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)

We have examined the molecular mechanism that enables the T4 bacteriophage DNA polymerase holoenzyme to synthesize DNA processively on the leading strand of the replication fork for many minutes, while allowing an identical holoenzyme on the lagging strand to recycle from one Okazaki fragment to the next in less than 4 s. We use a perfect hairpin helix of 15 base pairs to mimic the encounter of the polymerase with the end of a previously synthesized Okazaki fragment. Polymerase dissociation is monitored during the stall at the hairpin helix by the addition of excess T4 gene 32 protein (SSB protein), which rapidly melts the helix and allows a stalled polymerase molecule to continue DNA synthesis. In the accompanying paper, we show that polymerase holoenzyme dissociation is slow (half-life of 2.5 min) when this enzyme is stalled by nucleotide omission (Hacker, K. J., and Alberts, B. M. (1994) J. Biol. Chem. 269, 24209-24220). In contrast, the holoenzyme dissociates with a half-life of 1 s after hitting the hairpin helix, a rate sufficient to allow efficient polymerase recycling on the lagging strand in vivo. We conclude that, upon completing each Okazaki fragment, the holoenzyme senses an encounter with duplex DNA and then switches to a state that rapidly dissociates.
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PMID:The rapid dissociation of the T4 DNA polymerase holoenzyme when stopped by a DNA hairpin helix. A model for polymerase release following the termination of each Okazaki fragment. 792 78

Homologous recombination is a fundamental biological process. Biochemical understanding of this process is most advanced for Escherichia coli. At least 25 gene products are involved in promoting genetic exchange. At present, this includes the RecA, RecBCD (exonuclease V), RecE (exonuclease VIII), RecF, RecG, RecJ, RecN, RecOR, RecQ, RecT, RuvAB, RuvC, SbcCD, and SSB proteins, as well as DNA polymerase I, DNA gyrase, DNA topoisomerase I, DNA ligase, and DNA helicases. The activities displayed by these enzymes include homologous DNA pairing and strand exchange, helicase, branch migration, Holliday junction binding and cleavage, nuclease, ATPase, topoisomerase, DNA binding, ATP binding, polymerase, and ligase, and, collectively, they define biochemical events that are essential for efficient recombination. In addition to these needed proteins, a cis-acting recombination hot spot known as Chi (chi: 5'-GCTGGTGG-3') plays a crucial regulatory function. The biochemical steps that comprise homologous recombination can be formally divided into four parts: (i) processing of DNA molecules into suitable recombination substrates, (ii) homologous pairing of the DNA partners and the exchange of DNA strands, (iii) extension of the nascent DNA heteroduplex; and (iv) resolution of the resulting crossover structure. This review focuses on the biochemical mechanisms underlying these steps, with particular emphases on the activities of the proteins involved and on the integration of these activities into likely biochemical pathways for recombination.
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PMID:Biochemistry of homologous recombination in Escherichia coli. 796 21

Main properties of the single-stranded DNA-binding proteins (SSB-proteins) (type of the protein UP 1 from the calf thymus) have been analyzed. The combination of some properties allows one to say about the strong specificity of the term "SSB-proteins type UP 1" and their differences from the HMG-proteins and other DNA-binding proteins and enzymes. Basing on the data from literature and the author's data the hypothesis was made that eukaryotic SSB-proteins may be involved in DNA replication, though they have tight relationship with the proteins of heterogeneous nuclear ribonucleoprotein particles. The following fact evidence for the involvement of SSB-proteins in DNA replication: 1) preferential affinity to the single-strand DNA in comparison with the single-strand RNA; 2) the contact between DNA SSB-proteins in chromatin; 3) strong proportional dependence between the content of SSB-proteins in chromatin and the intensity of DNA synthesis; 4) the dependence between the content of SSB-proteins in the cells and genomic size in different organisms; 5) the capacity of SSB-proteins to specific stimulation of DNA replicative synthesis. Apparently SSB-proteins in chromatin modulate the replication with the aid of the unwinding of DNA, stabilizing of the single-strand region in this molecule and the activation of the DNA synthesis carried out by DNA polymerase.
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PMID:[Possibility of participation of proteins binding single-stranded DNA in eukaryotic DNA replication]. 799 35

Phage P4 DNA is replicated in cell-free extracts of Escherichia coli in the presence of partially purified P4 alpha protein [Krevolin and Calendar (1985), J. Mol. Biol. 182, 507-517]. Using a modified in vitro replication assay, we have further characterized this process. Analysis by agarose gel electrophoresis and autoradiography of in vitro replicated molecules demonstrates that the system yields supercoiled monomeric DNA as the main product. Electron microscopic analysis of in vitro generated intermediates indicates that DNA synthesis initiates in vitro mainly at ori, the origin of replication used in vivo. Replication proceeds from this origin bidirectionally, resulting in theta-type molecules. In contrast to the in vivo situation, no extensive single-stranded regions were found in these intermediates. The initiation proteins of the host, DnaB and DnaG, and the chaperones DnaJ and DnaK are not required for P4 replication, because polyclonal antibodies against those polypeptides do not inhibit the process. The reaction is inhibited by antibodies against the SSB protein, and by ara-CTP, a specific inhibitor of DNA polymerase III holoenzyme. Consistent with previous reports, P4 in vitro replication is independent of transcription by host RNA polymerase. Novobiocin, a DNA gyrase inhibitor, strongly inhibits P4 DNA synthesis, indicating that form I DNA is the required substrate.
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PMID:Phage P4 DNA replication in vitro. 802 13

Methyl-directed mismatch repair is initiated by the mismatch-provoked, MutHLS-dependent cleavage of the unmodified strand at a hemimethylated d(GATC) sequence. This reaction is independent of the polarity of the unmodified strand and can occur either 3' or 5' to the mismatch on the unmethylated strand (Au, K. G., Welsh, K., and Modrich, P. (1992) J. Biol. Chem. 267, 12142-12148). The overall repair reaction also occurs without regard to polarity of the unmethylated strand. Both hemimethylated configurations of a linear heteroduplex containing a single d(GATC) sequence are subject to methyl-directed correction in Escherichia coli extracts and in a purified repair system. Repair of both heteroduplex orientations requires MutH, MutL, MutS, DNA helicase II, SSB, and DNA polymerase III holoenzyme, but the two substrates differ with respect to exonuclease requirements for correction. When the unmethylated d(GATC) sequence that directs repair is located 5' to the mismatch on the unmodified strand, mismatch correction requires the 5'--> 3' hydrolytic activity of exonuclease VII or RecJ exonuclease. Repair directed by an unmodified d(GATC) sequence situated 3' to the mismatch depends on the 3'--> 5' activity of exonuclease I. Specific requirements for these activities are evident with circular heteroduplexes containing a single asymmetrically placed d(GATC) sequence, with the requirement for a 5'--> 3' or 3'--> 5' hydrolytic activity being determined by the orientation of the unmethylated strand along the shorter path joining the two sites in the DNA circle. This observation suggests that the methyl-directed repair system utilizes the proximal d(GATC) sequence to direct correction. To our knowledge, these experiments represent the first instance in which exonuclease I, exonuclease VII, and RecJ have been implicated in a particular DNA metabolic pathway.
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PMID:Methyl-directed mismatch repair is bidirectional. 838 65

We describe the purification to near homogeneity of a single-stranded DNA binding protein from 0-18-h embryos of Drosophila melanogaster. Drosophila SSB (D-SSB) is a heterotrimer with subunits of molecular weight of 70,000, 30,000, and 8000. It has a Stokes radius of 48.6 +/- 2 A and s20,w = 5.0 +/- 0.2 S. The interaction of D-SSB with ssDNA was examined by the quenching of intrinsic protein fluorescence. The binding site size was determined to be n = 22 +/- 4 nucleotides with a maximum quenching Qm = 35 +/- 3%. Equilibrium titrations indicate that D-SSB binds with low cooperativity, omega = 10-300, and high apparent affinity, K omega = (0.7-5) x 10(7) M-1, at 225 mM NaCl. Sedimentation of D-SSB bound to small oligonucleotides demonstrates that D-SSB does not require protein association for binding. D-SSB stimulates the extent and processivity of DNA synthesis of its cognate DNA polymerase alpha. On the basis of these properties, we conclude that D-SSB is the Drosophila cognate of the human and yeast SSB/RP-A proteins.
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PMID:A single-stranded DNA binding protein from Drosophila melanogaster: characterization of the heterotrimeric protein and its interaction with single-stranded DNA. 849 3

Mitochondrial DNA (mtDNA) is replicated by DNA polymerase gamma by a strand displacement mechanism involving mitochondrial single-stranded DNA-binding protein (mtSSB). mtSSB stimulates the overall rate of DNA synthesis on singly-primed M13 DNA mainly by stimulating the processivity of DNA synthesis rather than by stimulating primer recognition. We used electrophoretic mobility shift methods to study the effects of mtSSB on primer-template recognition by DNA pol gamma. Preliminary experiments showed that single mtSSB tetramers bind tightly to oligo(dT) single strands containing 32 to 48 residues. An oligonucleotide primer-template was designed with an 18-mer primer annealed to the 3'-portion of a 71-mer template containing 40 dT residues at its 5'-end as a binding site for mtSSB. DNA pol gamma bound to this primer-template either in the absence or presence of mtSSB in complexes that remained intact and enzymatically active following native gel electrophoresis. Association of mtSSB with the 5'-dT40-tail in the 18:71-mer primer-template reduced the binding of DNA polymerase gamma and the efficiency of primer extension. Binding of mtSSB to single-stranded DNA was also observed to block the action of the 3'-->5' exonuclease of DNA polymerase gamma. The size of fragments protected from 3'-->5' exonuclease trimming increases with increasing ionic strength in a manner consistent with the known salt dependence of the binding site size of Escherichia coli SSB.
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PMID:Effects of Xenopus laevis mitochondrial single-stranded DNA-binding protein on primer-template binding and 3'-->5' exonuclease activity of DNA polymerase gamma. 870 57

The herpes simplex virus type 1 (HSV) single-stranded DNA-binding protein (SSB, ICP8) stimulates the viral DNA polymerase (Pol) on an oligonucleotide-primed single-stranded DNA template. This stimulation is non-specific since other SSBs also increase Pol activity. However, only ICP8 was stimulatory when Pol activity was dependent upon priming by the viral helicase-primase complex. ICP8 also specifically stimulated the primer synthesis and ATPase activities of the helicase-primase. The mechanism of stimulation was different from that of Pol; helicase-primase stimulation required much lower amounts of ICP8 than the amount that saturates the DNA and optimally stimulates Pol. Furthermore, ICP8 did not act by removing secondary structure as stimulation also occurred on homopolymer templates. While the UL8 component of the helicase-primase is not required for enzymatic activities by a subassembly of the UL5 and UL52 proteins, only the holoenzyme (UL5/8/52) was stimulated by ICP8. These results identify a unique, functional interaction between the ICP8 SSB and the helicase-primase complex, mediated by the UL8 subunit.
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PMID:A functional interaction of ICP8, the herpes simplex virus single-stranded DNA-binding protein, and the helicase-primase complex that is dependent on the presence of the UL8 subunit. 912 59

We describe a novel promoter for E. coli RNA polymerase that functions efficiently only in the form of single-stranded DNA. Derived from the leading region of F plasmid, single-stranded Frpo sequence directs RNA polymerase to initiate transcription at a specific site within Frpo, and this specific transcription is highly stimulated by SSB. Prior denaturation activates transcription from otherwise inactive duplex DNA containing Frpo. Since RNAs synthesized on SSB-coated single-stranded Frpo are efficiently elongated into DNA chains by DNA polymerase III holoenzyme, transcription at Frpo serves also for priming DNA replication. A mode of recognition by RNA polymerase of a unique secondary structure within Frpo is proposed, and possible roles of this novel single-stranded promoter in expression and replication during conjugal transfer of F plasmid are discussed.
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PMID:Frpo: a novel single-stranded DNA promoter for transcription and for primer RNA synthesis of DNA replication. 920 Jun 8

Mutations in the dnaQ gene, which encodes the proofreading epsilon-subunit of the DNA polymerase III holoenzyme, lead to a mutator phenotype caused by enhanced error rates during DNA replication. In this paper, we studied the influence of ssb mutations on the dnaQ49 mutator, because of the involvement of SSB protein in DNA replication. We found that the ssb-113 mutation suppresses the mutator phenotype of dnaQ49. The suppression effect resulted from an enhanced expression of the dnaQ49 allele as determined by experiments with gene fusions. S1 nuclease analysis revealed that the increased dnaQ expression is based on transcriptional activation of the dnaQP2 promoter. This seems to be the consequence of an increased DNA supercoiling in the ssb-113 mutant, which also influenced further functions that are sensitive to alterations in DNA supercoiling. These results support the hypothesis that the expression of the epsilon-subunit of DNA polymerase III may additionally be modulated by DNA supercoiling, and suggest a possible role for DNA topology in mutagenesis.
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PMID:The ssb-113 allele suppresses the dnaQ49 mutator and alters DNA supercoiling in Escherichia coli. 928 36


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