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

---

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

In the absence of a growth factor or an appropriate extracellular matrix (ECM), cells are arrested in the G0/G1 phase. In this report, we demonstrate the evidence that TNF-alpha induced DNA synthesis of primary mouse hepatocytes in vitro by activating two distinct pathways. TNF-alpha induced drastic spreading of hepatocytes on hydrophobic plastic, while the adhesion was not influenced. The effect was time and dose dependent. The cell spreading was accompanied by the phosphorylation of paxillin, indicating the stimulation of focal adhesion molecules. TNF-alpha-induced spreading of hepatocytes was not transient, and kinetic analysis and morphologic observation suggest that the effect was different from epidermal growth factor- or hepatocyte growth factor-induced transient hepatocyte spreading. TNF-alpha-induced hepatocyte spreading was blocked by cytochalasin D, Arg-Gly-Asp peptides, cycloheximide, or anti-integrin beta1 Ab. Results of competitive PCR for ECM proteins demonstrated that TNF-alpha increased the expression of laminin alpha3 and gamma1 chains in hepatocytes. These data suggested that TNF-alpha induced cell anchorage for hepatocytes by up-regulating ECM production. More importantly, TNF-alpha, but neither epidermal growth factor nor hepatocyte growth factor, induced DNA synthesis following the spreading in primary hepatocytes on hydrophobic plastic, while mere cell spreading on collagen did not induce DNA synthesis. The DNA synthesis was blocked by the inhibition of either cell spreading or DNA polymerase, demonstrating that TNF-alpha induced DNA synthesis in primary hepatocytes by activating two distinct pathways, i.e., forming the scaffold and inducing growth signals. Taken together, TNF-alpha bifunctionally regulates the proliferation of primary hepatocytes, serving as both an ECM inducer and a growth factor.
...
PMID:TNF-alpha bifunctionally induces proliferation in primary hepatocytes: role of cell anchorage and spreading. 936 9

The DNA sequence of 9991 nt, corresponding to 18-51 map units of mouse adenovirus type 1 (MAV-1), was determined, completing the sequence of the Larsen strain of MAV-1. The length of the complete MAV-1 genome is 30,946 nucleotides, consistent with previous experimental estimates. The 18-51 map unit region encodes early region 2B proteins necessary for adenoviral replication as well as late region L1 and L2 structural and packaging proteins. Sequence comparison in this region with human adenoviruses indicates broad similarities, including colinear preservation of all recognized open reading frames (ORFs), with highest amino acid identity occurring in the DNA polymerase and polypeptide III (penton base subunit) ORFs. Virus-associated (VA) RNA is not encoded in the region where VA RNAs are found in the human adenoviruses, between E2B and L1, nor is it encoded anywhere in the entire MAV-1 genome. The MAV-1 polypeptide III lacks the arginine-glycine-aspartic acid (RGD) motif which is involved in an association with cell-surface integrins. Only one RGD sequence is found in an identified coding region in the entire MAV-1 genome. Similar to the porcine adenovirus, this RGD sequence is found in the C-terminus of the MAV-1 fiber protein.
...
PMID:Completion of the DNA sequence of mouse adenovirus type 1: sequence of E2B, L1, and L2 (18-51 map units). 938 95

The "runt domain" (RD) is a 128 amino acid region of the Drosophila pair-rule gene runt. This highly conserved region delineates the DNA-binding domain of a new family of transcription factors; the RD proteins. The family includes genes from Drosophila, chicken and mammals that are involved in a wide range of developmental processes, from sex determination and neurogenesis in Drosophila to hematopoiesis and osteoblast differentiation in mouse and human. The RD confers DNA binding ability and mediates the interaction of mammalian RD proteins with the beta-subunit (CBFbeta), which enhances the DNA binding. The primary sequence of RD shows no similarity to other known DNA-binding motifs and its three-dimensional (3D) structure is not known. We employed molecular modeling-based mutagenesis to generate a 3D model of RD. Fold recognition programs identified the palm subdomain of rat DNA polymerase beta as the most likely fold for RD. In the predicted model, the RD region which interacts with DNA contains two arginine residues, R130 and R135, which appear to be in close contact with the major groove of the DNA and to interact with the three essential guanine bases of the core DNA motif PyGPyGGT. We mutated these two R residues and demonstrated that mutations markedly reduced the binding of RD to DNA with no effect on RD interaction with CBFbeta. The data provide important clues about the possible 3D structure of the RD and its interaction with the core DNA motif.
...
PMID:Site-directed mutagenesis supports a three-dimensional model of the runt domain. 953 75

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.
...
PMID:Catalytic center of DNA polymerase beta for excision of deoxyribose phosphate groups. 957 63

The RGD (Arg-Gly-Asp) motif functions as a recognition site for adhesive proteins responsible for a number of cell-cell interactions. Certain viruses use this sequence as a receptor-binding site by interaction with cellular integrins. To elucidate the role of the RGD sequence of the phi29 terminal protein (TP), seven modified TPs were generated by site-directed mutagenesis. Most of the TP mutants were not efficiently used as primers, leading to a reduction of the TP-dAMP complex formation in the presence of the phi29 TP-DNA template. Moreover, these mutant TPs were poorly deoxyadenylylated by phi29 DNA polymerase in the absence of template. Analysis of primer TP/DNA polymerase complex formation showed that the modified TPs were affected in the formation of the heterodimeric complex. These results indicate that the RGD sequence present in phi29 TP is primarily involved in interaction with the viral DNA polymerase.
...
PMID:The RGD sequence in phage phi29 terminal protein is required for interaction with phi29 DNA polymerase. 970 51

To investigate the interactions that determine DNA polymerase accuracy, we have measured the fidelity of 26 mutants with amino acid substitutions in the polymerase domain of a 3'-5'-exonuclease-deficient Klenow fragment. Most of these mutant polymerases synthesized DNA with an apparent fidelity similar to that of the wild-type control, suggesting that fidelity at the polymerase active site depends on highly specific enzyme-substrate interactions and is not easily perturbed. In addition to the previously studied Y766A mutator, four novel base substitution mutators were identified; they are R668A, R682A, E710A, and N845A. Each of these five mutator alleles results from substitution of a highly conserved amino acid side chain located on the exposed surface of the polymerase cleft near the polymerase active site. Analysis of base substitution errors at four template positions indicated that each of the five mutator polymerases has its own characteristic error specificity, suggesting that the Arg-668, Arg-682, Glu-710, Tyr-766, and Asn-845 side chains may contribute to polymerase fidelity in a variety of different ways. We separated the contributions of the nucleotide insertion and mismatch extension steps by using a novel fidelity assay that scores base substitution errors during synthesis to fill a single nucleotide gap (and hence does not require mismatch extension) and by measuring the rates of polymerase-catalyzed mismatch extension reactions. The R682A, E710A, Y766A, and N845A mutations cause decreased fidelity at the nucleotide insertion step, whereas R668A results in lower fidelity in both nucleotide insertion and mismatch extension. Relative to wild type, several Klenow fragment mutants showed substantially more discrimination against extension of a T.G mismatch under the conditions of the fidelity assay, providing one explanation for the anti-mutator phenotypes of mutants such as R754A and Q849A.
...
PMID:Side chains that influence fidelity at the polymerase active site of Escherichia coli DNA polymerase I (Klenow fragment). 991 46

We developed an in vivo selection to identify 3'-azido-3'-deoxythymidine (AZT)-resistant mutants of rat DNA polymerase beta (pol beta). The selection utilizes pol beta's ability to substitute for Escherichia coli DNA polymerase I (pol I) in the SC18-12 strain, which lacks active pol I. pol beta allows SC18-12 cells to grow, but they depend on pol beta activity, so inhibition of pol beta by AZT kills them. We screened a library of randomly mutated pol beta cDNA for complementation of the pol I defect in the presence of AZT, and identified AZT-resistant mutants. We purified two enzymes with nonconservative mutations in the palm domain of the polymerase. The substitutions D246V and R253M result in reductions in the steady-state catalytic efficiency (Kcat/Km) of AZT-TP incorporation. The efficiency of dTTP incorporation was unchanged for the D246V enzyme, indicating that the substantial decrease in AZT-TP incorporation is responsible for its drug resistance. The R253M enzyme exhibits significantly higher Km(dTTP) and Kcat(dTTP) values, implying that the incorporation reaction is altered. These are the first pol beta mutants demonstrated to exhibit AZT resistance in vitro. The locations of the Asp-246 and Arg-253 side chains indicate that substrate specificity is influenced by residues distant from the nucleotide-binding pocket.
...
PMID:3'-Azido-3'-deoxythymidine-resistant mutants of DNA polymerase beta identified by in vivo selection. 992 Sep 40

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.
...
PMID:Repetitive use of a phosphate-binding module in DNA polymerase beta, Oct-1 POU domain and phage repressors. 1022 61

Comparison of the amino acid sequences of eucaryotic DNA primase and the family X polymerases indicates that primase shares significant sequence homology with this family. With the use of DNA polymerase beta (pol beta) as a paradigm for family X polymerases, these homologies include both the catalytic core domain/subunit of each enzyme (31 kDa domain of pol beta and p49 subunit of primase) as well as the accessory domain/subunit (8 kDa domain of pol beta and p58 subunit of primase). To further explore these homologies as well as provide insights into the mechanism of primase, we generated three mutants (R304K, R304Q, and R304A) of the p49 subunit at an arginine that is highly conserved between primase and the eukaryotic family X polymerases. These mutations significantly decreased the rate of primer synthesis, due primarily to a decreased rate of initiation, and the extent of impairment correlated with the severity of the mutation (A > Q > K). R304 also contributes to efficient utilization of the NTP that will become the 5'-terminus of the new primer, and these effects are at least partially mediated through interactions with the phosphates of this NTP. The implications of these results with respect to the structure and biological role of primase, as well as its relationship to the family X polymerases, are discussed.
...
PMID:Arg304 of human DNA primase is a key contributor to catalysis and NTP binding: primase and the family X polymerases share significant sequence homology. 1038 12

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.
...
PMID:Base substitution specificity of DNA polymerase beta depends on interactions in the DNA minor groove. 1040 11


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---