2.7.7.7 A329A not meaningfully associated with breast cancer risk; more likely to respond to Pt-based chemotherapy 761958 2.7.7.7 A408S A408S mutation results in a significant increase in both dNTP binding affinity and fidelity, kcat/Km for dATP is about 45% compared to the wild-type enzyme, D215A mutation results in inactivation of 3'-5'-exonuclease activity 721612 2.7.7.7 A467T naturally occuring mutation, the mutation is the most common POLG mutation and has been found to be associated with all of the disease symptoms analyzed. The A467T pol gamma possesses only 4% of the wild-type DNA polymerase activity and is compromised for its ability to interact with the p55 accessory subunit 723108 2.7.7.7 A471V moderate decrease in activity 761958 2.7.7.7 A957S naturally occuring mutation, involved in autosomal dominant progressive external ophthalmoplegia, the mutation is associated with motiif B in the active site 723108 2.7.7.7 D10A retains polymerase activity, reduced exonuclease activity, changes in dependency on metal activation of exonuclease activity 643653 2.7.7.7 D111A 94% loss of DNA polymerase activity 726027 2.7.7.7 D1122A loss of polymerization activity 722619 2.7.7.7 D1122E mutant enzyme with reduced polymerization activity, polymerization activity is 15% compared to wild-type activity, 3'-5' exonuclease activity remains, the mutant has lower Mg2+ affinity than does the wild-type 722619 2.7.7.7 D1124A loss of polymerization activity 722619 2.7.7.7 D1124E mutant enzyme with reduced polymerization activity, polymerization activity is 43% compared to wild-type activity, 722619 2.7.7.7 D1124N mutant enzyme with reduced polymerization activity, 3'-5' exonuclease activity remains, the mutant has lower Mg2+ affinity than does the wild-type 722619 2.7.7.7 D112A/E114A the mutant T4 DNA polymerases is defective in 3'-5' exonuclease activity and has a 1000fold increase in the development of spontaneous mutations compared to wild type polymerase 703192 2.7.7.7 D115A/E116A catalytically inactive 705693 2.7.7.7 D171A 96% loss of DNA polymerase activity 726027 2.7.7.7 D189G impaired for extension step of TLS 761958 2.7.7.7 D190A retains polymerase activity, reduced exonuclease activity, changes in dependency on metal activation of exonuclease activity 643652 2.7.7.7 D198A/E200A site-directed mutagenesis in the exonuclease domain resulting in loss of exonuclease activity 723108 2.7.7.7 D215A 3'-5'-exonuclease inactive mutant enzyme 721612 2.7.7.7 D219A the mutant T4 DNA polymerases is defective in 3'-5' exonuclease activity and has a 1000fold increase in the development of spontaneous mutations compared to wild type polymerase 703192 2.7.7.7 D259E moderate decrease of the polymerizing activity 722215 2.7.7.7 D259G moderate decrease of the polymerizing activity 722215 2.7.7.7 D259K the exonuclease activity of the mutant enzyme decreases drastically to 0.58% compared with that of the wild-type DNA polymerase 722215 2.7.7.7 D259N moderate decrease of the polymerizing activity 722215 2.7.7.7 D275A/E277A/D368A mutation in the catalytic subunit, exonuclease-deficient variant 761518 2.7.7.7 D324A the mutant T4 DNA polymerases is defective in 3'-5' exonuclease activity and has a 1000fold increase in the development of spontaneous mutations compared to wild type polymerase, the exonuclease activity of the D324A mutant is 100000-fold lower than wild type polymerase 703192 2.7.7.7 D349A the Mg2+-dependent DNA polymerase activity of the mutant is almost the same as that of wild type enzyme -, 705967 2.7.7.7 D362A the misincorporation frequency values of the D362A mutant are slightly higher (1.4–4.9fold) than those of the wild type protein 676558 2.7.7.7 D378A site-directed mutagenesis, the mutant is inactive in presence of Mg2+ 721996 2.7.7.7 D378E site-directed mutagenesis, the mutant is inactive in presence of Mg2+, it shows 35% of maximal activity in presenceof Mn2+ 721996 2.7.7.7 D380A site-directed mutagenesis, the mutant is inactive in presence of Mg2+ 721996 2.7.7.7 D380E site-directed mutagenesis, the mutant is inactive in presence of Mg2+ or Mn2+ 721996 2.7.7.7 D405A mutant enzyme loses 99.8% of DNA polymerizing activity and 90% of 3'->5' exonucleolytic activity 723691 2.7.7.7 D405E mutant enzyme loses 95.8% of DNA polymerizing activity and 90% of 3'->5' exonucleolytic activity 723691 2.7.7.7 D424A a proofreading deficient mutant of the Klenow fragment 721761 2.7.7.7 D424A mutation in polymerase active site, the polymerase activity is reduced more than 30fold compared to the wild-type activity -, 718921 2.7.7.7 D424A/D542A mutation in polymerase active site, complete inactivation of polymerase activity -, 718921 2.7.7.7 D473G wild-type variant Pfu-Pol(exo-) is is 60fold less accurate than Pfu-Pol(exo+) 662975 2.7.7.7 D529A the Mg2+-dependent DNA polymerase activity of the mutant is almost the same as that of wild type enzyme -, 705967 2.7.7.7 D531A site-directed mutagenesis, the mutant is inactive in presence of Mg2+ 721996 2.7.7.7 D531E site-directed mutagenesis, the mutant is inactive in presence of Mg2+, it shows 60% of maximal activity in presenceof Mn2+ 721996 2.7.7.7 D542A mutation in polymerase active site, the polymerase activity is reduced more than 50fold compared to the wild-type activity -, 718921 2.7.7.7 D907V mutant is less resistant to acyclovir and cidofovir than the wild type enzyme 690480 2.7.7.7 DELTA413–470 mutation in the spacer region of the alpha-subunit. Obtained in small amounts due to low solubility, mutant has barely detectable DNA polymerase activity 662379 2.7.7.7 DELTA483–533 expressed efficiently and purified as soluble holoenzyme complex associated with wild-type beta-subunit. The DNA polymerase activity of the mutant holoenzyme is reduced greatly as compared to wild type 662379 2.7.7.7 DELTA536–581 expressed efficiently and purified as soluble holoenzyme complex associated with wild-type beta-subunit. The DNA polymerase activity of the mutant enzyme is 80% of wild-type holoenzyme 662379 2.7.7.7 DELTA666–742 mutation in the spacer region of the alpha-subunit. Obtained in small amounts due to low solubility, mutant has barely detectable DNA polymerase activity 662379 2.7.7.7 DELTAH672-S775 mutant enzyme loses 99% of DNA polymerizing activity and 97% of 3'->5' exonucleolytic activity 723691 2.7.7.7 DELTAL717-S775 mutant enzyme loses 97% of DNA polymerizing activity and 97% of 3'->5' exonucleolytic activity 723691 2.7.7.7 DELTAL746-S775 mutant protein has DNA polymerizing activity with 2.3fold higher specific activity than that of the wild-type but retains only 10% of the 3'->5' exonucleolytic activity of the wild-type 723691 2.7.7.7 E113A 92% loss of DNA polymerase activity 726027 2.7.7.7 E1143G naturally occuring mutation, that is a frequent cause of ataxia-neuropathy syndrome, and found in 4% of European populations 723108 2.7.7.7 E128A 23% loss of DNA polymerase activity 726027 2.7.7.7 E170A complete loss of the 3'-5'exonuclease activity 721564 2.7.7.7 E200A exonuclease-deficient mutant 704422 2.7.7.7 E292K more active than wild-type enzyme 760878 2.7.7.7 E292K similar activity as wild-type enzyme 761958 2.7.7.7 E29K decreased insertion opposite abasic site (2-20) 761958 2.7.7.7 E413A the Mg2+-dependent DNA polymerase activity of the mutant is almost the same as that of wild type enzyme 705967 2.7.7.7 E419G 20fold decrease in kcat/Km on dG and 670fold decrease on N2-CH2-Anth-dG, extension defect 761958 2.7.7.7 E430G low activity on AP site 761958 2.7.7.7 E449K low activity on AP site, low fidelity 761958 2.7.7.7 E602V/A608V/I614M/E615G the mutant enzyme is able to incorporate both NTPs and dNTPs with the same catalytic efficiency as the wild-type enzyme incorporates dNTPs. The mutant enzyme allowed the generation of mixed RNA–DNA amplification products in PCR demonstrating DNA polymerase, RNA polymerase as well as reverse transcriptase activity within the same polypeptide. The mutant displays an expanded substrate spectrum towards other 2'-substituted nucleotides and is able to synthesize nucleic acid polymers in which each base bear a different 2'-substituent 675398 2.7.7.7 F12A GTP incorporation by the wild-type enzyme is about 1000fold slower than dGTP incorporation. The rate of GTP incorporation by the mutant enzyme F12A Dbh is 2-3fold slower than incorporation of dGTP. When making a deletion error, ribonucleotide discrimination by wild-type and F12A Dbh is the same as in normal DNA synthesis 720570 2.7.7.7 F12A mutant enzyme shows disproportionately reduced activity on the damaged template 676145 2.7.7.7 F12A mutant ribonucleoside triphosphates almost as efficiently as deoxyribonucleoside triphosphates, and, unlike analogous mutants in other polymerase families, shows no barrier to adding multiple ribonucleotides, suggesting that Dbh can readily accommodate a DNA/RNA duplex product 720570 2.7.7.7 F12A no detectable incorporation of ribonucleotide triphosphate is observed with the wild-type enzyme, the mutant form F12A efficiently incorporates and extends ribonucleotide triphosphate, even in the absence of Mn2+ ions -, 724848 2.7.7.7 F13V mutant enzyme is almost unable to carry out translesion synthesis over N2-furfuryl-dG, although its activity on undamaged DNA is unaffected. The F13V mutation has a modest effect on the ability of DinB to discriminate against ribonucleotides, increasing the frequency of their misincorporation from less than 0.00001 to 0.001 676145 2.7.7.7 F155S decreased activity on model abasic site 761958 2.7.7.7 F192C more active than wild-type enzyme 760878 2.7.7.7 F192C slight increase in activity with N2-furfuryl-dG-containing templates 761958 2.7.7.7 F388A site-directed mutagenesis, the mutant exhibits DNA-dependent and RNA-dependent DNA polymerase activities, while the wild-type enzyme is a DNA-depedent DNA polymerase -, 722837 2.7.7.7 F506G inactive mutant enzyme 662621 2.7.7.7 F506R complete loss of de novo DNA synthesis 662621 2.7.7.7 F576A mutation in alpha-subunit, mutant enzyme retains about 50% of wild-type activity 662379 2.7.7.7 F771A the mutation shows 30% decreased polymerase activity compared to the wild type enzyme 693076 2.7.7.7 G1123A mutant enzyme has 13% of the activity of the wild type enzyme 722619 2.7.7.7 G1123R mutant enzyme has 0.8% of the activity of the wild type enzyme 722619 2.7.7.7 G154E decreased activity opposite model abasic site, pathogenic 761958 2.7.7.7 G418K increased exonuclease activity 643660 2.7.7.7 G534R abscisic acid overly sensitive mutant, also hypersensitive to methyl methanesulfonate and UV-B light 706221 2.7.7.7 G575A mutation in alpha-subunit, mutant enzyme has nearly normal activity 662379 2.7.7.7 G575A/W576A/F578A mutation in alpha-subunit completely reduces DNA polymerase activity 662379 2.7.7.7 G848S naturally occuring mutation, involved in Alpers syndrome, the mutant shows compromised DNA binding ability that affects its overall functional efficiency 723108 2.7.7.7 G923D naturally occuring mutation, involved in autosomal dominant progressive external ophthalmoplegia, the mutation is associated with motiif B in the active site 723108 2.7.7.7 H145A 98% loss of DNA polymerase activity 726027 2.7.7.7 H190A 31% loss of DNA polymerase activity 726027 2.7.7.7 H329A mutation has little effect on template-dependent synthesis by Pol l from a paired primer terminus, but it reduces both template-independent and template-dependent synthesis during nonhomologous DNA end joining of intermediates whose 3' ends lack complementary template strand nucleotides 676140 2.7.7.7 H344A the Mg2+-dependent DNA polymerase activity of the mutant is almost the same as that of wild type enzyme -, 705967 2.7.7.7 H374A the Mg2+-dependent DNA polymerase activity of the mutant is almost the same as that of wild type enzyme -, 705967 2.7.7.7 H440A the Mg2+-dependent DNA polymerase activity of the mutant is almost the same as that of wild type enzyme 705967 2.7.7.7 H468A the Mg2+-dependent DNA polymerase activity of the mutant is almost the same as that of wild type enzyme 705967 2.7.7.7 H531A the Mg2+-dependent DNA polymerase activity of the mutant is almost the same as that of wild type enzyme 705967 2.7.7.7 H633D DNA polymerase activity of the mutant enzyme is higher than the polymerase activity of the wild-type enzyme. PCR efficiency of the H633D mutant is higher than that of the N565K mutant enzyme 729659 2.7.7.7 H633R significantly improved polymerase function compared to wild-type enzyme in terms of processivity (2-fold), extension rate (1.5fold) and PCR efficiency. The kcat value of the Twa H633R mutant is similar to that of wild-type, but the Km of the Twa H633R mutant is about 1.6fold lower than that of the wild-type 729659 2.7.7.7 I364Q binds the substrate with less efficiency than wild-type enzyme 643672 2.7.7.7 I364R unable binding of the substrate to the enzyme 643672 2.7.7.7 I39T more active than wild-type enzyme 760878 2.7.7.7 I39T similar activity to WT with several types of DNA damage 761958 2.7.7.7 K242R/I243K/P244S mutation of three linker amino acids, polymerase Dbh adopts the standard conformation of polymerase Dpo4. The interdomain linker also affects the single-base deletion frequency and the mispair extension efficiency of these polymerase -, 725547 2.7.7.7 K253E exonuclease activity of mutant increases 2.7fold compared to wild-type activity 722215 2.7.7.7 K253E/R255E exonuclease activity of mutant increases 1.8fold compared to wild-type activity 722215 2.7.7.7 K366T mutant enzyme shows a wild-type like phenotype in DNA-primed polymerisation in the presence of DNA as template, in terminal protein-primed reactions as initiation and transition it is impaired, especially in the presence of the Phi29 DNA-binding protein, protein p6 662613 2.7.7.7 K371T binds the substrate with the same efficiency as wild-type enzyme 643672 2.7.7.7 K557A mutation in alpha-subunit, mutant enzyme has nearly normal activity 662379 2.7.7.7 K65A 98% loss of DNA polymerase activity 726027 2.7.7.7 K66A 70% loss of DNA polymerase activity 726027 2.7.7.7 K687A/D688A/F689A mutation in alpha-subunit resulted in DNA polymerase activities that is 60–80% of wild-type enzyme 662379 2.7.7.7 K70A 19% loss of DNA polymerase activity 726027 2.7.7.7 L21F 30fold decrease in incorporation opposite N2-CH2-(9-anthracenyl)-dG (N2-CH2-Anth-dG) 761958 2.7.7.7 L21F more active than wild-type enzyme 760878 2.7.7.7 L329A site-directed mutagenesis -, 721508 2.7.7.7 L329A site-directed mutagenesis, the mutant exhibits DNA-dependent and RNA-dependent DNA polymerase activities, while the wild-type enzyme is a DNA-depedent DNA polymerase -, 722837 2.7.7.7 L329A/Q384A site-directed mutagenesis, the mutant exhibits DNA-dependent and RNA-dependent DNA polymerase activities, while the wild-type enzyme is a DNA-depedent DNA polymerase 722837 2.7.7.7 L329A/Y438A site-directed mutagenesis, the mutant exhibits DNA-dependent and RNA-dependent DNA polymerase activities, while the wild-type enzyme is a DNA-depedent DNA polymerase 722837 2.7.7.7 L391F defective in the in vitro replication initiation and DNA polymerase elongation assays and fails to recognize the viral replication origin if the protein is expressed at 37°C, expression at 32°C results in activities similar to wild-type enzyme 643641 2.7.7.7 L409I kcat/Km for dATP is about 20% compared to the wild-type enzyme 721612 2.7.7.7 L409M L409 mutation results in drastically reduced affinity for the correct dNTP, a much higher efficiency of both misincorporation and mismatch extension, and substantially lower fidelity as demonstrated by a PCR-based forward mutation assay, kcat/Km for dATP is about 155% compared to the wild-type enzyme, D215A mutation results in inactivation of 3'-5'-exonuclease activity 721612 2.7.7.7 L409V kcat/Km for dATP is about 35% compared to the wild-type enzyme 721612 2.7.7.7 L442F low activity on AP site 761958 2.7.7.7 L558A mutation in alpha-subunit, mutant enzyme retains about 50% of wild-type activity 662379 2.7.7.7 L561A the mutant has pre-steadystate Kd,app and kpol values for the incorporation of correct dNMPs that are comparable to or exhibits kpol values that are somewhat greater than wild type DNA polymerase 702328 2.7.7.7 L561A/Y567A the mutant has pre-steadystate Kd,app and kpol values for the incorporation of correct dNMPs that are comparable to or exhibits kpol values that are somewhat greater than wild type DNA polymerase 702328 2.7.7.7 L561A/Y567A/S565G the mutant has pre-steadystate Kd,app and kpol values for the incorporation of correct dNMPs that are comparable to or exhibits kpol values that are somewhat greater than wild type RB69 DNA polymerase 702328 2.7.7.7 L606G site-directed mutagenesis, the mutant shows increased polymerase activity and slightly reduced exonuclease activity compared to the wild-type enzyme, mutant pol delta L606G is highly error prone, incorporating single noncomplementary nucleotides at a high frequency during DNA synthesis 722686 2.7.7.7 L606K site-directed mutagenesis, the mutant shows increased polymerase activity and reduced exonuclease activity compared to the wild-type enzyme, mutant pol delta L606K is extremely accurate, with a higher fidelity of single nucleotide incorporation by the active site than that of wild-type pol delta, it does not catalyze detectable nucleotide mis-insertion even with nucleotide concentrations as high as 4 mM, but pol delta L606K mutant is impaired in the bypass of DNA adducts 722686 2.7.7.7 L778M mutant is less resistant to acyclovir and cidofovir than the wild type enzyme 690480 2.7.7.7 L823A the mutation shows 3% decreased polymerase activity compared to the wild type enzyme 693076 2.7.7.7 L831N/A814R truncated enzyme delta413-L813N/A814R has reduced temperature stability 643675 2.7.7.7 M408A site-directed mutagenesis, the mutant exhibits DNA-dependent and RNA-dependent DNA polymerase activities, while the wild-type enzyme is a DNA-depedent DNA polymerase 722837 2.7.7.7 M644F mutant enzyme has reduced fidelity resulting from strongly increased misinsertion rates 676222 2.7.7.7 M644L mutant enzyme synthesizes DNA with high fidelity 676222 2.7.7.7 M644W mutant enzyme synthesizes DNA with high fidelity 676222 2.7.7.7 molecular biology the high fidelty of the enzyme is suitable fo polymerase chain reaction (PCR), which requires accurate DNA amplification for gene cloning and diagnostic assay -, 721307 2.7.7.7 additional information a polX disruption mutant expressing 5'-deoxyribose phosphate lyase and a truncated polymerase domain is comparatively less sensitive to gamma-radiation at 10 kGy than a polX deletion mutant, both mutants show higher sensitivity to hydrogen peroxide -, 705565 2.7.7.7 additional information construction of a chimeric DNA polymerase derived from Thermus species Z05 and Thermotoga maritima DNA polymerases. These chimeric DNA polymerases are fashioned using structure-based tools to identify amino acid residues involved in the substrate-binding site of the exonuclease domain of a thermostable DNA polymerase. Mutation of some of these residues results in proteins in which DNA polymerase activity is unaffected, while proofreading activity ranges from 60% of the wild-type level to undetectable levels. Kinetic characterization of the exonuclease activity indicates that the mutations affects catalysis much more than binding. On the basis of their specificity constants (kcat/KM), the mutant enzymes have a 5-15-fold stronger preference for a double-stranded mismatched substrate over a single-stranded substrate than the wild-type DNA polymerase, a desirable attribute for RT/PCR -, 672087 2.7.7.7 additional information construction of Pol IV-defective dinB knockout mutants 680446 2.7.7.7 additional information deletion of the COOH-terminal zinc finger region (1289–1308) does not abolish the subunit interaction. Instead, while the polymerization of the deletion mutants remains, the 3'-5' exonuclease is completely inactivated. Deletion of DP1Pho(1–200) increases the exonuclease and DNA binding activities of PolDPho. Adding DP1Pho(1–200) to the truncated protein suppresses the elevated exonuclease activity 722631 2.7.7.7 additional information deletion of the dinB gene sensitized Pseudomonas aeruginosa to nitrofurazone and 4-nitroquinoline-1-oxide, consistent with a role for DinB in translesion DNA synthesis over N2-dG adducts, while the UV-inducible mutator phenotype is independent of dinB function 680476 2.7.7.7 additional information DNA binding, dNTP binding and catalytic activity of mutant enzyme in the presence of two metal ions, Mg2+ and Mn2+, overview 721996 2.7.7.7 additional information dyskinetoplastid bloodstream form parasites produced during RNAi are not viable, disruption of network-free minicircle replication precedes parasite death 722149 2.7.7.7 additional information engineered DNA polymerases are obtained by the flexible attachment of helix-hairpin-helix DNA binding domains of topoisomerase V of Methanopyrus kandleri to catalytic domains of DNA polymerases, domain fusion using the Taq and Pfu DNA polymerases or the phi29 DNA polymerase. The chimeric polymerases retain high processivity at high concentrations of salts and other inhibitors of DNA synthesis, such as phenol, blood, and DNA intercalating dyes, domain attachment has a potential to greatly increase the thermostability of chimeric DNA polymerases 721660 2.7.7.7 additional information fusion of RB69 DNA polymerase with its cognate single-stranded DNA binding protein via a short six amino acid linker increases affinity for primer-template DNA by 6fold and subsequently increases processivity by 7fold while maintaining fidelity 677053 2.7.7.7 additional information generaion of chimeras of Sulfolobus solfataricus DNA polymerase Dpo4 and Sulfolobus acidocaldarius DNA polymerase Dbh in which their little finger domains have been interchanged. Interestingly, by replacing the little finger domain of Dbh with that of Dpo4, the enzymatic properties of the chimeric enzyme are more Dpo4-like in that the enzyme is more processive, can bypass an abasic site and a thymine-thymine cyclobutane pyrimidine dimer, and predominantly makes base pair substitutions when replicating undamaged DNA. The converse is true for the Dpo4-LF-Dbh chimera, which is more Dbh-like in its processivity and ability to bypass DNA adducts and generate single-base deletion errors. The unique but variable little finger domain of Y-family polymerases plays a major role in determining the enzymatic and biological properties of each individual Y-family member -, 725361 2.7.7.7 additional information generation of a mutated thermostable DNA polymerase, Taq M1, from Thermus aquaticus that exhibits an increased reverse transcriptase activity. The Taq polymerase mutant Taq M1 has similar PCR sensitivity and nuclease activity as the respective Taq wild-type DNA polymerase, but Taq M1 exhibits a significantly increased reverse transcriptase activity especially at high temperatures compared to the wild-type 721895 2.7.7.7 additional information generations of deletion umuDC operon mutants, pol V Mut shows altered requirements of accessory factors compared to the wild-type enzyme, overview 722100 2.7.7.7 additional information insertions in the DNA polymerase III epsilon subunit gene (dnaQ) cause a specific defect in SOS induction by nalidixic acid but not by mitomycin C -, 704278 2.7.7.7 additional information null mutations in Arabidopsis POL2a are embryonic lethal 706221 2.7.7.7 additional information parasites overexpressing Tcpolbeta shows reduced levels of 7,8-dihydro-8-oxoguanine in kDNA and an increased survival after treatment with hydrogen peroxide when compared to control cells. The resistance is lost after treating Tcpolbeta overexpressors with methoxiamine, a potent base-excision repair inhibitor 723132 2.7.7.7 additional information POLPs have two extra sequences in the polymerase domain that are absent in prokaryotic PolIs. Deletion of either insert from OsPOLP1 causes a decrease in DNA synthetic activity, processivity, and DNA binding activity 676558 2.7.7.7 additional information reconstitution of replicase activity by co-expression of essential components of DNA polymerase holoenzyme from Pseudomonas aeruginosa: expression of processivity factor, i.e. beta-subunit, single-stranded DNA-binding protein, a complex containing the polymerase, i.e. alpha-subunit, and exonuclease, i.e. epsilon-subunit, and the essential components of the DnaX complex tau3deltadelta', Pseudomonas aeruginosa alphaepsilon can substitute completely for Escherichia coli polymerase III in Escherichia coli holoenzyme reconstitution assays, addition of purified Escherichia coli chi and psi, components of the DnaX complex, increases the apparent specific activity of the Pseudomonas tau3deltadelta' complex about 10-fold and enables the reconstituted enzyme to function better under physiological salt conditions, overview 680635 2.7.7.7 additional information the (G)-PYF box is located in a hydrophobic region close to the active site. The (G)-PYF box mutants exhibit altered DNA binding properties. In addition, the thermal stability of all mutants is reduced compared to that of wild type, and this effect could be attributed to increased exposure of the hydrophobic region 722960 2.7.7.7 additional information the DNA coding sequence of KOD from Pyrococcus sp. KOD1 is optimized based on the codon usage bias of Pichia pastoris and synthesized by overlapping PCR, and the nonspecific DNA-binding protein Sso7d from Sulfolobus solfataricus is fused to the C-terminus of KOD. The resulting novel gene is cloned into a pHBM905A vector and introduced into Pichia pastoris GS115 for secretory expression. The yield of the target protein reaches approximately 250 mg/l after a 6 day induction with 1% (v/v) methanol in shake flasks. This yield is much higher than those of other DNA polymerases expressed heterologously in Escherichia coli. The recombinant enzyme expressed in Pichia pastoris exhibits excellent thermostability, extension rate and fidelity. When Sso7d is fused to the enzyme, a significant enhancement of processivity is achieved regardless of the starting processivity of the enzyme 732681 2.7.7.7 additional information the intervening domain of the thermostable Thermus aquaticus DNA polymerase (TAQ: polymerase), which has no catalytic activity, is exchanged for the 3'-5' exonuclease domain of the homologous mesophile Escherichia coli DNA polymerase I (Escherichia coli pol I) and the homologous thermostable Thermotoga neapolitana DNA polymerase (TNE: polymerase). Three chimeric DNA polymerases are constructed using the three-dimensional (3D) structure of the Klenow fragment of the Escherichia coli pol I and 3D models of the intervening and polymerase domains of the TAQ: polymerase and the TNE: polymerase: chimera TaqEc1 (exchange of residues 292-423 from TAQ: polymerase for residues 327-519 of Escherichia coli pol I), chimera TaqTne1 (exchange of residues 292-423 of TAQ: polymerase for residues 295-485 of TNE: polymerase) and chimera TaqTne2 (exchange of residues 292-448 of TAQ: polymerase for residues 295-510 of TNE: polymerase). The chimera TaqEc1 shows characteristics from both parental polymerases at an intermediate temperature of 50°C: high polymerase activity, processivity, 3'-5' exonuclease activity and proof-reading function The chimeras TaqTne1 and TaqTne2 show no significant 3'-5' exonuclease activity and no proof-reading function. The chimera TaqTne1 shows an optimum temperature at 60°C, decreased polymerase activity compared with the TAQ: polymerase and reduced processivity. The chimera TaqTne2 shows high polymerase activity at 72°C, processivity and less reduced thermostability compared with the chimera TaqTne1 728727 2.7.7.7 additional information the wild-type enzyme is modified by deletion of the N-terminal 5' to 3' exonuclease domain, by fusion with the DNA-binding protein Sso7d and by introduction of four known effective point mutations from other DNA polymerase mutants, and codon optimization to reduce the GC content. A mutant is obtained that provides higher product yields than the conventional Taq pol without decreased fidelity. Next, four rounds of compartmentalized self-replication (CSR) selection are performed with a randomly mutated library of this modified Tth pol and mutants are obtainwed that provide higher product yields in fewer cycles of emulsion PCR than the parent Tth pol as well as the conventional Taq pol 760489 2.7.7.7 additional information Tpa-S DNA polymerase (the fusion of the Sso7d protein to the C-terminus of Tpa DNA polymerase) improves the performance of the Tpa DNA polymerase to make it suitable for long and rapid polymerase chain reaction -, 722133 2.7.7.7 N176A 2% increase of DNA polymerase activity 726027 2.7.7.7 N188W kcat/Km for dCTP is 2.9fold compared to kcat/Km of wild-type enzyme -, 725385 2.7.7.7 N210D the mutant enzyme shows very weak 3'-5'-exonuclease activity compared to the wild-type enzyme (0.1%). No significant difference in DNA polymerase activity as compared with that of the wild-type enzyme. Mutation frequency in PCR becomes higher as 3'–5' exonuclease activity decreases 725590 2.7.7.7 N249Y exhibits increased catalytic activity when compared to the wild-type enzyme, the mutation decreases the affinity for NAD(H) cofactor -, 725713 2.7.7.7 N483Q/S486Q/T539N/Y545Q/D547T/P548Q/A570Q/D578Q/A597T/W604R /S612N/V730L/R736Q/S739N/M747R selection of a polymerase with 15 mutations, mostly located at the template binding interface, by directed evolution of Thermus aquaticus DNA polymerase I, the mutant enzyme is a single variant of the Stoffel fragment of Taq DNA polymerase I, the enzyme shows broad template specificity and is a thermostable DNA-dependent and RNA-dependent DNA-polymerase, see also EC 2.7.7.49 723163 2.7.7.7 N565K DNA polymerase activity of the mutant enzyme is higher than the polymerase activity of the wild-type enzyme. PCR efficiency of the H633D mutant is higher than that of the N565K mutant enzyme 729659 2.7.7.7 N678A no change in polymerase activity, increased mismatch-directed exonuclease activity 643674 2.7.7.7 P169T more active than wild-type enzyme 760878 2.7.7.7 P169T slight decrease in activity on undamaged DNA 761958 2.7.7.7 P556A mutation in alpha-subunit, mutant enzyme has nearly normal activity 662379 2.7.7.7 P556A/K557A/L558A mutation in alpha-subunit completely reduces DNA polymerase activity 662379 2.7.7.7 P680G reduced kcat, no change in relative DNA binding affinity or Km, nearly complete loss in the processive mode of DNA synthesis 643674 2.7.7.7 P680Q reduced kcat, no change in relative DNA binding affinity or Km, nearly complete loss in the processive mode of DNA synthesis 643674 2.7.7.7 Q342A the Mg2+-dependent DNA polymerase activity of the mutant is almost the same as that of wild type enzyme -, 705967 2.7.7.7 Q384A site-directed mutagenesis, the mutant exhibits DNA-dependent and RNA-dependent DNA polymerase activities, while the wild-type enzyme is a DNA-depedent DNA polymerase -, 722837 2.7.7.7 Q384A/Y438A site-directed mutagenesis, the mutant exhibits DNA-dependent and RNA-dependent DNA polymerase activities, while the wild-type enzyme is a DNA-depedent DNA polymerase 722837 2.7.7.7 Q507E increased exonuclease activity 643660 2.7.7.7 Q667A polymerase defective, no change in exonuclease activity 643674 2.7.7.7 R113A mutation reduces viral yield (2fold), alters the kinetics of viral DNA replication, and decreases the fidelity of DNA replication 675709 2.7.7.7 R182A mutation reduces viral yield (2fold), alters the kinetics of viral DNA replication, and decreases the fidelity of DNA replication 675709 2.7.7.7 R219I less active than wild-type enzyme 760878 2.7.7.7 R219I slight decrease in activity 761958 2.7.7.7 R246X 5-10fold less active with 8-oxo-dG-, N2-CH2-Anth-dG-, O6-Me-dG- and abasic-containing templates 761958 2.7.7.7 R255D exonuclease activity of mutant increases 2.9fold compared to wild-type activity 722215 2.7.7.7 R258A | mutant of Pol beta has a facilitated subdomain-reopening step. Rate of single-nucleotide incorporation catalyzed by R258A is identical to that of wild-type 672254 2.7.7.7 R279A mutation reduces viral yield (5fold), alters the kinetics of viral DNA replication, and decreases the fidelity of DNA replication 675709 2.7.7.7 R280A mutation reduces viral yield (5fold), alters the kinetics of viral DNA replication, and decreases the fidelity of DNA replication 675709 2.7.7.7 R298H less active than wild-type enzyme, markedly reduced thermal stability 760878 2.7.7.7 R298H less active than wild-type on several different lesions 761958 2.7.7.7 R322H the His332 mutant exhibits faster forward rate constants relative to wild-type Dpo4. The kpol values for the His332 mutant incorporation opposite G and 8-oxoG are 3.6- and 4.6fold faster than for wild-type Dpo4. The nucleotide binding affinity trend is opposite that of wild-type Dpo4, Glu332, and Leu332, with tighter dCTP binding during bypass of G. As in the case of Ala332, the kinetic analysis indicates that His332 inserts dCTP opposite G with slightly greater efficiency than opposite 8-oxoG -, 719841 2.7.7.7 R322L kpol (forward rate of polymerization) values for the Leu332 mutant incorporation opposite G and 8-oxoG are 4.1- and 1.9fold higher than those for wild-type Dpo4. The Leu332 mutant is about 2fold more efficient at incorporation of dCTP opposite 8-oxoG compared with G -, 719841 2.7.7.7 R331A/R322A mutant has lower forward rate constants relative to wild-type enzyme for both G and 8-oxoG. The double mutant-catalyzed insertion of dCTP opposite 8-oxoG is about 4fold higher than dCTP insertion opposite G. The measured binding affinity of dCTP is tighter than that of wild-type Dpo4 for unmodified DNA, but the binding affinity of dCTP opposite 8-oxoG is similar to that observed for wild-type enzyme. The catalytic efficiency for dCTP incorporation increases about 4fold for unmodified DNA and decreases about 2fold for 8-oxoG-modified DNA. The mutant fails to incorporate dATP opposite 8-oxoG in the presteady-state experiments. It inserts dCTP opposite 8-oxoG with an about 200fold greater efficiency than it does dATP and the steady-state efficiency of dATP incorporation is decreased about 27fold relative to the wild-type enzyme -, 719841 2.7.7.7 R332A mutant enzyme displays a higher affinity (lower KD,dCTP) for dCTP when bound to the unmodified DNA compared with the KD,dCTP measured for mutant-catalyzed incorporation of dCTP opposite 8-oxoG. Wild-type enzyme shows a greater affinity for dCTP opposite to 8-oxoG-modified DNA. The catalytic efficiency of the mutant is 23fold higher at incorporation of C opposite G and 1.2fold lower than wild-type enzyme in dCTP incorporation opposite 8-oxoG -, 719841 2.7.7.7 R332E mutant enzyme retains fidelity against bypass of 7,8-dihydro-8-oxodeoxyguanosine (8-oxoG) that is similar to wild enzyme. A crystal structure of the mutant and 8-oxoG:C pair reveals water-mediated hydrogen bonds between Glu332 and the O-8 atom of 8-oxoG. The kpol (forward rate of polymerization) value for dCTP incorporation opposite G is 4.8fold higher than for wild-type enzyme. The kpol (forward rates of polymerization) value for dCTP incorporation opposite 8-oxoG is 3.5fold higher than wild-type enzyme insertion of dCTP opposite 8-oxoG. The catalytic efficiency of the Glu332 mutant is 2.3fold greater than wild-type Dpo4 for dCTP incorporation opposite G but 1.7fold less efficient than wild-type Dpo4 for incorporation opposite 8-oxoG -, 719841 2.7.7.7 R512W decreased activity on undamaged and damaged DNA 761958 2.7.7.7 R61A the mutations decreases the ability of the enzyme to distinguish between a thymine-thymine dimer lesion and undamaged DNA. Mutation does not impact the global structure and equilibrium motions of DNA polymerase eta. Only local conformational changes around the mutation sites are observed 761729 2.7.7.7 R61K the mutations decreases the ability of the enzyme to distinguish between a thymine-thymine dimer lesion and undamaged DNA. Mutation does not impact the global structure and equilibrium motions of DNA polymerase eta. Only local conformational changes around the mutation sites are observed 761729 2.7.7.7 R821A the mutation shows 19% increased polymerase activity compared to the wild type enzyme 693076 2.7.7.7 R822A the mutation shows 5% increased polymerase activity compared to the wild type enzyme 693076 2.7.7.7 R822A/Y824A the mutation shows 36% increased polymerase activity compared to the wild type enzyme 693076 2.7.7.7 R852C naturally occuring mutation, involved in Alpers syndrome, the mutant shows compromised DNA binding ability that affects its overall functional efficiency 723108 2.7.7.7 R853Q naturally occuring mutation, involved in Alpers syndrome, the mutant shows compromised DNA binding ability that affects its overall functional efficiency 723108 2.7.7.7 R943H naturally occuring mutation, involved in autosomal dominant progressive external ophthalmoplegia, the mutation is associated with motiif B in the active site, the mutant retains less than 1% of the wild-type polymerase activity and displays a severe decrease in processivity 723108 2.7.7.7 R964C mutation identified in a patient with lactic acidosis. Recombinant R964C Pol gamma shows only 14% activity of wild-type enzyme. The mutation could be associated with the severe lactic acidosis induced by long-term use of nucleoside reverse-transcriptase inhibitors 675228 2.7.7.7 R964C naturally occuring mutation 723108 2.7.7.7 R964C the mutation demonstrates a 33% decrease in dTTP incorporation efficiency and a 3fold lower d4TTP discrimination relative to that of the wild type enzyme 701733 2.7.7.7 S184A 46% loss of DNA polymerase activity 726027 2.7.7.7 S423R 1.6fold more effcient than wild-type, 2fold increased DNA binding affinity, pathogenic 761958 2.7.7.7 S62A the mutations decreases the ability of the enzyme to distinguish between a thymine-thymine dimer lesion and undamaged DNA. Mutation does not impact the global structure and equilibrium motions of DNA polymerase eta. Only local conformational changes around the mutation sites are observed 761729 2.7.7.7 S62L the mutations decreases the ability of the enzyme to distinguish between a thymine-thymine dimer lesion and undamaged DNA. Mutation does not impact the global structure and equilibrium motions of DNA polymerase eta. Only local conformational changes around the mutation sites are observed 761729 2.7.7.7 S719A/Y720A/W721 mutation in alpha-subunit completely reduces DNA polymerase activity 662379 2.7.7.7 T134A 23% loss of DNA polymerase activity 726027 2.7.7.7 T139A 83% loss of DNA polymerase activity 726027 2.7.7.7 T239W kcat/Km for dCTP is 3.6fold compared to kcat/Km of wild-type enzyme -, 725385 2.7.7.7 T239W the frameshift deletion is selective for purines and involves normal conformational change followed by slow phosphodiester bond formation -, 719853 2.7.7.7 T239W the mutant enzyme is used to analyze conformational changes associated with the addition of dCTP opposite N2-alkylG adducts -, 720233 2.7.7.7 T326A site-directed mutagenesis, the mutant exhibits DNA-dependent and RNA-dependent DNA polymerase activities, while the wild-type enzyme is a DNA-depedent DNA polymerase -, 722837 2.7.7.7 T44M lesion-specific reduction in activity. Reduced activity with N2-CH2-Anth-dG, O6-Me-dG and abasic sites 761958 2.7.7.7 T471A less accurate, by factors of 1.6 than the wild-type variant Pfu-Pol(exo-) 662975 2.7.7.7 T471G less accurate, by factors of 1.2 than the wild-type variant Pfu-Pol(exo-) 662975 2.7.7.7 T473A decreased activity on undamaged and damaged DNA 761958 2.7.7.7 T851A naturally occuring mutation, involved in Alpers syndrome, the mutant shows compromised DNA binding ability that affects its overall functional efficiency 723108 2.7.7.7 V130I mutant has relaxed discrimination against the major groove adduct N6-furfuryl-dA 761958 2.7.7.7 W576A mutation in alpha-subunit, mutant enzyme is nearly inactive 662379 2.7.7.7 W748S naturally occuring mutation that has intrinsic lower polymerase activity as well as a demonstrated lower affinity for DNA compared to the wild-type enzyme 723108 2.7.7.7 W748S/E1143G naturally occuring mutation, the E1143G single-nucleotide polymorphism can modulate the deleterious effect of the W748S mutation 723108 2.7.7.7 Y12A active-site mutation Y12A in Dpo4, causes both a dramatic loss of ribonucleotide discrimination and a decrease in nucleotide incorporation efficiency -, 725713 2.7.7.7 Y12A mutation causes an average increase of 220fold in matched ribonucleotide incorporation efficiency and an average decrease of 9fold in correct deoxyribonucleotide incorporation efficiency, leading to an average reduction of 2000fold in sugar selectivity. The mutant incorporates more than 20 consecutive ribonucleotides into primer/template (DNA/DNA) duplexes, suggesting that this mutant protein possesses both aDNA-dependent DNA polymerase activity and a DNA-dependent RNA polymerase activity -, 718858 2.7.7.7 Y147A 19% loss of DNA polymerase activity 726027 2.7.7.7 Y178A 40% loss of DNA polymerase activity 726027 2.7.7.7 Y271G the mutant is rationally designed to provide flexibility to the steric gate backbone carboxyl of Tyr-271 in pol beta 761516 2.7.7.7 Y311F the mutant enzyme shows very weak 3'-5'-exonuclease activity compared to the wild-type enzyme (0.01%). No significant difference in DNA polymerase activity as compared with that of the wild-type enzyme 725590 2.7.7.7 Y410I kcat/Km for dATP is about 20% compared to the wild-type enzyme 721612 2.7.7.7 Y410L kcat/Km for dATP is about 45% compared to the wild-type enzyme 721612 2.7.7.7 Y410V Y410V mutation results in high fidelity in both misincorporation assays and forward mutation assays, but displays a substantially higher Km than wild-type enzyme, kcat/Km for dATP is about 10% compared to the wild-type enzyme, D215A mutation results in inactivation of 3'-5'-exonuclease activity 721612 2.7.7.7 Y419A/E420A/D421A mutation in alpha-subunit resulted in DNA polymerase activities that is 60–80% of wild-type enzyme 662379 2.7.7.7 Y432S less active on undamaged and damaged DNA, extension defect, decreased DNA binding affinity 761958 2.7.7.7 Y432S less active than wild-type enzyme, markedly reduced thermal stability 760878 2.7.7.7 Y438A site-directed mutagenesis, the mutant exhibits DNA-dependent and RNA-dependent DNA polymerase activities, while the wild-type enzyme is a DNA-depedent DNA polymerase -, 722837 2.7.7.7 Y46A 2% loss of DNA polymerase activity 726027 2.7.7.7 Y505A N-[6-N-(2,4-dinitrophenyl)aminohexanoyl]-2-aminoethyl triphosphate, N-(2,4-dinitro-5-fluorophenyl)-2-aminoethyl triphosphate, N-(2,4-dinitro-5-imidazolylphenyl)-4-aminobutyl triphosphate and N-(2,4-dinitro-5-imidazolylphenyl)-2-aminoethyl triphosphate inhibit mutant enzyme Y505A and are inactive against wild-type enzyme. DNA polymerase lambda and its mutant Y505A show different abilities of incorporating NNTPs in the presence of an abasic site on the template strand 676226 2.7.7.7 Y505A slightly less active in de novo DNA synthesis when compared with wild-type enzyme 662621 2.7.7.7 Y567A the mutant has pre-steadystate Kd,app and kpol values for the incorporation of correct dNMPs that are comparable to or exhibits kpol values that are somewhat greater than wild type DNA polymerase 702328 2.7.7.7 Y708A mutation of pol delta, exhibits slow growth, sensitivity to hydroxyurea and strong mutator phenotype for frameshifts and base substitutions 643664 2.7.7.7 Y824A the mutation shows 25% increased polymerase activity compared to the wild type enzyme 693076 2.7.7.7 Y831A mutation of pol epsilon, slight sensitivity to hydroxyurea, semidominant mutator phenotype for frameshifts and base substitutions 643664 2.7.7.7 Y869A mutation of pol alpha, strain is viable, exhibits slow growth, sensitivity to hydroxyurea and spontaneous mutator phenotype for frameshifts and base substitutions 643664 2.7.7.7 Y955C naturally occuring mutation, involved in autosomal dominant progressive external ophthalmoplegia, the mutation is associated with motiif B in the active site, the mutant retains less than 1% of the wild-type polymerase activity and displays a severe decrease in processivity, the mutation increases nucleotide misinsertion replication errors 10-100 fold in the absence of exonucleolytic proofreading 723108