Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A lysine residue, contained in the motif "Kx2h", has been invariantly found in the eukaryotic-type (family B) class of DNA-dependent DNA polymerases with a proofreading function. The importance of this lysine has been assessed by site-directed mutagenesis in the corresponding residue (Lys143) of phi29 DNA polymerase. Substitution of this residue either by arginine or isoleucine severely impaired the catalytic efficiency of the 3'-5' exonuclease activity, giving a characteristic distributive pattern that contrasts with the processive pattern displayed by the wild-type phi29 DNA polymerase. Exonuclease assays carried out in the presence of a DNA trap, together with direct analysis of enzyme/ssDNA interaction, allowed us to conclude that this altered pattern was due to a reduction in the catalytic rate of these mutants, but not to a weakened association with ssDNA. These phenotypes indicate that the lysine residue of motif Kx2h plays an auxiliary role in catalysis of the exonuclease reaction, in very good agreement with recent crystallographic data showing that the lysine homologue of T4 DNA polymerase is indirectly involved in metal binding at the 3'-5' exonuclease active site. In agreement with a critical role in proofreading, substitution of Lys143 of phi29 DNA polymerase by arginine or isoleucine produced mutator enzymes that displayed a high frequency of misincorporation. Mutants at Lys143 also showed a reduced DNA polymerization capacity, but only when DNA synthesis was coupled to strand-displacement, an intrinsic property of phi29 DNA polymerase that is specifically affected by mutations at residues directly or indirectly involved in metal binding at the 3'-5' exonuclease active site.
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PMID:An invariant lysine residue is involved in catalysis at the 3'-5' exonuclease active site of eukaryotic-type DNA polymerases. 923 1

The molecular basis for the DNA repair dysfunction observed in mutant Chinese hamster ovary cell lines of X-ray repair cross complementing group 1 (XRCC1) is unknown and the exact role of the XRCC1 protein remains unclear. To help clarify the role of the XRCC1 gene we analyzed four mutant cell lines of this complementation group and a revertant cell line for XRCC1 protein content and for sequence alterations in the XRCC1 coding region. Immunoblot analysis of cellular extracts indicated that each of four mutant lines was lacking XRCC1 protein, whereas the repair-proficient revertant line derived from one of these mutants contained a normal level of XRCC1. Although each of these cell lines expressed XRCC1 mRNA, we found in all cases a distinct point mutation resulting in crucial alterations in the encoded XRCC1 protein sequence of 633 amino acids. Two of the mutations cause non-conservative amino acid changes, Glu102-->Lys and Cys390-->Tyr, at positions that are invariant among hamster, mouse and human XRCC1 sequences and are located in putative functional domains. A third debilitating mutation disrupts RNA splicing, generating multiple transcripts of different length that contain deletions spanning a region of >100 amino acids in the midsection of the XRCC1 coding sequence. A fourth mutation results in a termination codon that shortens the open reading frame to 220 amino acids, however, in the revertant cell line a further mutation in the same codon, Stop221-->Leu, permits translation of a full-length functional variant protein. These mutational data indicate the importance of the putative functional regions in XRCC1, such as the BRCA1 C-terminal (BRCT) domain found in common with BRCA1 and other DNA repair and cell cycle checkpoint proteins, and also regions necessary for interaction with DNA polymerase beta and DNA ligase III.
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PMID:Mutations in hamster single-strand break repair gene XRCC1 causing defective DNA repair. 946 64

The amino-terminal 8-kDa domain of vertebrate DNA polymerase beta (pol beta) has an activity to excise deoxyribose phosphate (dRP) groups from 5'-incised apurinic/apyrimidinic (AP) sites during base excision repair. The excision reaction proceeds via a beta-elimination reaction following formation of a Schiff base between an aldehyde group of the AP site and an amino group of the enzyme. Here we report that the Lys-72 residue of this enzyme is the catalytic center for dRP excision. Substitutions of Lys-72 with Arg or Gln reduced the dRP excision activity to less than 1% of the wild-type 8-kDa domain, while substitutions of Lys-35, Lys-68, or Lys-84 did not abolish its activity. The Lys-72 mutations also significantly decreased Schiff base intermediates trapped by reduction with sodium borohydride. The 8-kDa domain alone was able to bind preferentially to a single-nucleotide gap or 5'-incised synthetic AP site on double-stranded DNA. The Lys-72 mutations did not affect this damage-specific DNA binding activity. When introduced into the intact enzyme, a mutation of Lys-72 to Arg did not affect DNA synthesis activity of pol beta, but eliminated the repair activity. Addition of the wild-type 8-kDa domain to this reaction restored the repair activity. These results indicate a specific role of Lys-72 of pol beta in the dRP excision during base excision repair.
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PMID:Catalytic center of DNA polymerase beta for excision of deoxyribose phosphate groups. 957 63

The Crithidia fasciculata KAP1 gene encodes a small basic protein (p21) associated with kinetoplast DNA. The p21 protein has a nine amino acid cleavable presequence closely related to those of several other proteins targeted to the kinetoplast and binds non-specifically to kinetoplast minicircle DNA. The p21 protein also has a calculated pI of 13 with two amino acids (lysine and alanine) accounting for more than 50% of the residues and with 25 out of 28 lysine residues contained in the C-terminal half of the protein. Immunolocalization of p21 shows that the protein is found exclusively in the kinetoplast with a localization distinctly different from the antipodal localization of kinetoplast DNA topoisomerase and DNA polymerase. The KAP11 gene is a single copy gene and the KAP1 mRNA is present at a constant level throughout the cell cycle. This highly basic protein may play a role in the condensation or segregation of the kinetoplast DNA.
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PMID:The Crithidia fasciculata KAP1 gene encodes a highly basic protein associated with kinetoplast DNA. 971 9

The genetic characterization of a foscarnet-resistant strain of varicella-zoster virus (VZV) that was isolated from a patient with AIDS is reported. Compared with the sequence of the Dumas reference strain, this strain, which was isolated from a patient in whom foscarnet treatment failed, had two point mutations. The emergence of one of the mutations, which includes a change from a glutamic acid to a lysine at position 512 in the DNA polymerase, suggests that this mutation is implicated in the VZV foscarnet resistance. The other mutation, which replaces serine 863 by a glycine, is also present in 2 susceptible strains--Oka and a wild-type isolate.
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PMID:Single amino acid change in DNA polymerase is associated with foscarnet resistance in a varicella-zoster virus strain recovered from a patient with AIDS. 985 75

We have isolated spontaneous rifampicin-resistant mutants from Escherichia coli that showed allele-specific suppression of the copy-number phenotype of ColE1 high-copy-number mutants in vivo. The key step in the regulatory circuitry of the initiation of ColE1 DNA replication is the formation of the persistent hybrid between the primer RNA and the DNA template around the replication origin. Three host-encoded enzymes, RNase H, DNA polymerase I, and RNA polymerase, are essential to the replication initiation in vitro. To decide whether the activity of RNA polymerase is involved directly in the formation of the persistent hybrid, we screened rifampicin-resistant colonies for suppressors of ColE1 copy-number mutants. Suppressor strain YY572 (rpoB572) changes the 572 residue of the beta subunit of RNA polymerase, encoded by the rpoB gene, from isoleucine to leucine. Another suppressor, YY513 (rpoB513), changes the 513 residue from glutamine to lysine. The other known rifampicin-resistant alleles located at residue 513, rpoB8 and rpoB101, did not show a significant suppression of the copy number of those ColE1 copy-number mutants as rpoB513. The suppression by rpoB513 on different ColE1 copy-number mutants showed allelic specificity. The possible roles of RNA polymerase in control of ColE1 copy number are discussed.
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PMID:Allele-specific suppression of ColE1 high-copy-number mutants by a rpoB mutation of Escherichia coli. 988 6

The Streptomyces coelicolor dnaE gene, encoding the catalytic alpha-subunit of DNA polymerase III (pol III) was isolated by genetic complementation of a temperature-sensitive DNA replication mutant, S. coelicolor ts-38. The deduced protein sequence (1179 residues) is highly similar to the Escherichia coli-type pol III alpha-subunit, rather than to the PolC-type alpha-subunit that is known to be essential for replication in the 'low G + C' Gram-positive bacteria such as Bacillus subtilis. The dnaE gene is able to restore replication to a 'slow stop' mutant (ts-38) and a 'fast stop' mutant (ts-114); the dnaE gene of ts-38 carries a single amino acid substitution (Glu-802 to Lys), and the mutation in ts-114 has been mapped between codons 697 and 1062 of dnaE. Mutant ts-38 is considered to be defective in assembly of the multisubunit pol III holoenzyme and, hence, in initiation of replication, whereas ts-114 is defective in chain elongation. This study provides the first evidence that a DnaE-type pol III is essential for replication in a Gram-positive bacterium. In addition, the complementation studies suggest that the C-terminal 117 residues are not essential for DnaE function in S. coelicolor. When integrated at a distant site on the chromosome, a fragment containing the 3' half of dnaE(codons 697-1179) is capable of rescuing ts-38 (but not ts-114) at the restrictive temperature; it was demonstrated that homogenotization was responsible for this phenomenon.
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PMID:A 'gram-negative-type' DNA polymerase III is essential for replication of the linear chromosome of Streptomyces coelicolor A3(2). 1004 37

Motifs for sequence specific-protein-DNA interactions, such as helix-turn-helix, zinc finger and leucine zipper, are now better understood as a result of extensive studies of three-dimensional (3D) structures of transcription factors. On the other hand, little attention has been paid to motifs for sequence nonspecific binding, namely DNA-phosphate binding. To address the question whether different transcription factors and DNA manipulation enzymes, that is enzymes that work on DNA, share a similar mode of phosphate binding, we surveyed interactions between DNA and protein module, a structural unit of a globular protein. We analyzed the modular organization of DNA polymerase beta and found that residues making contact with DNA phosphates were localized to five modules. Structural comparison of these phosphate-binding modules against others in transcription factors and DNA manipulation enzymes revealed that DNA polymerase beta, the Oct-1 POU domain, 434 Cro and the Arc repressor have a phosphate-binding module with 3D structures similar to one another. This newly detected module, the phosphate-binding helix-turn-helix (pbHTH) module, named for its function and 3D structure, interacts with DNA by (i) making hydrogen bonds between a DNA phosphodiester oxygen and an amino hydrogen of the main chain located at the N-terminus of a C-terminal alpha-helix, and (ii) making electrostatic interactions between DNA phosphates and side chains of lysine or arginine. Finding structurally and functionally similar phosphate-binding units in different transcription factors and DNA manipulation enzymes suggests that shuffling of modules is not limited to the DNA base-recognition motif. Phosphate-binding modules are apparently also shuffled in DNA-binding proteins.
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PMID:Repetitive use of a phosphate-binding module in DNA polymerase beta, Oct-1 POU domain and phage repressors. 1022 61

To examine the hypothesis that interactions between a DNA polymerase and the DNA minor groove are critical for accurate DNA synthesis, we studied the fidelity of DNA polymerase beta mutants at residue Arg(283), where arginine, which interacts with the minor groove at the active site, is replaced by alanine or lysine. Alanine substitution, removing minor groove interactions, strongly reduces polymerase selectivity for all single-base mispairs examined. In contrast, the lysine substitution, which retains significant interactions with the minor groove, has wild-type-like selectivity for T.dGMP and A.dGMP mispairs but reduced selectivity for T.dCMP and A.dCMP mispairs. Examination of DNA crystal structures of these four mispairs indicates that the two mispairs excluded by the lysine mutant have an atom (N2) in an unfavorable position in the minor groove, while the two mispairs permitted by the lysine mutant do not. These results suggest that unfavorable interactions between an active site amino acid side chain and mispair-specific atoms in the minor groove contribute to DNA polymerase specificity.
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PMID:Base substitution specificity of DNA polymerase beta depends on interactions in the DNA minor groove. 1040 11

The 3' --> 5' exonuclease activity of proofreading DNA polymerases requires two divalent metal ions, metal ions A and B. Mutational studies of the 3' --> 5' exonuclease active center of the bacteriophage T4 DNA polymerase indicate that residue Asp-324, which binds metal ion A, is the single most important residue for the hydrolysis reaction. In the absence of a nonenzymatic source of hydroxide ions, an alanine substitution for residue Asp-324 reduced exonuclease activity 10-100-fold more than alanine substitutions for the other metal-binding residues, Asp-112 and Asp-219. Thus, exonuclease activity is reduced 10(5)-fold for the D324A-DNA polymerase compared with the wild-type enzyme, while decreases of 10(3)- to 10(4)-fold are detected for the D219A- and D112A/E114A-DNA polymerases, respectively. Our results are consistent with the proposal that a water molecule, coordinated by metal ion A, forms a metal-hydroxide ion that is oriented to attack the phosphodiester bond at the site of cleavage. Residues Glu-114 and Lys-299 may assist the reaction by lowering the pK(a) of the metal ion-A coordinated water molecule, whereas residue Tyr-320 may help to reorient the DNA from the binding conformation to the catalytically active conformation.
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PMID:Mutational and pH studies of the 3' --> 5' exonuclease activity of bacteriophage T4 DNA polymerase. 1045 97


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