Showing papers on "Primase published in 1997"
TL;DR: Results suggest that this helicase may be involved in the initiation of DNA replication as a DNA unwinding enzyme.
520 citations
TL;DR: Crystal structures of binary and ternary complexes of the E. coli Rep helicase bound to single-stranded DNA or ssDNA and ADP were determined to a resolution of 3.0 A to suggest that these may be coupled to translocation of the Rep dimer along DNA.
490 citations
TL;DR: An extended structure-based alignment of eukaryotic DNA polymerase sequences provides structural insights that should be applicable to most eukarian DNA polymerases.
426 citations
TL;DR: DNA replication very likely proceeds in two phases: an initial phase of theta replication, initiated at one or more of the origins, followed by a rolling-circle mode of replication, which generates concatemers that are cleaved and packaged into infectious viral particles.
Abstract: The Herpesviridae comprise a large class of animal viruses of considerable public health importance. Of the Herpesviridae, replication of herpes simplex virustype-1 (HSV-1) has been the most extensively studied. The linear 152-kbp HSV-1 genome contains three origins of DNA replication and approximately 75 open-reading frames. Of these frames, seven encode proteins that are required for originspecific DNA replication. These proteins include a processive heterodimeric DNA polymerase, a single-strand DNA-binding protein, a heterotrimeric primosome with 5'-3' DNA helicase and primase activities, and an origin-binding protein with 3'-5' DNA helicase activity. HSV-1 also encodes a set of enzymes involved in nucleotide metabolism that are not required for viral replication in cultured cells. These enzymes include a deoxyuridine triphosphatase, a ribonucleotide reductase, a thymidine kinase, an alkaline endo-exonuclease, and a uracil-DNA glycosylase. Host enzymes, notably DNA polymerase alpha-primase, DNA ligase I, and topoisomerase II, are probably also required. Following circularization of the linear viral genome, DNA replication very likely proceeds in two phases: an initial phase of theta replication, initiated at one or more of the origins, followed by a rolling-circle mode of replication. The latter generates concatemers that are cleaved and packaged into infectious viral particles. The rolling-circle phase of HSV-1 DNA replication has been reconstituted in vitro by a complex containing several of the HSV-1 encoded DNA replication enzymes. Reconstitution of the theta phase has thus far eluded workers in the field and remains a challenge for the future.
355 citations
TL;DR: Data is shown that the product of the Werner's syndrome gene is indeed a DNA helicase, and Expressions in Sf21, as well as in HeLa cells, showed that the WRN DNA helic enzyme is exclusively transported to the nucleoplasm, which is consistent with its function in DNA metabolism.
Abstract: The gene responsible for Werner’s syndrome (WRN) contains a region homologous to the Escherichia coli RecQ type DNA helicase and was thought to code for a DNA helicase belonging to this helicase family. However, no evidence has been shown before to substantiate this prediction. Here, we show data that the product of the WRN gene is indeed a DNA helicase. The gene product, a polypeptide with a relative molecular mass of 170 kDa, expressed in the insect Spodoptera frugiperda (Sf21) cell and purified by affinity column chromatography contained both the ATPase and DNA unwinding activities characteristic of DNA helicase. Expressions in Sf21, as well as in HeLa cells, showed that the WRN DNA helicase is exclusively transported to the nucleoplasm, which is consistent with its function in DNA metabolism. Our studies on strand displacement suggest that WRN helicase can unwind not only a duplex DNA, but also an RNA‐DNA heteroduplex, while the latter reaction seems less efficient. Enzymological features learned from the purified WRN helicase are discussed with respect to the biological function, which remains to be clarified.
202 citations
TL;DR: There are five well-characterized nuclear DNA polymerases in eukaryotes (DNA polymerases alpha, beta, delta, epsilon and zeta) and this short review summarizes the current knowledge concerning the participation of each in DNA-repair.
Abstract: There are five well-characterized nuclear DNA polymerases in eukaryotes (DNA polymerases alpha, beta, delta, epsilon and zeta) and this short review summarizes our current knowledge concerning the participation of each in DNA-repair. The three major DNA excision-repair pathways involve a DNA synthesis step that replaces altered bases or nucleotides removed during repair. Base excision-repair removes many modified bases and abasic sites, and in mammalian cells this mainly involves DNA polymerase beta. An alternative means for completion of base excision-repair, involving DNA polymerases delta or epsilon, may also operate and be even more important in yeast. Nucleotide excision-repair uses DNA polymerases delta or epsilon to resynthesize the bases removed during repair of pyrimidine dimers and other bulky adducts in DNA. Similarly, mismatch-repair of replication errors appears to involve DNA polymerases delta or epsilon. DNA polymerase alpha is required for semi-conservative replication of DNA but not for repair of DNA. A more recently discovered enzyme, DNA polymerase zeta, appears to be involved in the bypass of damage, without excision, and occurs during DNA replication of a damaged template.
162 citations
TL;DR: The DDC1 gene was identified, together with MEC3 and other checkpoint genes, during a screening for mutations causing synthetic lethality when combined with a conditional allele altering DNA primase, and is involved in all the known DNA damage checkpoints.
Abstract: The DDC1 gene was identified, together with MEC3 and other checkpoint genes, during a screening for mutations causing synthetic lethality when combined with a conditional allele altering DNA primase. Deletion of DDC1 causes sensitivity to UV radiation, methyl methanesulfonate (MMS) and hydroxyurea (HU). ddc1Delta mutants are defective in delaying G1-S and G2-M transition and in slowing down the rate of DNA synthesis when DNA is damaged during G1, G2 or S phase, respectively. Therefore, DDC1 is involved in all the known DNA damage checkpoints. Conversely, Ddc1p is not required for delaying entry into mitosis when DNA synthesis is inhibited. ddc1 and mec3 mutants belong to the same epistasis group, and DDC1 overexpression can partially suppress MMS and HU sensitivity of mec3Delta strains, as well as their checkpoint defects. Moreover, Ddc1p is phosphorylated periodically during a normal cell cycle and becomes hyperphosphorylated in response to DNA damage. Both phosphorylation events are at least partially dependent on a functional MEC3 gene.
146 citations
TL;DR: Results indicate that NCp7 plays major roles in proviral DNA synthesis within the virion core due to its ability to promote prime-specific proviralDNA synthesis while concurrently inhibiting non-specific reverse transcription of viral and cellular RNAs.
122 citations
TL;DR: The results show how enzymes can use RNA and protein subunits cooperatively to recognize specific sequences in double‐stranded DNA.
Abstract: Group II introns use intron-encoded reverse transcriptase, maturase and DNA endonuclease activities for site-specific insertion into DNA. Remarkably, the endonucleases are ribonucleoprotein complexes in which the excised intron RNA cleaves the sense strand of the recipient DNA by reverse splicing, while the intron-encoded protein cleaves the antisense strand. Here, studies with the yeast group II intron aI2 indicate that both the RNA and protein components of the endonuclease contribute to recognition of an ~30 bp DNA target site. Our results lead to a model in which the protein component first recognizes specific nucleotides in the most distal 59 exon region of the DNA target site (E2‐21 to ‐11). Binding of the protein then leads to DNA unwinding, enabling the intron RNA to base pair to a 13 nucleotide DNA sequence (E2‐12 to E3F1) for reverse splicing. Antisense-strand cleavage requires additional interactions of the protein with the 39 exon DNA (E3F 1t o F10). Our results show how enzymes can use RNA and protein subunits cooperatively to recognize specific sequences in doublestranded DNA.
118 citations
TL;DR: The results suggest that DNA primase plays an essential role in a subset of the Rad53p‐dependent checkpoint pathways controlling cell cycle progression in response to DNA damage.
Abstract: The temperature-sensitive yeast DNA primase mutant pri1-M4 fails to execute an early step of DNA replication and exhibits a dominant, allele-specific sensitivity to DNA-damaging agents. pri1-M4 is defective in slowing down the rate of S phase progression and partially delaying the G1–S transition in response to DNA damage. Conversely, the G2 DNA damage response and the S–M checkpoint coupling completion of DNA replication to mitosis are unaffected. The signal transduction pathway leading to Rad53p phosphorylation induced by DNA damage is proficient in pri1-M4, and cell cycle delay caused by Rad53p overexpression is counteracted by the pri1-M4 mutation. Altogether, our results suggest that DNA primase plays an essential role in a subset of the Rad53p-dependent checkpoint pathways controlling cell cycle progression in response to DNA damage.
117 citations
TL;DR: It is unambiguously demonstrated that the T7 gene 4 protein is a 5'-to-3' helicase and imply that during DNA unwinding the 5'- to- 3' strand transverses the center of the ring while the 3'-to the 5-to-5' strand is excluded from the centerof the ring.
Abstract: The bacteriophage T7 DNA helicase/primase (gene 4 protein) is a ring-like hexamer that encircles ssDNA and requires forked DNA to catalyze DNA unwinding. We report that optimal rates of unwinding of forked DNA require ssDNA tails of 55 nucleotides on the 5‘-to-3‘ strand and 15 nucleotides on the 3‘-to-5‘ strand. Surprisingly, streptavidin bound to a biotinylated 3‘-end fully substitutes for the 3‘-to-5‘ ssDNA tail. This suggests that excluding the 3‘-to-5‘ DNA strand from the center of the helicase is an essential aspect of the mechanism of hexameric helicase-catalyzed DNA unwinding. We also report that streptavidin bound to a biotinylated dT within the 5‘-to-3‘ strand of the duplexed region abolishes DNA unwinding; whereas, streptavidin bound to a biotinylated dT within the duplexed region of the other strand has no effect. These results unambiguously demonstrate that the T7 gene 4 protein is a 5‘-to-3‘ helicase and imply that during DNA unwinding the 5‘-to-3‘ strand transverses the center of the ring wh...
TL;DR: An exclusion model of DNA unwinding is proposed in which T7 helicase hexamer interacts mainly with the ssDNA strands duringDNA unwinding, encircling the 5′-strand and excluding the 3′-Strand from the hole.
TL;DR: A model is suggested in which the flanking domains influence and regulate the unwinding properties of NDH II, a full-length human nuclear DNA helicase II was cloned and overexpressed in a baculovirus-derived expression system.
TL;DR: This work examines the 5′ ends of true replicate of human mitochondrial DNA at one nucleotide resolution in vivo by making use of ligation-mediated polymerase chain reaction, finding the distribution and relative amounts of origins of the true replicate are exactly the same as those of total newly synthesized heavy strands.
TL;DR: The postulate that the stalled products of the DNA polymerases are caused by the existence of an unusual DNA conformation(s) within the TRS, during the in vitro DNA synthesis, enhancing the DNA slippages and the hairpin formations in the T RS due to primer realignment.
TL;DR: It is shown that a carboxyl-terminal domain of the gene 4 protein is required for interaction with T7 DNA polymerase during leading strand DNA synthesis, indicating that for lagging strand synthesis a different set of helicase/primase-DNA polymerase interactions are involved.
TL;DR: The relative mismatch extension efficiency is strongly decreased by substituting the alpha-thiotriphosphate analog for the next correct natural deoxynucleotide after the 3'-mismatch termini, and the possibility of using the technique for studies of mismatch extension kinetics for two polymerases lacking exonucleolytic activity is shown.
TL;DR: The highly conserved DNA polymerase alpha-primase complex (pol-prism) is the only eukaryotic DNA polymerases that can initiate DNA synthesis de novo.
TL;DR: This analysis represents the first comprehensive structure-function analysis of the conserved motifs in helicase superfamily 1 and proposes that mutations in motifs III, IV, V, and VI may be involved in the coupling of these two activities to the process of DNA unwinding.
TL;DR: Results show that mutations in two highly conserved regions within the pLS1‐ssoA seemed to function as a terminator for primer RNA synthesis, while the latter may be a binding site for RNA polymerase.
Abstract: Plasmid rolling circle replication involves generation of single-stranded DNA (ssDNA) intermediates. ssDNA released after leading strand synthesis is converted to a double-stranded form using solely host proteins. Most plasmids that replicate by the rolling circle mode contain palindromic sequences that act as the single strand origin, sso. We have investigated the host requirements for the functionality of one such sequence, ssoA, from the streptococcal plasmid pLS1. We used a new cell-free replication system from Streptococcus pneumoniae to investigate whether host DNA polymerase I was required for lagging strand synthesis. Extracts from DNA polymerase I-deficient cells failed to replicate, but this was corrected by adding purified DNA polymerase I. Efficient DNA synthesis from the pLS1-ssoA required the entire DNA polymerase I (polymerase and 5'-3' exonuclease activities). ssDNA containing the pLS1-ssoA was a substrate for specific RNA polymerase binding and a template for RNA polymerase-directed synthesis of a 20 nucleotide RNA primer. We constructed mutations in two highly conserved regions within the ssoA: a six nucleotide conserved sequence and the recombination site B. Our results show that the former seemed to function as a terminator for primer RNA synthesis, while the latter may be a binding site for RNA polymerase.
TL;DR: The results shown in this paper demonstrate that the DNA polymerase–primer TP heterodimer is not dissociated immediately after initiation, and probably reflects the polymerase requirement for a DNA primer of a minimum length to efficiently catalyze DNA elongation.
Abstract: Phage φ29 from Bacillus subtilis is a paradigm of the protein‐primed replication mechanism, in which a single‐subunit DNA polymerase is involved in both the specific protein‐primed initiation step and normal DNA elongation. To start φ29 DNA replication, the viral DNA polymerase must interact with a free molecule of the viral terminal protein (TP), to prime DNA synthesis once at each φ29 DNA end. The results shown in this paper demonstrate that the DNA polymerase–primer TP heterodimer is not dissociated immediately after initiation. On the contrary, there is a transition stage in which the DNA polymerase synthesizes a five nucleotide‐long DNA molecule while complexed with the primer TP, undergoes some structural change during replication of nucleotides 6–9, and finally dissociates from the primer protein when nucleotide 10 is inserted onto the nascent DNA chain. This behaviour probably reflects the polymerase requirement for a DNA primer of a minimum length to efficiently catalyze DNA elongation. The significance of such a limiting transition stage is supported by the finding of abortive replication products consisting of the primer TP linked up to eight nucleotides, detected during in vitro replication of φ29 TP‐DNA particularly under conditions that decrease the strand‐displacement capacity of φ29 DNA polymerase.
TL;DR: The biochemical characteristics of the HCV NS3 DNA helicase activity are deciphered and the possible multifunctional nature of the NS3 protein is discussed and compared with that of Simian virus 40 large T antigen.
Abstract: Hepatitis C virus (HCV) nonstructural protein 3 (NS3) is a known RNA helicase, an enzyme that unwinds RNA · DNA and RNA · RNA duplexes. We have now deciphered the biochemical characteristics of the HCV NS3 DNA helicase activity. Recombinant NS3 was expressed in Escherichia coli, purified to near homogeneity, and tested for DNA helicase activity. The optimal conditions for DNA unwinding (for example, the preferred pH and magnesium ion concentration) were similar to those for RNA unwinding. The DNA helicase activity was very sensitive to potassium ion concentration, while DNA binding and DNA-stimulated ATPase activities were not. The direction of DNA unwinding was determined to be 3′ to 5′. All four ribonucleoside triphosphates (ATP, GTP, CTP, UTP) and deoxynucleoside triphosphates (dATP, dGTP, dCTP, dTTP) could serve as energy sources, but GTP and dGTP were less efficient than the others. When nucleotide analog inhibitors were added to the DNA helicase reaction, the overall order of inhibitory capacity was: adenosine 5′-O-(3-thiotriphosphate) > adenylyl-imidodiphosphate and adenylyl-(β,γ-methylene)-diphosphate > AMP. DNA helicase activity was inhibited strongly by ssDNA and ssRNA, but was little affected by dsDNA. The ATPase activity was stimulated greatly by ssDNA and ssRNA, but not by dsDNA. The NS3 protein could unwind up to 500 base pairs of duplex DNA. The possible multifunctional nature of the NS3 protein is discussed and compared with that of Simian virus 40 large T antigen.
TL;DR: The results suggest that overproduced RecG inhibits the initiation of replication by prematurely resolving the R‐loops formed at the replication origin region of these plasmids with its unique helicase activity.
Abstract: The RecG protein of Escherichia coli is a DNA helicase that promotes branch migration of the Holliday junctions. We found that overproduction of RecG protein drastically decreased copy numbers of ColE1-type plasmids, which require R-loop formation between the template DNA and a primer RNA transcript (RNA II) for the initiation of replication. RecG efficiently inhibited in vitro ColE1 DNA synthesis in a reconstituted system containing RNA polymerase, RNase HI and DNA polymerase I. RecG promoted dissociation of RNA II from the R-loop in a manner that required ATP hydrolysis. These results suggest that overproduced RecG inhibits the initiation of replication by prematurely resolving the R-loops formed at the replication origin region of these plasmids with its unique helicase activity. The possibility that RecG regulates the initiation of a unique mode of DNA replication, oriC-independent constitutive stable DNA replication, by its activity in resolving R-loops is discussed.
TL;DR: It is demonstrated here that the UL8 protein is required for the helicase activity of the UL5/52 subassembly on long duplex DNA substrates with a single-stranded DNA loading site fully coated with the virus-encoded single strand DNA binding protein, ICP8.
TL;DR: 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.
TL;DR: It is suggested that DNA primase is one of targets of sphingosine, an effector molecule in apoptosis, and their potencies of inhibition in vitro were compared with their in vivo effects on human leukemic cells.
Abstract: Sphingosine is a potent inhibitor of a mammalian DNA primase in vitro (Simbulan et al., Biochemistry 33, 9007-9012, 1994). Here we measured the inhibition of DNA primase in vitro by 9 sphingosine-analogues with respect to RNA primer synthesis and DNA primase-dependent DNA synthesis, and their potencies of inhibition in vitro were compared with their in vivo effects on human leukemic cells. Sphingosine, phytosphingosine and N, N-dimethylsphingosine strongly inhibited the activity of purified calf thymus DNA primase, and also inhibited the growth of human leukemic cell line HL-60, exerting strong cytotoxicity. Dihydrosphingosine and cis-sphingosine, which showed more subtle inhibition of DNA primase in vitro, moderately inhibited the cell growth in vivo and caused cell death. In contrast, N-acyl-, N-octyl-, and N-acetylsphingosine (ceramides) showing little inhibition of DNA primase suppressed cell growth only slightly. HL 60 cell was arrested at Go/G1 phase by exogenously added sphingosine. From these results, it is suggested that DNA primase is one of targets of sphingosine, an effector molecule in apoptosis.
TL;DR: Results suggest that 5-foU produced in DNA has mutagenic potential leading to T-->G transversions during DNA synthesis, which is similar to that experienced by 3'-->5' exonuclease activity.
Abstract: 5-Formyluracil (5-foU) is a major lesion of thymine produced in DNA by ionizing radiation and various chemical oxidants. To assess its biochemical effects on DNA replication, 22mer oligonucleotide templates containing an internal 5-foU at defined sites were synthesized by the phosphoramidite method and examined for ability to serve as a template for various DNA polymerases in vitro . Klenow fragments with and without 3'-->5'exonuclease of DNA polymerase I, Thermus thermophilus DNA polymerase (exonuclease-deficient) and Pyrococcus furiosus DNA polymerase (exonuclease-proficient) read through the site of 5-foU in the template. Primer extension assays revealed that the 5-foU directed not only incorporation of dAMP but also dCMP opposite the lesion during DNA synthesis. Misincorporation opposite 5-foU was unaffected by 3'-->5' exonuclease activity. DNA polymerases had different dissociation rates from a dCMP/T mispair and from a dCMP/5-foU mispair. The incorporation of an 'incorrect' nucleotide was dependent on the sequence context and DNA polymerase used. These results suggest that 5-foU produced in DNA has mutagenic potential leading to T-->G transversions during DNA synthesis.
TL;DR: The RepA protein of the mobilizable broad host range plasmid RSF1010 has a key function in its replication and forms a homohexamer of 29,896-Da subunits, which is one of the smallest known helicases.
TL;DR: The findings suggest that the UL8 protein may be involved in recruiting HSV-1 DNA polymerase into the viral DNA replication complex and also identify a potential new target for antiviral therapy.
Abstract: The herpes simplex virus type 1 (HSV-1) UL8 DNA replication protein is a component of a trimeric helicase-primase complex. Sixteen UL8-specific monoclonal antibodies (MAbs) were isolated and characterized. In initial immunoprecipitation experiments, one of these, MAb 804, was shown to coprecipitate POL, the catalytic subunit of the HSV-1 DNA polymerase, from extracts of insect cells infected with recombinant baculoviruses expressing the POL and UL8 proteins. Coprecipitation of POL was dependent on the presence of UL8 protein. Rapid enzyme-linked immunosorbent assays (ELISAs), in which one protein was bound to microtiter wells and binding of the other protein was detected with a UL8- or POL-specific MAb, were developed to investigate further the interaction between the two proteins. When tested in the ELISAs, five of the UL8-specific MAbs consistently inhibited the interaction, raising the possibility that these antibodies act by binding to epitopes at or near a site(s) on UL8 involved in its interaction with POL. The epitopes recognized by four of the inhibitory MAbs were approximately located by using a series of truncated UL8 proteins expressed in mammalian cells. Three of these MAbs recognized an epitope near the C terminus of UL8, which was subjected to fine mapping with a series of overlapping peptides. The C-terminal peptides were then tested in the ELISA for their ability to inhibit the POL-UL8 interaction: the most potent exhibited a 50% inhibitory concentration of approximately 5 microM. Our findings suggest that the UL8 protein may be involved in recruiting HSV-1 DNA polymerase into the viral DNA replication complex and also identify a potential new target for antiviral therapy.
TL;DR: Limited proteolysis of bacteriophage T7 primase/helicase with endoproteinase Glu-C produces several proteolytic fragments, which were prepared and shown to retain helicase activity, which supports a model in which the gene 4 proteins consist of functionally separable domains.
Abstract: Limited proteolysis of bacteriophage T7 primase/helicase with endoproteinase Glu-C produces several proteolytic fragments. One of these fragments, which is derived from the C-terminal region of the protein, was prepared and shown to retain helicase activity. This result supports a model in which the gene 4 proteins consist of functionally separable domains. Crystals of this C-terminal fragment of the protein have been obtained that are suitable for X-ray diffraction studies.