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)

The coordinated action of many enzymatic activities is required at the DNA replication fork to ensure the error-free, efficient, and simultaneous synthesis of the leading and lagging strands of DNA. In order to define the essential protein-protein interactions and model the regulatory pathways that control Okazaki fragment synthesis, we have reconstituted the replication fork of Escherichia coli in vitro in a rolling circle-type DNA replication system. In this system, in the presence of the single-stranded DNA binding protein, the helicase/primase function on the lagging-strand template is provided by the primosome, and the synthesis of DNA strands is catalyzed by the DNA polymerase III holoenzyme. These reconstituted replication forks synthesize equivalent amounts of leading- and lagging-strand DNA, move at rates comparable to those measured in vivo (600-800 nucleotides/s at 30 degrees C), and can synthesize leading strands in the range of 150-500 kilobases in length. Using this system, we have studied the cycle of Okazaki fragment synthesis at the replication fork. This cycle is likely to have several well defined decision points, steps in the cycle where incorrect execution by the enzymatic machinery will result in an alteration in the product of the reaction, i.e. in the size of the Okazaki fragments. Since identification of these decision points should aid in the determination of which of the enzymes acting at the replication fork control the cycle, we have endeavored to identify those reaction parameters that, when varied, alter the size of the Okazaki fragments synthesized. Here we demonstrate that some enzymes, such as the DnaB helicase, remain associated continuously with the fork while others, such as the primase, must be recruited from solution each time synthesis of an Okazaki fragment is initiated. We also show that variation of the concentration of the ribonucleoside triphosphates and the deoxyribonucleoside triphosphates affects Okazaki fragment size, that the control mechanisms acting at the fork to control Okazaki fragment size are not fixed at the time the fork is assembled but can be varied during the lifetime of the fork, and that alteration in the rate of the leading-strand DNA polymerase cannot account for the effect of the deoxyribonucleoside triphosphates.
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PMID:Coordinated leading- and lagging-strand synthesis at the Escherichia coli DNA replication fork. I. Multiple effectors act to modulate Okazaki fragment size. 174 Apr 51

We have used a partially reconstituted replication system consisting of T7 DNA polymerase and T7 gene 4 protein to examine the effect of benzo[a]pyrene (B[a]P) adducts on DNA synthesis and gene 4 protein activities. The gene 4 protein is required for T7 DNA replication because of its ability to act as both a primase and helicase. We show here that total synthesis decreases as the level of adducts per molecule of DNA increases, suggesting that the B[a]P adducts are blocking an aspect of the replication process. Polyacrylamide gels indicate that a shorter DNA product is produced on modified templates and this is confirmed by determining the average chain lengths from the ratio of chain initiations to chain elongation. Gene 4 protein primed synthesis reactions display a greater sensitivity to the presence of B[a]P adducts than do oligonucleotide-primed reactions. By challenging synthesis on oligonucleotide-primed B[a]P-modified DNA with unmodified DNA, we present evidence that the T7 DNA polymerase freely dissociates after encountering an adduct. Prior studies [Brown, W. C., & Romano, L. J. (1989) J. Biol. Chem. 264, 6748-6754] have shown that the gene 4 protein alone does not dissociate from the template during translocation upon encountering an adduct. However, when gene 4 protein primed DNA synthesis is challenged, we observe an increase in synthesis but to lesser extent than observed on oligonucleotide-primed synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effects of benzo[a]pyrene-DNA adducts on a reconstituted replication system. 184 52

Herpesviruses are helper viruses for productive adeno-associated virus (AAV) replication. To analyze the herpes simplex virus type 1 (HSV-1) functions mediating helper activity, we coinfected HeLa cells with AAV type 2 (AAV-2) and different HSV-1 mutants defective in individual HSV replication genes. AAV replication was fully accomplished in the absence of HSV DNA replication and thus did not require expression of late HSV genes. In addition, HSV mutants lacking either the origin-binding protein or the functional DNA polymerase fully maintained the capacity to replicate AAV. Cotransfection of the cloned, replication-competent AAV-2 genome together with the seven HSV replication genes (UL5, UL8, UL9, UL29, UL30, UL42, and UL52) led to productive AAV replication. Cotransfections with different combinations of these genes demonstrated that a subset of four of them, coding for the HSV helicase-primase complex (UL5, UL8, UL52) and the major DNA-binding protein (UL29), was already sufficient to mediate the helper effect. Thus, the HSV helper activity for productive AAV replication seems to consist of DNA replication functions. This appears to be different from the helper effect provided by adenovirus, which predominantly modulates AAV gene regulation.
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PMID:A subset of herpes simplex virus replication genes provides helper functions for productive adeno-associated virus replication. 185 24

Many of the proteins that operate at the replication fork in Escherichia coli have been defined genetically. These include some of the subunits of the DNA polymerase III holoenzyme, the DnaB replication fork helicase, and the DnaG primase. The multiprotein primosome (which includes the DnaB and DnaG proteins), defined biochemically on the basis of its requirement during bacteriophage phi X174 complementary-strand synthesis, could serve as the helicase-primase replication machine on the lagging-strand template. In order to determine if this is the case, we have begun an investigation of the phenotypes of mutants with mutations priA, priB, and priC, which encode the primosomal proteins factor Y (protein n'), n, and n", respectively. Inactivation of priA by insertional mutagenesis resulted in the induction of the SOS response, as evinced by induction of a resident lambda prophage, extreme filamentation, and derepression of an indicator operon in which beta-galactosidase production was controlled by the dinD1 promoter. In addition, the copy numbers of resident pBR322 plasmids were reduced four- to fivefold in these strains, and production of phi X174 phage was delayed considerably. These results are discussed in the context of existing models for SOS induction and possible roles for the PriA protein at the replication fork in vivo.
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PMID:Inactivation of the Escherichia coli priA DNA replication protein induces the SOS response. 193 75

In this paper we examine the role of the DNA polymerase accessory proteins in modulating the processivity of DNA synthesis by the bacteriophage T4-coded five protein "holoenzyme" replication complex in vitro. Primed single-stranded DNA was used as a template for the DNA synthesis reactions, and buffer conditions were chosen to mimic in vivo salt concentrations. We find that the accessory proteins significantly increase the DNA-bound lifetime of the holoenzyme complex but that the maximum lifetime of the complex is still less than 10 s at 22 degrees C. The accessory proteins greatly enhance the processivity of the holoenzyme relative to that of the polymerase alone. ATP hydrolysis catalyzed by the accessory proteins complex is required to achieve this enhancement. We have investigated the temporal relationship between ATP hydrolysis by the accessory proteins and primer elongation by the holoenzyme and find that ATPase activity is required for initial assembly of the holoenzyme complex but not for elongation per se. Thus we conclude that the increased processivity displayed by the holoenzyme in moving through regions of template secondary structure reflects the high intrinsic processivity of the holoenzyme complex itself rather than a requirement for a concomitant ATPase-driven helicase activity during elongation. We have also measured the ATPase activity of the accessory proteins as a function of polymerase concentration and find that the rate of ATP hydrolysis catalyzed by this complex decreases significantly when the accessory proteins are assembled (with polymerase and gene 32 protein) into the five-protein holoenzyme and coupled to primer elongation. Based on these results we discuss mechanisms by which the ATPase activity of the polymerase accessory proteins might stimulate the overall processivity of the holoenzyme.
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PMID:Stimulation of the processivity of the DNA polymerase of bacteriophage T4 by the polymerase accessory proteins. The role of ATP hydrolysis. 198 49

Nucleotide excision is initiated by the UvrABC endonuclease system in which the initial DNA interaction is with UvrA which was dimerized in the presence of ATP. Nucleoprotein formation most likely takes place on undamaged regions of DNA by (UvrA)2 which has been dimerized in the presence of ATP. Topological unwinding of DNA, driven by ATP binding, is increased by the presence of UvrB to approximately a single helical turn. The Uvr(A)2B complex translocates to a damaged site by the combined Uvr(A)2B helicase in which the driving force is provided by the UvrB-associated ATPase. The dual incision reaction is initiated by the binding of the UvrC protein to the Uvr(A)2B-nucleoprotein complex. The proteins in this post-incision nucleoprotein complex do not turn over and require the presence of the UvrD protein and DNA polymerase I under polymerizing conditions. The final integrity of the DNA strands is restored with polynucleotide ligase.
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PMID:The UvrABC endonuclease system of Escherichia coli--a view from Baltimore. 214 12

A priming mechanism requiring dnaA, dnaB, and dnaC proteins operates on a single-stranded DNA coated with single-stranded DNA-binding protein. This novel priming, referred to as "ABC-priming," requires a specific hairpin structure whose stem carries a dnaA protein recognition sequence (dnaA box). In conjunction with primase and DNA polymerase III holoenzyme, ABC-priming can efficiently convert single-stranded DNA into the duplex replicative form. dnaA protein specifically recognizes and binds the single-stranded hairpin and permits the loading of dnaB protein to form a prepriming protein complex containing dnaA and dnaB proteins which can be physically isolated. ABC-priming can replace phi X174 type priming on the lagging strand template of pBR322 in vitro, suggesting a possible function of ABC-priming for the lagging strand synthesis and duplex unwinding. Similar to the phi X174 type priming, a mobile nature of ABC-priming was indicated by helicase activity in the presence of ATP of a prepriming protein complex formed at the hairpin. The implications of this novel priming in initiation of replication at the chromosomal origin, oriC, and in its contribution to the replication fork are discussed.
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PMID:The ABC-primosome. A novel priming system employing dnaA, dnaB, dnaC, and primase on a hairpin containing a dnaA box sequence. 216 1

Plasmid ColE1 can replicate using RNAase H and DNA polymerase I. However, it can also replicate in the absence of these enzymes. In this case, formation of a persistent hybrid between a transcript (RNA II) and the DNA indirectly activates subsequent DNA synthesis, instead of providing a primer as it does in the presence of these enzymes. To activate DNA synthesis, a certain length is required for the hybridized region and the region of minimum length cannot include a palindrome. These results show that the single-stranded region of DNA displaced by the hybridization is responsible for the activation. A single-stranded region was identified on the nontranscribed strand by its enhanced reactivity to dimethyl sulfate. The necessary length for the single-stranded region is at least 40 nucleotides. The region probably provides a site for initial binding of a helicase that further unwinds the template DNA for initiation of DNA synthesis.
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PMID:Transcriptional activation of ColE1 DNA synthesis by displacement of the nontranscribed strand. 244 75

The bacteriophage T4 gene 41 protein is a 5' to 3' DNA helicase which unwinds DNA ahead of the growing replication fork and, together with the T4 gene 61 protein, also functions as a primase to initiate DNA synthesis on the lagging strand. Proteolytic cleavage by trypsin approximately 20 amino acids from the COOH terminus of the 41 protein produces 41T, a 51,500-dalton fragment (possibly still associated with small COOH-terminal fragments) which still retains the ssDNA-stimulated GTPase (ATPase) activity, the 61 protein-stimulated DNA helicase activity, and the ability to act with 61 protein to synthesize pentaribonucleotide primers. In the absence of the T4 gene 32 ssDNA binding protein, the primase-helicase composed of the tryptic fragment (41T) and 61 proteins efficiently primes DNA synthesis on circular ssDNA templates by the T4 DNA polymerase and the three T4 polymerase accessory proteins. In contrast, the 41T protein is defective as a helicase or a primase component on 32 protein-covered DNA. Thus, unlike the intact protein, 41T does not support RNA-dependent DNA synthesis on 32 protein-covered ssDNA and does not stimulate strand displacement DNA synthesis on a nicked duplex DNA template. High concentrations of 32 protein strongly inhibit RNA primer synthesis with either 41 T or intact 41 protein. The 44/62 and 45 polymerase accessory proteins (and even the 44/62 proteins to some extent) substantially reverse the 32 protein inhibition of RNA primer synthesis with intact 41 protein but not with 41T protein. We propose that the COOH-terminal region of the 41 protein is required for its interaction with the T4 polymerase accessory proteins, permitting the synthesis and utilization of RNA primers and helicase function within the T4 replication complex. When this region is altered, as in 41T protein, the protein is unable to assemble a functional primase-helicase in the replication complex. An easy and rapid purification of T4 41 protein produced by a plasmid encoding this gene (Hinton, D. M., Silver, L. L., and Nossal, N. G. (1985) J. Biol. Chem. 260, 12851-12857) is also described.
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PMID:Trypsin cleavage in the COOH terminus of the bacteriophage T4 gene 41 DNA helicase alters the primase-helicase activities of the T4 replication complex in vitro. 246 40

Cloning of the phi X174 viral origin of replication into phage M13mp8 produced an M13-phi X174 chimera, the DNA of which directed efficient replicative-form----single-strand rolling-circle replication in vitro. This replication assay was performed with purified phi X174-encoded gene A protein, Escherichia coli rep helicase, single-stranded DNA-binding protein, and DNA polymerase III holoenzyme. The nicking of replicative-form I (RFI) DNA by gene A protein was essentially unaffected by the presence of UV lesions in the DNA. However, unwinding of UV-irradiated DNA by the rep helicase was inhibited twofold as compared with unwinding of the unirradiated substrate. UV irradiation of the substrate DNA caused a strong inhibition in its ability to direct DNA synthesis. However, even DNA preparations that contained as many as 10 photodimers per molecule still supported the synthesis of progeny full-length single-stranded DNA. The appearance of full-length radiolabeled products implied at least two full rounds of replication, since the first round released the unlabeled plus viral strand of the duplex DNA. Pretreatment of the UV-irradiated DNA substrate with purified pyrimidine dimer endonuclease from Micrococcus luteus, which converted photodimer-containing supercoiled RFI DNA into relaxed, nicked RFII DNA and thus prevented its replication, reduced DNA synthesis by 70%. Analysis of radiolabeled replication products by agarose gel electrophoresis followed by autoradiography revealed that this decrease was due to a reduction in the synthesis of progeny full-length single-stranded DNA. This implies that 70 to 80% of the full-length DNA products produced in this system were synthesized on molecules that carried photodimers. Thus, similarly to its activity on UV-irradiated single-stranded DNA, DNA polymerase III holenzyme can bypass pyrimidine photodimers in the more complex replicative form --->single-strand replication, which involves, in addition to the polymerizing activity, the unwinding of the duplex by the rep helicase and the participation of a more complex multiprotein replisome.
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PMID:Rolling-circle replication of UV-irradiated duplex DNA in the phi X174 replicative-form----single-strand replication system in vitro. 252 71


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