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)

We have purified to homogeneity the primer recognition proteins (PRP) from human HeLa cells. PRP is associated with DNA polymerase alpha complex in HeLa cells. Purified PRP is free of DNA polymerases alpha, beta, and delta, deoxyribonuclease, DNA primase, ATPase, topoisomerase, and DNA ligase activities. The protein structure of the PRP was defined by sodium dodecyl sulfate gel electrophoresis, which revealed two polypeptides of 36,000 Da (PRP 1) and 41,000 Da (PRP 2). The two polypeptides are associated in a complex in the native state. The Stokes radius of the PRP complex by gel filtration is 40.5 A and the sedimentation coefficient in glycerol gradients is 5.7 S. Purified PRP, which exhibits no DNA polymerase activity, completely restores the activity of DNA polymerase alpha on templates with low primer to template ratios such as heat-denaturated DNA, poly(dA)-oligo(dT), and singly primed M13 single-stranded DNA. Experiments using various amounts of PRP, DNA polymerase alpha, and DNA indicate that a concentration dependence exists between these components in the DNA replication process. Amino acid composition analysis indicates that the PRP is rich in hydrophobic amino acids.
...
PMID:Purification and characterization of primer recognition proteins from HeLa cells. 236 57

We report a simple, two-step method (phosphocellulose and immunoaffinity column chromatographies) for purification of the mouse DNA polymerase alpha-primase complex. The advantages of this method over other procedures are its simplicity and rapidity, with little loss by proteolysis. Sedimentation analysis in a glycerol density gradient of the immunoaffinity-purified fraction revealed that four polypeptides with molecular weights of 180,000, 68,000, 54,000 and 46,000 in the enzyme fraction form a physical complex. Peptide mapping by reversed phase-high performance liquid chromatography demonstrated unequivocally that these four polypeptides constituting the complex are different entities.
...
PMID:Peptide mapping of the four subunits of the mouse DNA polymerase alpha-primase complex. 238 57

We have purified yeast DNA polymerase II to near homogeneity as a 145-kDa polypeptide. During the course of this purification we have detected and purified a novel form of DNA polymerase II that we designate as DNA polymerase II. The most highly purified preparations of DNA polymerase II are composed of polypeptides with molecular masses of 200, 80, 34, 30, and 29 kDa. Immunological analysis and peptide mapping of DNA polymerase II and the 200-kDa subunit of DNA polymerase II indicate that the 145-kDa DNA polymerase II polypeptide is derived from the 200-kDa polypeptide of DNA polymerase II. Activity gel analysis shows that the 145- and the 200-kDa polypeptides have catalytic function. The polypeptides present in the DNA polymerase II preparation copurify with the polymerase activity with a constant relative stoichiometry during chromatography over five columns and co-sediment with the activity during glycerol gradient centrifugation, suggesting that this complex may be a holoenzyme form of DNA polymerase II. Both forms of DNA polymerase II possess a 3'-5' exonuclease activity that remains tightly associated with the polymerase activity during purification. DNA polymerase II is similar to the proliferating cell nuclear antigen (PCNA)-independent form of mammalian DNA polymerase delta in its resistance to butylpheny-dGTP, template specificity, stimulation of polymerase and exonuclease activity by KCl, and high processivity. Although calf thymus PCNA does not stimulate the activity of DNA polymerase II on poly(dA):oligo(dT), possibly due to the limited length of the template, the high processivity of yeast DNA polymerase II on this template can be further increased by the addition of PCNA, suggesting that conditions may exist for interactions between PCNA and yeast DNA polymerase II.
...
PMID:Purification and characterization of DNA polymerase II from the yeast Saccharomyces cerevisiae. Identification of the catalytic core and a possible holoenzyme form of the enzyme. 240 68

It has been shown that DNA primase activity is tightly associated with 10S DNA polymerase alpha from calf thymus (Yoshida, S. et al. (1983) Biochim. Biophys. Acta 741, 348-357). In the present study, the primase activity was separated from DNA polymerase alpha by treating purified 10S DNA polymerase alpha with 3.4 M urea followed by a fast column chromatography (Pharmacia FPLC, Mono Q column equilibrated with 2 M urea). Ten to 20 % of the primase activity was separated from 10S DNA polymerase alpha by this procedure but 80-90% remained in the complex. The separated primase activity sedimented at 5.6S through a gradient of glycerol. The separated primase was strongly inhibited by araATP (Ki = 10 microM) and was also sensitive to salts such as KCl (50% inhibition at 30 mM). The primase used poly(dT) or poly(dC) as templates efficiently, but showed little activity with poly(dA) or poly(dI). These properties agree well with those of the primase activity in the DNA polymerase alpha-primase complex (10S DNA polymerase alpha). These results indicate that the calf thymus primase may be a part of the 10S DNA polymerase alpha and its enzymological characters are preserved after separation from the complex.
...
PMID:Characterization of DNA primase separated from DNA polymerase alpha-DNA primase complex of calf thymus. 242 5

The reverse transcriptase polymerase of the human T-cell lymphotropic virus/lymphadenopathy-associated virus has been cloned into an expression vector and expressed in Escherichia coli. Two polypeptides of 66 and 51 kDa molecular mass are detectable in polymerase-expressing bacterial lysates with human patient sera. They are processed from a short-lived 120-kDa polyprotein precursor equivalent to a region consisting of polymerase, protease, and endonuclease. The 51 kDa protein appears to originate from the 66-kDa molecule; additional processing products are 32- and 15-kDa proteins. The bacterially expressed polymerase is enzymatically active and exhibits the template specificities, ion requirements, and response to inhibitors of the authentic enzyme. It was purified by DEAE-cellulose-, phosphocellulose-, and poly(rC)-agarose column chromatography followed by glycerol density gradient centrifugation. It copurifies with an RNase H activity, suggesting the existence of a virus-coded DNA polymerase-RNase H complex. The purified bacterial enzyme allows a safe large-scale screening for inhibitors of both activities.
...
PMID:RNase H activity associated with bacterially expressed reverse transcriptase of human T-cell lymphotropic virus III/lymphadenopathy-associated virus. 244 62

The protective effect of Mr 70,000 heat shock protein (HSP-70) during thermotolerance has been previously observed. However, it is not known what cellular processes or components may be protected by this protein during the tolerance state. In the studies reported here, the protective effects of purified HSP-70, the nonspecific heat-stable proteins fetuin and trypsin inhibitor (ovomucoid), and other proteins and agents such as bovine serum albumin, D2O, or glycerol on protein and DNA synthesis during heating were investigated in vitro. In vitro protein synthesis at 30, 40, and 42 degrees C was measured by globin mRNA translation. Protein synthesis was inhibited 40 to 70% when incubated for 60 min at 40 and 42 degrees C. However, protein synthesis was protected when either fetuin or ovomucoid was present during protein synthesis at elevated temperatures. The protection was concentration dependent. The HSP-70 purified from Chinese hamster (HA-1) cells was also able to confer protection to the translation system, but at much lower concentrations than either fetuin or ovomucoid. Other proteins, such as bovine serum albumin, or other agents, such as D2O or glycerol which are known protectors of cellular survival during heating, did not protect the translation system. Similar experiments were performed with DNA synthesis in vitro. Purified DNA polymerase alpha was added to the activated calf thymus DNA in an in vitro replication system. A temperature of 46 degrees C for 60 min inhibited replication by 40%. Addition of heat-stable proteins, purified HSP-70, bovine serum albumin, D2O, or glycerol did not confer protection to the replication system. These studies provide new evidence that HSP-70 may confer protection to a component of the protein synthesis machinery during thermotolerance.
...
PMID:Effects of heat shock proteins (Mr 70,000) on protein and DNA synthesis at elevated temperatures in vitro. 246 56

Human immunodeficiency virus (HIV) reverse transcriptase has been purified from yeast transformed by an autoreplicating plasmid containing the retroviral DNA polymerase gene. The previously described purification procedure for the yeast-expressed reverse transcriptase [Barr, P.J., Power, M.D., Chun Ting Lee-Ng, Gibson, H. & Luciw, P. (1987) Bio/Technology 5, 486-489] has been substantially modified, leading to an increased yield and a higher degree of purity. Several biochemical properties of the enzyme are described (template specificity, effect of DNA synthesis inhibitors); interestingly, HIV reverse transcriptase is highly resistant to N-ethylmaleimide. A complex between the human retroviral enzyme and the bovine tRNALys was shown, using a direct approach, by glycerol gradient centrifugation, as well as by the protective and specific effect of the tRNALys against enzyme inactivation by thermal denaturation and trypsin digestion. A competitive type of inhibition of HIV reverse transcriptase by tRNALys, but not by tRNAVal, is observed when viral RNA or activated DNA are used as templates.
...
PMID:Human immunodeficiency virus reverse transcriptase expressed in transformed yeast cells. Biochemical properties and interactions with bovine tRNALys. 247 48

The introduction of a replication-inhibiting lesion into the DNA of Escherichia coli produces a marked elevation in mutation rate. The mutation pathway is a component of the induced, multigene SOS response. SOS mutagenesis is a tightly regulated process dependent on two RecA-mediated proteolytic events: cleavage of the LexA repressor to induce the UmuC and UmuD mutagenesis proteins, and cleavage of UmuD to UmuD' to activate the mutation pathway. To investigate the protein-protein interactions responsible for SOS mutagenesis, we have studied the interaction of UmuC, UmuD, and UmuD'. To probe intracellular interaction, we have used immunoprecipitation techniques with antibodies against UmuC or UmuD and UmuD'. We have found that antibody to UmuC precipitates UmuD' from cell extracts, and antibody to UmuD and UmuD' precipitates UmuC. Thus we conclude that UmuC probably associates tightly with UmuD' in cells. For biochemical studies, we have purified the UmuC and UmuD' proteins to use with the previously purified UmuD. UmuC associates strongly with an affinity column of UmuD and UmuD', eluting only under strongly dissociating conditions (2 M urea or 1.5 M KSCN). UmuC also associates efficiently with UmuD or UmuD' in solution, as judged by velocity sedimentation in a glycerol gradient. The likely stoichiometry is one UmuC with a dimeric UmuD or UmuD'. From these experiments and previous work, we infer that SOS mutagenesis depends on the action of the UmuC-UmuD' complex and probably RecA to rescue a stalled DNA polymerase III holoenzyme at the DNA lesion.
...
PMID:UmuC mutagenesis protein of Escherichia coli: purification and interaction with UmuD and UmuD'. 255 36

DNA polymerase delta from calf thymus was purified under conditions that minimized proteolysis to a specific activity of 27,000 units/mg. The four step isolation procedure included phosphocellulose, hydroxyapatite, heparin-Sepharose and FPLC-MonoS. This enzyme consists of four polypeptides with Mr of 140, 125, 48 and 40 kilodaltons. Velocity gradient sedimentation in glycerol removed the 48 kDa polypeptide while the other three sedimented with the DNA polymerase activity. The biochemical properties of the three subunit enzyme and the copurification of 3'----5' exonuclease activity were typical for a bona fide DNA polymerase delta. Tryptic peptide analysis showed that the 140 kDa polypeptide was different from the catalytic 180 kDa polypeptide of calf thymus DNA polymerase alpha. Both high Mr polypeptides (140 and 125 kDa) were catalytically active as analysed in an activity gel. Four templates were used by DNA polymerase delta with different preferences, namely poly(dA)/oligo(dT)12-18 much much greater than activated DNA greater than poly(dA-dT) greater than primed single-stranded M13DNA. Calf thymus proliferating cell nuclear antigen (PCNA) could not stimulated this DNA polymerase delta in any step of the isolation procedure. If tested on poly(dA)/oligo(dT)12-18 (base ratio 10:1), PCNA had no stimulatory effect on DNA polymerase delta when tested with low enzyme DNA ratio nor did it change the kinetic behaviour of the enzyme. DNA polymerase delta itself did not contain PCNA. The enzyme had an intrinsic processivity of several thousand bases, when tested either on the homopolymer poly(dA)/oligo(dT)12-18 (base ratio 64:1) or on primed single-stranded M13DNA. Contrary to DNA polymerase alpha, no pausing sites were seen with DNA polymerase delta. Under optimal in vitro replication conditions the enzyme could convert primed single-stranded circular M13 DNA of 7,200 bases to its double-stranded form in less than 10 min. This supports that a PCNA independent DNA polymerase delta exists in calf thymus in addition to a PCNA dependent enzyme (Lee, M.Y.W.T. et al. (1984) Biochemistry 23, 1906-1913).
...
PMID:Calf thymus DNA polymerase delta independent of proliferating cell nuclear antigen (PCNA). 256 61

A DNA polymerase alpha-primase complex, which had been purified by means of immunoaffinity column chromatography, showed little activity in a reaction mixture composed of Tris-HCl buffer, but showed full activity in potassium phosphate buffer. It was found that potassium ion is required for the reaction by the immunoaffinity-purified enzyme. On the other hand, the DNA polymerase alpha purified by the orthodox biochemical method showed full activity in both buffer systems. A protein factor, which could restore the activity of immunoaffinity-purified DNA polymerase alpha-primase complex in the potassium-free reaction mixture, was separated from biochemically purified DNA polymerase alpha. The factor, designated as factor T, was stable to heat up to 70 degrees C, but was sensitive to trypsin. It sedimented at about 4S through a glycerol gradient. SDS-polyacrylamide gel electrophoresis revealed two polypeptide bands at 56 and 54 kDa. By immunoprecipitation, the factor T was shown to be physically associated with DNA polymerase alpha-primase complex. The stimulation was also observed with poly[d(A-T)], primed M13 DNA, and heat-denatured DNA.
...
PMID:A novel stimulating protein of mammalian DNA polymerase alpha. 260 93


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

---