The Experts below are selected from a list of 285 Experts worldwide ranked by ideXlab platform
Paul E. Boehmer - One of the best experts on this subject based on the ideXlab platform.
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Ligand induced stabilization of the melting temperature of the HSV-1 Single-Strand DNA binding Protein using the thermal shift assay
Biochemical and biophysical research communications, 2014Co-Authors: Kanchi Ravi Rupesh, Aaron Smith, Paul E. BoehmerAbstract:We have adapted the thermal shift assay to measure the ligand binding properties of the herpes simplex virus-1 Single-Strand DNA binding Protein, ICP8. By measuring SYPRO Orange fluorescence in microtiter plates using a fluorescence-enabled thermal cycler, we have quantified the effects of oligonucleotide ligands on the melting temperature of ICP8. We found that Single-Stranded oligomers raise the melting temperature of ICP8 in a length- and concentration-dependent manner, ranging from 1°C for (dT)5 to a maximum of 9°C with oligomers ⩾10 nucleotides, with an apparent Kd of
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Escherichia coli RecA promotes strand invasion with cisplatin-damaged DNA.
Biochimie, 2005Co-Authors: A.v. Nimonkar, Giuseppe Villani, N. Tanguy Le Gac, Paul E. BoehmerAbstract:The antitumor drug cisplatin causes intrastrand cross-linking of adjacent guanine residues that severely distorts the DNA backbone. These DNA adducts impede the progress of the replisome and may result in replication fork arrest. In Escherichia coli, the response to cisplatin involves the action of the prototypic recombinase RecA. Here we show that RecA can utilize, albeit at reduced levels, oligonucleotides that bear site-specific cisplatin-induced 1,2 d(GpG) intrastrand cross-links in strand invasion reactions. Binding of RecA to cisplatin-damaged oligonucleotides was not affected, indicating that the impediment was in the pairing step. The cognate E. coli Single-Strand DNA-binding Protein specifically stimulated strand invasion particularly with cisplatin-damaged DNA. These results indicate that RecA is capable of processing the major cisplatin-induced lesion via a recombination mechanism.
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On the mechanism of strand assimilation by the herpes simplex virus type‐1 single‐strand DNA‐binding Protein (ICP8)
Nucleic acids research, 2003Co-Authors: Amitabh V. Nimonkar, Paul E. BoehmerAbstract:ICP8, the herpes simplex virus type-1 encoded Single-Strand DNA (ssDNA)-binding Protein, promotes the assimilation of a Single-Stranded DNA molecule into a homologous duplex plasmid resulting in the formation of a displacement loop. Here we examine the mechanism of this process. In contrast to the RecA-type recombinases that catalyze strand invasion via an active search for homology, ICP8 acts by a salt-dependent strand annealing mechanism. The active species in this reaction is a ssDNA:ICP8 nucleoProtein filament. There appears to be no requirement for ICP8 to interact with the acceptor DNA. At higher concentrations, ICP8 promotes the reverse reaction, presumably owing to its helix destabilizing activity. ICP8-mediated strand assimilation imparts Single-Stranded character onto the acceptor DNA, consistent with the formation of a displacement loop. These data suggest that the recombination activity of ICP8 is similar to the mechanism of eukaryotic Rad52.
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Reconstitution of recombination-dependent DNA synthesis in herpes simplex virus 1
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Amitabh V. Nimonkar, Paul E. BoehmerAbstract:The repair of double-strand DNA breaks by homologous recombination is essential for the maintenance of genome stability. In herpes simplex virus 1, double-strand DNA breaks may arise as a consequence of replication fork collapse at sites of oxidative damage, which is known to be induced upon viral infection. Double-strand DNA breaks are also generated by cleavage of viral a sequences by endonuclease G during genome isomerization. We have reconstituted a system using purified Proteins in which strand invasion is coupled with DNA synthesis. In this system, the viral Single-Strand DNA-binding Protein promotes assimilation of Single-Stranded DNA into a homologous supercoiled plasmid, resulting in the formation of a displacement loop. The 3' terminus of the invading DNA serves as a primer for long-chain DNA synthesis promoted by the viral DNA replication Proteins, including the polymerase and helicase-primase. Efficient extension of the invading primer also requires a DNA-relaxing enzyme (eukaryotic topoisomerase I or DNA gyrase). The viral polymerase by itself is insufficient for DNA synthesis, and a DNA-relaxing enzyme cannot substitute for the viral helicase-primase. The viral Single-Strand DNA-binding Protein, in addition to its role in the invasion process, is also required for long-chain DNA synthesis. Form X, a topologically distinct, positively supercoiled form of displacement-loop, does not serve as a template for DNA synthesis. These observations support a model in which recombination and replication contribute toward maintaining viral genomic stability by repairing double-strand breaks. They also account for the extensive branching observed during viral replication in vivo.
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The Herpes Simplex Virus Type-1 Single-Strand DNA-binding Protein (ICP8) Promotes Strand Invasion
The Journal of biological chemistry, 2003Co-Authors: Amitabh V. Nimonkar, Paul E. BoehmerAbstract:ICP8, the herpes simplex virus type-1 Single-Strand DNA-binding Protein, was recently shown to promote strand exchange in conjunction with the viral replicative helicase (Nimonkar, A. V., and Boehmer, P. E. (2002) J. Biol. Chem. 277, 15182-15189). Here we show that ICP8 also catalyzes strand invasion in an ATP-independent manner. Thus, ICP8 promotes the assimilation of a Single-Stranded donor molecule into a homologous plasmid, resulting in the formation of a displacement loop. Invasion of a homologous duplex by Single-Stranded DNA requires homology at either 3' or 5' end of the invading strand. The reaction is dependent on the free energy of supercoiling and alters the topology of the acceptor plasmid. Hence, strand invasion products formed by ICP8 are resistant to the action of restriction endonucleases that cleave outside of the area of pairing. The ability to catalyze strand invasion is a novel activity of ICP8 and the first demonstration of a eukaryotic viral Single-Strand DNA-binding Protein to promote this reaction. In this regard ICP8 is functionally similar to the prototypical prokaryotic recombinase RecA and its eukaryotic homologs. This strand invasion activity of ICP8 coupled with DNA synthesis may explain the high prevalence of branched DNA structures during viral replication.
I. R. Lehman - One of the best experts on this subject based on the ideXlab platform.
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An initial ATP-independent step in the unwinding of a herpes simplex virus type I origin of replication by a complex of the viral origin-binding Protein and Single-Strand DNA-binding Protein
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: I. R. LehmanAbstract:Using a spectrophotometric assay that measures the hyperchromicity that accompanies the unwinding of a DNA duplex, we have identified an ATP-independent step in the unwinding of a herpes simplex virus type 1 (HSV-1) origin of replication, Ori(s), by a complex of the HSV-1 origin binding Protein (UL9 Protein) and the HSV-1 Single-Strand DNA binding Protein (ICP8). The sequence unwound is the 18-bp A + T-rich segment that links the two high-affinity UL9 Protein binding sites, boxes I and II of Ori(s). P1 nuclease sensitivity of Ori(s) and Single-Strand DNA-dependent ATPase measurements of the UL9 Protein indicate that, at 37 degrees C, the A + T-rich segment is sufficiently single stranded to permit the binding of ICP8. Binding of the UL9 Protein to boxes I and II then results in the formation of the UL9 Protein-ICP8 complex, that can, in the absence of ATP, promote unwinding of the A + T-rich segment. On addition of ATP, the helicase activity of the UL9 Protein-ICP8 complex can unwind boxes I and II, permitting access of the replication machinery to the Ori(s) sequences.
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Leading and lagging strand DNA synthesis in vitro by a reconstituted herpes simplex virus type 1 replisome.
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Maria Falkenberg, I. R. Lehman, Per EliasAbstract:The synthesis of double-stranded DNA by a rolling circle mechanism was reconstituted in vitro with a replisome consisting of the DNA polymerase-UL42 complex and the heterotrimeric helicase-primase encoded by herpes simplex virus type 1. Okazaki fragments 3 kilobases in length and leading strands that may exceed 10 kilobases are produced. Lagging strand synthesis is stimulated by ribonucleoside triphosphates. DNA replication appears to be processive because it resists competition with an excess of (dT)(150)/(dA)(20). The Single-Strand DNA binding Protein ICP8 is not required, and high concentrations of ICP8 can, in fact, inhibit lagging strand synthesis. The inhibition can, however, be overcome by the addition of an excess of the UL8 component of the helicase-primase. Rolling circle replication by the herpesvirus and bacteriophage T7 replisomes appears to proceed by a similar mechanism.
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The Herpes Simplex Virus Type 1 Helicase-primase ANALYSIS OF HELICASE ACTIVITY
Journal of Biological Chemistry, 1998Co-Authors: Maria Falkenberg, Per Elias, I. R. LehmanAbstract:The rate of unwinding of duplex DNA by the herpes simplex virus type 1 (HSV-1)-encoded helicase-primase (primosome) was determined by measuring the rate of appearance of single strands from a circular duplex DNA containing a 40-nucleotide 5' Single-Stranded tail, i.e. a preformed replication fork, in the presence of the HSV-1 single strand DNA-binding Protein, infected cell Protein 8 (ICP8). With this substrate, the rate at low ionic strength was highly sensitive to Mg2+ concentration. The Mg2+ dependence was a reflection of both the requirement for ICP8 for helicase activity and the ability of ICP8 to reverse the helicase reaction as a consequence of its capacity to anneal homologous single strands at Mg2+ concentrations in excess of 3 mM. The rate of unwinding of duplex DNA by the HSV-1 primosome was also determined indirectly by measuring the rate of leading strand synthesis with a preformed replication fork as template in the presence of the T7 DNA polymerase. The value of 60-65 base pairs unwound/s by both methods is consistent with the rate of 50 base pairs/s estimated for the rate of fork movement in vivo during replication of pseudorabies virus, another herpesvirus. Interaction with the helicase-primase did not increase its helicase activity.
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the ul8 subunit of the heterotrimeric herpes simplex virus type 1 helicase primase is required for the unwinding of single strand dna binding Protein icp8 coated dna substrates
Journal of Biological Chemistry, 1997Co-Authors: Maria Falkenberg, Per Elias, David A Bushnell, I. R. LehmanAbstract:Abstract The Herpes simplex virus type 1 primosome consists of three subunits that are the products of theUL5, UL8, and UL52 genes. The heterotrimeric enzyme has DNA-dependent ATPase, helicase, and primase activities. Earlier studies show that a subassembly consisting of the UL5 and UL52 gene products was indistinguishable from the heterotrimeric enzyme in its helicase and primase activities. We demonstrate here that the UL8 Protein is required for the helicase activity of the UL5/52 subassembly on long duplex DNA substrates (>30 nucleotides) with a Single-Stranded DNA loading site fully coated with the virus-encoded single strand DNA binding Protein, ICP8. The Escherichia coli single strand DNA binding Protein cannot substitute for ICP8, suggesting a specific physical interaction between ICP8 and the UL8 Protein. Surface plasmon resonance measurements demonstrated an interaction between ICP8 and the UL5/52/8 heterotrimer but not with the UL5/52 subassembly or the UL8 Protein alone. At a subsaturating level of ICP8, the UL5/52 subassembly does show helicase activity, suggesting that the subassembly can bind to Single-Stranded DNA but not to ICP8-coated DNA.
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Unwinding of the box I element of a herpes simplex virus type 1 origin by a complex of the viral origin binding Protein, Single-Strand DNA binding Protein, and Single-Stranded DNA
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Sam S. K. Lee, I. R. LehmanAbstract:The herpes simplex virus type 1 (HSV-1) genome contains three origins of replication: oriL and two copies of oriS. These origins contain specific sequences, box I and box II, linked by an AT-rich segment, that are recognized by an HSV-1-encoded origin binding Protein (UL9 Protein) which also possesses DNA helicase activity. Despite its intrinsic helicase activity, the UL9 Protein is unable to unwind oriS or the box I element of oriS, either in the presence or absence of the HSV-1-encoded Single-Strand DNA binding Protein, ICP8. However, a complex of the UL9 Protein and ICP8 can unwind box I if it contains a 3′ Single-Stranded tail at least 18 nt in length positioned downstream of box I. These findings suggest a model for the initiation of HSV-1 DNA replication in which a complex consisting of the UL9 Protein bound to box I, and ICP8 bound to Single-Stranded DNA generated at the A+T rich linker, perhaps as a consequence of transcription, unwinds an HSV-1 origin of replication to provide access to the replication machinery with the consequent initiation of viral DNA replication. This mode of unwinding is distinct from that observed for other animal viruses—e.g., simian virus 40 or bovine papilloma virus—in which the initiator Protein, T antigen, or E1 Protein alone, unwinds elements of the origin sequence, and the Single-Strand DNA binding Protein serves only to keep the separated strands apart.
Per Elias - One of the best experts on this subject based on the ideXlab platform.
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ul52 primase interactions in the herpes simplex virus 1 helicase primase are affected by antiviral compounds and mutations causing drug resistance
Journal of Biological Chemistry, 2014Co-Authors: Isabella Muylaert, Zhiyuan Zhao, Per EliasAbstract:Abstract Herpes simplex virus 1 (HSV-1) UL5/8/52 helicase-primase complex is required for DNA unwinding at the replication fork and synthesis of primers during virus replication, and it has become a promising novel target for antiviral therapy. Using molecular cloning, we have identified three separate domains of UL52. Co-immunoprecipitation experiments in extracts from cells transiently expressing HA-tagged UL5, FLAG-UL8, and enhanced GFP-tagged UL52 domains revealed that the N-terminal domain of UL52 primase binds UL5 helicase and the middle domain interacts with the UL8 accessory Protein. In addition, an interaction between the single strand DNA-binding Protein ICP8 and the UL52 middle domain was observed. The complex between UL5 and UL52 was stabilized by the antiviral compound BAY 54-6322, and mutations providing resistance to the drug obliterate this effect. Our results also suggest a mechanism for accommodating conformational strain resulting from movement of UL5 and UL52 in opposite directions on the lagging strand template, and they identify molecular complexes that can be further examined by structural biology techniques to resolve the mechanism of primer synthesis during herpesvirus replication. Finally, they help to explain the mechanism of action of a novel class of antiviral compounds currently being evaluated in clinical trials.
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Leading and lagging strand DNA synthesis in vitro by a reconstituted herpes simplex virus type 1 replisome.
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Maria Falkenberg, I. R. Lehman, Per EliasAbstract:The synthesis of double-stranded DNA by a rolling circle mechanism was reconstituted in vitro with a replisome consisting of the DNA polymerase-UL42 complex and the heterotrimeric helicase-primase encoded by herpes simplex virus type 1. Okazaki fragments 3 kilobases in length and leading strands that may exceed 10 kilobases are produced. Lagging strand synthesis is stimulated by ribonucleoside triphosphates. DNA replication appears to be processive because it resists competition with an excess of (dT)(150)/(dA)(20). The Single-Strand DNA binding Protein ICP8 is not required, and high concentrations of ICP8 can, in fact, inhibit lagging strand synthesis. The inhibition can, however, be overcome by the addition of an excess of the UL8 component of the helicase-primase. Rolling circle replication by the herpesvirus and bacteriophage T7 replisomes appears to proceed by a similar mechanism.
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The Herpes Simplex Virus Type 1 Helicase-primase ANALYSIS OF HELICASE ACTIVITY
Journal of Biological Chemistry, 1998Co-Authors: Maria Falkenberg, Per Elias, I. R. LehmanAbstract:The rate of unwinding of duplex DNA by the herpes simplex virus type 1 (HSV-1)-encoded helicase-primase (primosome) was determined by measuring the rate of appearance of single strands from a circular duplex DNA containing a 40-nucleotide 5' Single-Stranded tail, i.e. a preformed replication fork, in the presence of the HSV-1 single strand DNA-binding Protein, infected cell Protein 8 (ICP8). With this substrate, the rate at low ionic strength was highly sensitive to Mg2+ concentration. The Mg2+ dependence was a reflection of both the requirement for ICP8 for helicase activity and the ability of ICP8 to reverse the helicase reaction as a consequence of its capacity to anneal homologous single strands at Mg2+ concentrations in excess of 3 mM. The rate of unwinding of duplex DNA by the HSV-1 primosome was also determined indirectly by measuring the rate of leading strand synthesis with a preformed replication fork as template in the presence of the T7 DNA polymerase. The value of 60-65 base pairs unwound/s by both methods is consistent with the rate of 50 base pairs/s estimated for the rate of fork movement in vivo during replication of pseudorabies virus, another herpesvirus. Interaction with the helicase-primase did not increase its helicase activity.
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the ul8 subunit of the heterotrimeric herpes simplex virus type 1 helicase primase is required for the unwinding of single strand dna binding Protein icp8 coated dna substrates
Journal of Biological Chemistry, 1997Co-Authors: Maria Falkenberg, Per Elias, David A Bushnell, I. R. LehmanAbstract:Abstract The Herpes simplex virus type 1 primosome consists of three subunits that are the products of theUL5, UL8, and UL52 genes. The heterotrimeric enzyme has DNA-dependent ATPase, helicase, and primase activities. Earlier studies show that a subassembly consisting of the UL5 and UL52 gene products was indistinguishable from the heterotrimeric enzyme in its helicase and primase activities. We demonstrate here that the UL8 Protein is required for the helicase activity of the UL5/52 subassembly on long duplex DNA substrates (>30 nucleotides) with a Single-Stranded DNA loading site fully coated with the virus-encoded single strand DNA binding Protein, ICP8. The Escherichia coli single strand DNA binding Protein cannot substitute for ICP8, suggesting a specific physical interaction between ICP8 and the UL8 Protein. Surface plasmon resonance measurements demonstrated an interaction between ICP8 and the UL5/52/8 heterotrimer but not with the UL5/52 subassembly or the UL8 Protein alone. At a subsaturating level of ICP8, the UL5/52 subassembly does show helicase activity, suggesting that the subassembly can bind to Single-Stranded DNA but not to ICP8-coated DNA.
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The DNA ligands influence the interactions between the herpes simplex virus 1 origin binding Protein and the single strand DNA-binding Protein, ICP-8.
The Journal of biological chemistry, 1995Co-Authors: Claes M. Gustafsson, Maria Falkenberg, Stina Simonsson, Hadi Valadi, Per EliasAbstract:The herpes simplex virus type 1 (HSV-1) origin binding Protein, OBP, is a DNA helicase specifically stimulated by the viral single strand DNA-binding Protein, ICP-8. The stimulation is dependent on direct Protein-Protein interactions between the C-terminal domain of OBP, delta OBP, and ICP 8 (Boehmer, P.E., Craigie, M.C., Stow, N.D., and Lehman, I.R. (1994) J. Biol. Chem. 269, 29329-29334). We have now observed that this interaction is dramatically influenced by the nature of the DNA ligand. Stable complexes between delta OBP, ICP 8, and double-stranded DNA, presented either as a specific duplex oligonucleotide or a restriction fragment containing the HSV-1 origin of replication, oriS, can be detected by gel chromatography and gel electrophoresis. In contrast, a Single-Stranded oligonucleotide, oligo(dT)65, will completely disrupt the complex between delta OBP and ICP 8. We therefore suggest that the interaction between delta OBP and ICP 8 serves to position the single strand DNA-binding Protein with high precision onto Single-Stranded DNA at a replication fork or at an origin of DNA replication.
Maria Falkenberg - One of the best experts on this subject based on the ideXlab platform.
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Leading and lagging strand DNA synthesis in vitro by a reconstituted herpes simplex virus type 1 replisome.
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Maria Falkenberg, I. R. Lehman, Per EliasAbstract:The synthesis of double-stranded DNA by a rolling circle mechanism was reconstituted in vitro with a replisome consisting of the DNA polymerase-UL42 complex and the heterotrimeric helicase-primase encoded by herpes simplex virus type 1. Okazaki fragments 3 kilobases in length and leading strands that may exceed 10 kilobases are produced. Lagging strand synthesis is stimulated by ribonucleoside triphosphates. DNA replication appears to be processive because it resists competition with an excess of (dT)(150)/(dA)(20). The Single-Strand DNA binding Protein ICP8 is not required, and high concentrations of ICP8 can, in fact, inhibit lagging strand synthesis. The inhibition can, however, be overcome by the addition of an excess of the UL8 component of the helicase-primase. Rolling circle replication by the herpesvirus and bacteriophage T7 replisomes appears to proceed by a similar mechanism.
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The Herpes Simplex Virus Type 1 Helicase-primase ANALYSIS OF HELICASE ACTIVITY
Journal of Biological Chemistry, 1998Co-Authors: Maria Falkenberg, Per Elias, I. R. LehmanAbstract:The rate of unwinding of duplex DNA by the herpes simplex virus type 1 (HSV-1)-encoded helicase-primase (primosome) was determined by measuring the rate of appearance of single strands from a circular duplex DNA containing a 40-nucleotide 5' Single-Stranded tail, i.e. a preformed replication fork, in the presence of the HSV-1 single strand DNA-binding Protein, infected cell Protein 8 (ICP8). With this substrate, the rate at low ionic strength was highly sensitive to Mg2+ concentration. The Mg2+ dependence was a reflection of both the requirement for ICP8 for helicase activity and the ability of ICP8 to reverse the helicase reaction as a consequence of its capacity to anneal homologous single strands at Mg2+ concentrations in excess of 3 mM. The rate of unwinding of duplex DNA by the HSV-1 primosome was also determined indirectly by measuring the rate of leading strand synthesis with a preformed replication fork as template in the presence of the T7 DNA polymerase. The value of 60-65 base pairs unwound/s by both methods is consistent with the rate of 50 base pairs/s estimated for the rate of fork movement in vivo during replication of pseudorabies virus, another herpesvirus. Interaction with the helicase-primase did not increase its helicase activity.
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the ul8 subunit of the heterotrimeric herpes simplex virus type 1 helicase primase is required for the unwinding of single strand dna binding Protein icp8 coated dna substrates
Journal of Biological Chemistry, 1997Co-Authors: Maria Falkenberg, Per Elias, David A Bushnell, I. R. LehmanAbstract:Abstract The Herpes simplex virus type 1 primosome consists of three subunits that are the products of theUL5, UL8, and UL52 genes. The heterotrimeric enzyme has DNA-dependent ATPase, helicase, and primase activities. Earlier studies show that a subassembly consisting of the UL5 and UL52 gene products was indistinguishable from the heterotrimeric enzyme in its helicase and primase activities. We demonstrate here that the UL8 Protein is required for the helicase activity of the UL5/52 subassembly on long duplex DNA substrates (>30 nucleotides) with a Single-Stranded DNA loading site fully coated with the virus-encoded single strand DNA binding Protein, ICP8. The Escherichia coli single strand DNA binding Protein cannot substitute for ICP8, suggesting a specific physical interaction between ICP8 and the UL8 Protein. Surface plasmon resonance measurements demonstrated an interaction between ICP8 and the UL5/52/8 heterotrimer but not with the UL5/52 subassembly or the UL8 Protein alone. At a subsaturating level of ICP8, the UL5/52 subassembly does show helicase activity, suggesting that the subassembly can bind to Single-Stranded DNA but not to ICP8-coated DNA.
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The DNA ligands influence the interactions between the herpes simplex virus 1 origin binding Protein and the single strand DNA-binding Protein, ICP-8.
The Journal of biological chemistry, 1995Co-Authors: Claes M. Gustafsson, Maria Falkenberg, Stina Simonsson, Hadi Valadi, Per EliasAbstract:The herpes simplex virus type 1 (HSV-1) origin binding Protein, OBP, is a DNA helicase specifically stimulated by the viral single strand DNA-binding Protein, ICP-8. The stimulation is dependent on direct Protein-Protein interactions between the C-terminal domain of OBP, delta OBP, and ICP 8 (Boehmer, P.E., Craigie, M.C., Stow, N.D., and Lehman, I.R. (1994) J. Biol. Chem. 269, 29329-29334). We have now observed that this interaction is dramatically influenced by the nature of the DNA ligand. Stable complexes between delta OBP, ICP 8, and double-stranded DNA, presented either as a specific duplex oligonucleotide or a restriction fragment containing the HSV-1 origin of replication, oriS, can be detected by gel chromatography and gel electrophoresis. In contrast, a Single-Stranded oligonucleotide, oligo(dT)65, will completely disrupt the complex between delta OBP and ICP 8. We therefore suggest that the interaction between delta OBP and ICP 8 serves to position the single strand DNA-binding Protein with high precision onto Single-Stranded DNA at a replication fork or at an origin of DNA replication.
Amitabh V. Nimonkar - One of the best experts on this subject based on the ideXlab platform.
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On the mechanism of strand assimilation by the herpes simplex virus type‐1 single‐strand DNA‐binding Protein (ICP8)
Nucleic acids research, 2003Co-Authors: Amitabh V. Nimonkar, Paul E. BoehmerAbstract:ICP8, the herpes simplex virus type-1 encoded Single-Strand DNA (ssDNA)-binding Protein, promotes the assimilation of a Single-Stranded DNA molecule into a homologous duplex plasmid resulting in the formation of a displacement loop. Here we examine the mechanism of this process. In contrast to the RecA-type recombinases that catalyze strand invasion via an active search for homology, ICP8 acts by a salt-dependent strand annealing mechanism. The active species in this reaction is a ssDNA:ICP8 nucleoProtein filament. There appears to be no requirement for ICP8 to interact with the acceptor DNA. At higher concentrations, ICP8 promotes the reverse reaction, presumably owing to its helix destabilizing activity. ICP8-mediated strand assimilation imparts Single-Stranded character onto the acceptor DNA, consistent with the formation of a displacement loop. These data suggest that the recombination activity of ICP8 is similar to the mechanism of eukaryotic Rad52.
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Reconstitution of recombination-dependent DNA synthesis in herpes simplex virus 1
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Amitabh V. Nimonkar, Paul E. BoehmerAbstract:The repair of double-strand DNA breaks by homologous recombination is essential for the maintenance of genome stability. In herpes simplex virus 1, double-strand DNA breaks may arise as a consequence of replication fork collapse at sites of oxidative damage, which is known to be induced upon viral infection. Double-strand DNA breaks are also generated by cleavage of viral a sequences by endonuclease G during genome isomerization. We have reconstituted a system using purified Proteins in which strand invasion is coupled with DNA synthesis. In this system, the viral Single-Strand DNA-binding Protein promotes assimilation of Single-Stranded DNA into a homologous supercoiled plasmid, resulting in the formation of a displacement loop. The 3' terminus of the invading DNA serves as a primer for long-chain DNA synthesis promoted by the viral DNA replication Proteins, including the polymerase and helicase-primase. Efficient extension of the invading primer also requires a DNA-relaxing enzyme (eukaryotic topoisomerase I or DNA gyrase). The viral polymerase by itself is insufficient for DNA synthesis, and a DNA-relaxing enzyme cannot substitute for the viral helicase-primase. The viral Single-Strand DNA-binding Protein, in addition to its role in the invasion process, is also required for long-chain DNA synthesis. Form X, a topologically distinct, positively supercoiled form of displacement-loop, does not serve as a template for DNA synthesis. These observations support a model in which recombination and replication contribute toward maintaining viral genomic stability by repairing double-strand breaks. They also account for the extensive branching observed during viral replication in vivo.
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The Herpes Simplex Virus Type-1 Single-Strand DNA-binding Protein (ICP8) Promotes Strand Invasion
The Journal of biological chemistry, 2003Co-Authors: Amitabh V. Nimonkar, Paul E. BoehmerAbstract:ICP8, the herpes simplex virus type-1 Single-Strand DNA-binding Protein, was recently shown to promote strand exchange in conjunction with the viral replicative helicase (Nimonkar, A. V., and Boehmer, P. E. (2002) J. Biol. Chem. 277, 15182-15189). Here we show that ICP8 also catalyzes strand invasion in an ATP-independent manner. Thus, ICP8 promotes the assimilation of a Single-Stranded donor molecule into a homologous plasmid, resulting in the formation of a displacement loop. Invasion of a homologous duplex by Single-Stranded DNA requires homology at either 3' or 5' end of the invading strand. The reaction is dependent on the free energy of supercoiling and alters the topology of the acceptor plasmid. Hence, strand invasion products formed by ICP8 are resistant to the action of restriction endonucleases that cleave outside of the area of pairing. The ability to catalyze strand invasion is a novel activity of ICP8 and the first demonstration of a eukaryotic viral Single-Strand DNA-binding Protein to promote this reaction. In this regard ICP8 is functionally similar to the prototypical prokaryotic recombinase RecA and its eukaryotic homologs. This strand invasion activity of ICP8 coupled with DNA synthesis may explain the high prevalence of branched DNA structures during viral replication.
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in vitro strand exchange promoted by the herpes simplex virus type 1 single strand dna binding Protein icp8 and dna helicase primase
Journal of Biological Chemistry, 2002Co-Authors: Amitabh V. Nimonkar, Paul E. BoehmerAbstract:The genome of herpes simplex virus type-1 undergoes a high frequency of homologous recombination in the absence of a virus-encoded RecA-type Protein. We hypothesized that viral homologous recombination is mediated by the combined action of the viral single strand DNA-binding Protein (ICP8) and helicase-primase. Our results show that ICP8 catalyzes the formation of recombination intermediates (joint molecules) between circular Single-Stranded acceptor and linear duplex donor DNA. Joint molecules formed by invasion of a 3'-terminal strand displaces the non-complementary 5'-terminal strand, thereby creating a loading site for the helicase-primase. Helicase-primase acts on these joint molecules to promote ATP-dependent branch migration. Finally, we have reconstituted strand exchange by the synchronous action of ICP8 and helicase-primase. Based on these data, we present a recombination mechanism for a eukaryotic DNA virus in which a single strand DNA-binding Protein and helicase cooperate to promote homologous pairing and branch migration.