ring fragmentation product of thymidine C5-hydrate onphosphodiesterases and klenow (exo-) fragment.
• Nucleic Acids Res. 1995; 23: 4642-8
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• N-(2-Deoxy-beta-D-erythro-pentofuranosyl)-N-3-(2R-hydroxyisobutyricacid)urea (alpha-R-hydroxy-beta-ureidoisobutyric acid, 8) was sitespecifically incorporated into a series of oligonucleotides via theammonolysis of biopolymers containing 5R-thymidine C5-hydrate (3).alpha-R-hydroxy-beta-ureidoisobutyric acid (8) inhibits snake venomphosphodiesterase, lambda exonuclease and Klenow (exo-) fragment. Kineticmeasurements for insertion of nucleotides opposite 8 by Klenow (exo-)fragment indicate that this lesion is instructive.

• Tucker PA, Tsernoglou D, Tucker AD, Coenjaerts FE, Leenders H, van der Vliet PC
• Crystal structure of the adenovirus DNA binding protein reveals a hook-onmodel for cooperative DNA binding.
• EMBO J. 1994; 13: 2994-3002
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• The adenovirus single-stranded DNA binding protein (Ad DBP) is amultifunctional protein required, amongst other things, for DNAreplication and transcription control. It binds to single- anddouble-stranded DNA, as well as to RNA, in a sequence-independent manner.Like other single-stranded DNA binding proteins, it binds ssDNA,cooperatively. We report the crystal structure, at 2.6 A resolution, ofthe nucleic acid binding domain. This domain is active in DNA replication.The protein contains two zinc atoms in different, novel coordinations. Thezinc atoms appear to be required for the stability of the protein foldrather than being involved in direct contacts with the DNA. The crystalstructure shows that the protein contains a 17 amino acid C-terminalextension which hooks onto a second molecule, thereby forming a proteinchain. Deletion of this C-terminal arm reduces cooperativity in DNAbinding, suggesting a hook-on model for cooperativity. Based on thisstructural work and mutant studies, we propose that DBP forms a proteincore around which the single-stranded DNA winds.

• Oakley MG, Turnbull KD, Dervan PB
• Synthesis of a hybrid protein containing the iron-binding ligand ofbleomycin and the DNA-binding domain of Hin.
• Bioconjug Chem. 1994; 5: 242-7
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• The iron-binding and oxygen-activating domain of the natural productbleomycin [pyrimidoblamic acid-beta-hydroxy-L-histidine (PBA-beta-OH-His)]was attached to the NH2 terminus of the DNA binding domain of Hinrecombinase (residues 139-190). This hybrid 54-residue proteinPBA-beta-OH-His-Hin-(139-190) binds specifically to DNA at four distinctHin binding sites with affinities comparable to those of the unmodifiedHin(139-190). In the presence of dithiothreitol,Fe(II).PBA-beta-OH-His-Hin-(139-190) cleaves DNA with specificityremarkably similar to that of Fe(II).EDTA-Hin(139-190). Analysis of thecleavage patterns suggests that site-specific DNA cleavage is mediated bya localized diffusible species, in contrast with cleavage by bleomycin,which occurs through a nondiffusible oxidant. This has implications forthe design of second-generation artificial sequence specific DNA cleavingproteins and defines limitations in current efforts to createatom-specific chemistry on DNA.

• Katayanagi K, Okumura M, Morikawa K
• Crystal structure of Escherichia coli RNase HI in complex with Mg2+ at 2.8A resolution: proof for a single Mg(2+)-binding site.
• Proteins. 1993; 17: 337-46
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• To obtain more precise insight into the Mg(2+)-binding site essential forRNase HI catalytic activity, we have determined the crystal structure ofE. coli RNase HI in complex with Mg2+. The analyzed cocrystal, which isnot isomorphous with the Mg(2+)-free crystal previously refined at 1.48 Aresolution, was grown at a high MgSO4 concentration more than 100 mM sothat even weakly bound Mg2+ sites could be identified. The structure wassolved by the molecular replacement method, using the Mg(2+)-free crystalstructure as a search model, and was refined to give a final R-value of0.190 for intensity data from 10 to 2.8 A, using the XPLOR and PROLSQprograms. The backbone structures are in their entirety very similar toeach other between the Mg(2+)-bound and the metal-free crystals, exceptfor minor regions in the enzyme interface with the DNA/RNA hybrid. Theactive center clearly revealed a single Mg2+ atom located at a positionalmost identical to that previously found by the soaking method. Althoughthe two metal-ion mechanism had been suggested by another group (Yang, W.,Hendrickson, W.A., Crouch, R.J., Satow, Y. Science 249:1398-1405, 1990)and partially supported by the crystallographic study of inactive HIV-1 RTRNase H fragment (Davies, J.F., II, Hostomska, Z., Hostomsky, Z., Jordan,S.R., Matthews, D. Science 252:88-95, 1991), the present result excludesthe possibility that RNase HI requires two metal-binding sites foractivity. In contrast to the features in the metal-free enzyme, the sidechains of Asn-44 and Glu-48 are found to form coordinate bonds with Mg2+in the metal-bound crystal.

• Murphy KC, Casey L, Yannoutsos N, Poteete AR, Hendrix RW
• Localization of a DNA-binding determinant in the bacteriophage P22 Erfprotein.
• J Mol Biol. 1987; 194: 105-17
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• Four amber fragments of the recombination-promoting P22 Erf protein werecharacterized. The intact Erf monomer contains 204 amino acids. The ambermutations produce fragments of 190, 149, 130 and 95 amino acid residues,all of which are inactive in vivo. The 190 residue fragment is moresusceptible to proteolysis in cell extracts than is intact Erf. It breaksdown to a stable remnant that is slightly larger than the 149 residuefragment. The 149 and 130 residue fragments are stable; electronmicroscopy of the purified fragments reveals that they have similarmorphologies, retaining the ring-like oligomeric structure, but lackingthe tooth-like protruding portions of intact Erf. Intact Erf and the 149residue fragment have similar affinities for single-stranded DNA; theaffinity of the 130 residue fragment is 40-fold lower in low salt at pH6.0. The 95 residue fragment is unstable in vivo. These observations,combined with previous observations, are interpreted as suggesting thatthe boundary of the amino-terminal domain of the protein lies betweenresidues 96 and 130, that certain residues between 131 and 149 form partof an interdomain DNA-binding segment of the protein, that the boundary ofthe carboxy-terminal domain lies to the C-terminal side of residue 149,and that the carboxy-terminal domain is not necessary for assembly of thering oligomer, although it is essential for Erf activity in vivo.

• Kabat D
• Fibrous complexes of deoxyribonucleic acid with certain globular proteins.Role of divalent metal ions in the organization of nucleoproteinstructures.
• Biochemistry. 1967; 6: 3443-58