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Sliding Clamp (sliding + clamp)
Selected AbstractsOn the mechanism of loading the PCNA sliding clamp by RFCMOLECULAR MICROBIOLOGY, Issue 1 2008Isabelle Dionne Summary Sliding clamps play central roles in a broad range of DNA replication and repair processes. The clamps form circular molecules that must be opened and resealed around DNA by the clamp loader complex to fulfil their function. While most eukaryotes and many archea possess a homo-trimeric PCNA, the PCNA of Sulfolobus solfataricus is a heterotrimer. Here, we exploit the asymmetry of S. solfataricus PCNA to create a series of circularly permuted PCNA subunit fusions, thereby covalently closing defined interfaces within the heterotrimer. Using these concatamers, we investigate the requirements for loading the clamp onto DNA and reveal that a single defined interface within the heterotrimer is opened during the loading process. Subunit,specific interactions between S. solfataricus RFC clamp loader and PCNA permit us to superimpose our data upon the structure of yeast RFC,PCNA complex, thereby presenting a general model for PCNA loading by RFC in archaea and eukaryotes. [source] Molecular mechanism of DNA replication-coupled inactivation of the initiator protein in Escherichia coli: interaction of DnaA with the sliding clamp-loaded DNA and the sliding clamp-Hda complexGENES TO CELLS, Issue 6 2004Masayuki Su'etsugu In Escherichia coli, the ATP-DnaA protein initiates chromosomal replication. After the DNA polymerase III holoenzyme is loaded on to DNA, DnaA-bound ATP is hydrolysed in a manner depending on Hda protein and the DNA-loaded form of the DNA polymerase III sliding clamp subunit, which yields ADP-DnaA, an inactivated form for initiation. This regulatory DnaA-inactivation represses extra initiation events. In this study, in vitro replication intermediates and structured DNA mimicking replicational intermediates were first used to identify structural prerequisites in the process of DnaA-ATP hydrolysis. Unlike duplex DNA loaded with sliding clamps, primer RNA-DNA heteroduplexes loaded with clamps were not associated with DnaA-ATP hydrolysis, and duplex DNA provided in trans did not rescue this defect. At least 40-bp duplex DNA is competent for the DnaA-ATP hydrolysis when a single clamp was loaded. The DnaA-ATP hydrolysis was inhibited when ATP-DnaA was tightly bound to a DnaA box-bearing oligonucleotide. These results imply that the DnaA-ATP hydrolysis involves the direct interaction of ATP-DnaA with duplex DNA flanking the sliding clamp. Furthermore, Hda protein formed a stable complex with the sliding clamp. Based on these, we suggest a mechanical basis in the DnaA-inactivation that ATP-DnaA interacts with the Hda-clamp complex with the aid of DNA binding. [source] On the mechanism of loading the PCNA sliding clamp by RFCMOLECULAR MICROBIOLOGY, Issue 1 2008Isabelle Dionne Summary Sliding clamps play central roles in a broad range of DNA replication and repair processes. The clamps form circular molecules that must be opened and resealed around DNA by the clamp loader complex to fulfil their function. While most eukaryotes and many archea possess a homo-trimeric PCNA, the PCNA of Sulfolobus solfataricus is a heterotrimer. Here, we exploit the asymmetry of S. solfataricus PCNA to create a series of circularly permuted PCNA subunit fusions, thereby covalently closing defined interfaces within the heterotrimer. Using these concatamers, we investigate the requirements for loading the clamp onto DNA and reveal that a single defined interface within the heterotrimer is opened during the loading process. Subunit,specific interactions between S. solfataricus RFC clamp loader and PCNA permit us to superimpose our data upon the structure of yeast RFC,PCNA complex, thereby presenting a general model for PCNA loading by RFC in archaea and eukaryotes. [source] Expression, purification and preliminary X-ray analysis of proliferating cell nuclear antigen from the archaeon Thermococcus thioreducensACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2009Miranda L. Byrne-Steele Proliferating cell nuclear antigen (PCNA) is a DNA sliding clamp which confers processivity on replicative DNA polymerases. PCNA also acts as a sliding platform that enables the association of many DNA-processing proteins with DNA in a non-sequence-specific manner. In this investigation, the PCNA from the hyperthermophilic archaeon Thermococcus thioreducens (TtPCNA) was cloned, overexpressed in Escherichia coli and purified to greater than 90% homogeneity. TtPCNA crystals were obtained by sitting-drop vapor-diffusion methods and the best ordered crystal diffracted to 1.86,Å resolution using synchrotron radiation. The crystals belonged to the hexagonal space group P63, with unit-cell parameters a = b = 89.0, c = 62.8,Å. Crystals of TtPCNA proved to be amenable to complete X-ray analysis and future structure determination. [source] Crystallographic study of G178S mutant of human proliferating cell nuclear antigenACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2008Asami Hishiki Proliferating cell nuclear antigen (PCNA) is an evolutionarily conserved protein that forms a ring-shaped homotrimer that functions as a sliding clamp for DNA replication. The rev6-1 mutation of Saccharomyces cerevisiae, which inactivates both translesion DNA synthesis and damage-avoidance pathways while having little effect on normal cell growth, has a G178S substitution in the PCNA protein. Human PCNA protein carrying the G178S substitution was crystallized. Two crystal forms were obtained under similar conditions. Crystal forms I and II belong to space groups P21, with unit-cell parameters a = 84.1, b = 130.2, c = 97.8,Å, , = 113.4°, and P212121, with unit-cell parameters a = 68.1, b = 100.2, c = 131.2,Å, respectively. Structural analyses by molecular replacement are now in progress. [source] Structure of the heterotrimeric PCNA from Sulfolobus solfataricusACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 10 2006Gareth J. Williams PCNA is a ring-shaped protein that encircles DNA, providing a platform for the association of a wide variety of DNA-processing enzymes that utilize the PCNA sliding clamp to maintain proximity to their DNA substrates. PCNA is a homotrimer in eukaryotes, but a heterotrimer in crenarchaea such as Sulfolobus solfataricus. The three proteins are SsoPCNA1 (249 residues), SsoPCNA2 (245 residues) and SsoPCNA3 (259 residues). The heterotrimeric protein crystallizes in space group P21, with unit-cell parameters a = 44.8, b = 78.8, c = 125.6,Å, , = 100.5°. The crystal structure of this heterotrimeric PCNA molecule has been solved using molecular replacement. The resulting structure to 2.3,Å sheds light on the differential stabilities of the interactions observed between the three subunits and the specificity of individual subunits for partner proteins. [source] Molecular mechanism of DNA replication-coupled inactivation of the initiator protein in Escherichia coli: interaction of DnaA with the sliding clamp-loaded DNA and the sliding clamp-Hda complexGENES TO CELLS, Issue 6 2004Masayuki Su'etsugu In Escherichia coli, the ATP-DnaA protein initiates chromosomal replication. After the DNA polymerase III holoenzyme is loaded on to DNA, DnaA-bound ATP is hydrolysed in a manner depending on Hda protein and the DNA-loaded form of the DNA polymerase III sliding clamp subunit, which yields ADP-DnaA, an inactivated form for initiation. This regulatory DnaA-inactivation represses extra initiation events. In this study, in vitro replication intermediates and structured DNA mimicking replicational intermediates were first used to identify structural prerequisites in the process of DnaA-ATP hydrolysis. Unlike duplex DNA loaded with sliding clamps, primer RNA-DNA heteroduplexes loaded with clamps were not associated with DnaA-ATP hydrolysis, and duplex DNA provided in trans did not rescue this defect. At least 40-bp duplex DNA is competent for the DnaA-ATP hydrolysis when a single clamp was loaded. The DnaA-ATP hydrolysis was inhibited when ATP-DnaA was tightly bound to a DnaA box-bearing oligonucleotide. These results imply that the DnaA-ATP hydrolysis involves the direct interaction of ATP-DnaA with duplex DNA flanking the sliding clamp. Furthermore, Hda protein formed a stable complex with the sliding clamp. Based on these, we suggest a mechanical basis in the DnaA-inactivation that ATP-DnaA interacts with the Hda-clamp complex with the aid of DNA binding. [source] | ||||||||||