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Saleem A. Khan - One of the best experts on this subject based on the ideXlab platform.
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Rolling-Circle Replication
Plasmid Biology, 2014Co-Authors: Saleem A. KhanAbstract:Plasmids that replicate by a Rolling-Circle (RC) mechanism are ubiquitous in gram-positive bacteria and are also found in gram-negative bacteria as well as in Archaea. This chapter discusses the general anatomy of Rolling-Circle replicating (RCR) plasmids, architecture of the double-strand origin (dso), the single-strand origin (sso), the initiator proteins and their structure-function relationship, key events during the initiation and termination process, and the role of host proteins in plasmid RC Replication. It highlights the gaps in the current understanding of the Replication of RCR plasmids and possible future lines of research that may uncover these gaps. The first of the RCR plasmids to be identified were native to the gram-positive bacterium, Staphylococcus aureus. The Rep proteins of the pT181 family act as dimers and utilize Tyr-191 of the two monomers in the initiation and termination events. Biochemical analyses using heterodimers of the pT181 RepC protein have provided insights into the role of individual monomers in plasmid RC Replication. Elucidation of the three-dimensional structure of plasmid Rep proteins should considerably increase the understanding of the mechanistic aspects of plasmid RC Replication. The availability of crystal structures of the initiators of various plasmid families should provide major insights into the mechanisms of initiation and termination of plasmid RC Replication.
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Plasmid Rolling-Circle Replication: highlights of two decades of research
Plasmid, 2005Co-Authors: Saleem A. KhanAbstract:This review provides a historical perspective of the major findings that contributed to our current understanding of plasmid Rolling-Circle (RC) Replication. Rolling-Circle-replicating (RCR) plasmids were discovered approximately 20 years ago. The first of the RCR plasmids to be identified were native to Gram-positive bacteria, but later such plasmids were also identified in Gram-negative bacteria and in archaea. Further studies revealed mechanistic similarities in the Replication of RCR plasmids and the single-stranded DNA bacteriophages of Escherichia coli, although there were important differences as well. Three important elements, a gene encoding the initiator protein, the double strand origin, and the single strand origin, are contained in all RCR plasmids. The initiator proteins typically contain a domain involved in their sequence-specific binding to the double strand origin and a domain that nicks within the double strand origin and generates the primer for DNA Replication. The double strand origins include the start-site of leading strand synthesis and contain sequences that are bound and nicked by the initiator proteins. The single strand origins are required for synthesis of the lagging strand of RCR plasmids. The single strand origins are non-coding regions that are strand-specific, and contain extensive secondary structures. This minireview will highlight the major findings in the study of plasmid RC Replication over the past twenty years. Regulation of Replication of RCR plasmids will not be included since it is the subject of another review.
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DNA-protein interactions during the initiation and termination of plasmid pT181 Rolling-Circle Replication.
Progress in nucleic acid research and molecular biology, 2003Co-Authors: Saleem A. KhanAbstract:Initiation of DNA Replication requires the generation of a primer at the origin of Replication that can be utilized by a DNA polymerase for DNA synthesis. This can be accomplished by several means, including the synthesis of an RNA primer by a DNA primase or RNA polymerase, by nicking of one strand of the DNA to generate a free 3'-OH end that can be used as a primer, and by the utilization of the OH group present in an amino acid such as serine within an initiation protein as a primer. Furthermore, some single-stranded DNA genomes can utilize a snap-back 3'-OH end generated due to self-complementarity as a primer for DNA Replication. The different modes of initiation require the generation of highly organized DNA-protein complexes at the origin that trigger the initiation of Replication. A large majority of small, multicopy plasmids of Gram-positive bacteria and some of Gram-negative bacteria replicate by a Rolling-Circle (RC) mechanism (for previous reviews, see Refs.). More than 200 Rolling-Circle replicating (RCR) plasmids have so far been identified and, based on sequence homologies in their Replication regions, can be grouped into approximately seven families (Refs., and http://www.essex.ac.uk/bs/staff/osborn/DPR-home.htm). This review will focus on plasmids of the pT181 family that replicate by an RC mechanism. So far, approximately 25 plasmids have been identified as belonging to this family based on the sequence homology in their double-strand origins (dsos) and the genes encoding the initiator (Rep) proteins. This review will highlight our current understanding of the structural features of the origins of Replication, and the DNA-protein and protein-protein interactions that result in the generation of a Replication-initiation complex that triggers Replication. It will discuss the molecular events that result in the precise termination of Replication once the leading-strand DNA synthesis has been completed. This review will also discuss the various biochemical activities of the initiator proteins encoded by the plasmids of the pT181 family and the mechanism of inactivation of the Rep activity after supporting one round of leading-strand Replication. Finally, the review will outline the mechanism of Replication of the lagging strand of the pT181 plasmid as well as the limited information that is available on the role of host proteins in pT181 leading- and lagging-strand Replication.
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Biochemical characterization of the Staphylococcus aureus PcrA helicase and its role in plasmid Rolling Circle Replication
The Journal of biological chemistry, 2002Co-Authors: Tseh-ling Chang, Asma Naqvi, Syam P. Anand, M. Gabriela Kramer, Rajan Munshi, Saleem A. KhanAbstract:Previous genetic studies have suggested that a putative chromosome-encoded helicase, PcrA, is required for the Rolling Circle Replication of plasmid pT181 in Staphylococcus aureus. We have overexpressed and purified the staphylococcal PcrA protein and studied its biochemical properties in vitro. Purified PcrA helicase supported the in vitro Replication of plasmid pT181. It had ATPase activity that was stimulated in the presence of single-stranded DNA. Unlike many replicative helicases, PcrA was highly active as a 5' --> 3' helicase and had a weaker 3' --> 5' helicase activity. The RepC initiator protein encoded by pT181 nicks at the origin of Replication and becomes covalently attached to the 5' end of the DNA. The 3' OH end at the nick then serves as a primer for displacement synthesis. PcrA helicase showed an origin-specific unwinding activity with supercoiled plasmid pT181 DNA that had been nicked at the origin by RepC. We also provide direct evidence for a protein-protein interaction between PcrA and RepC proteins. Our results are consistent with a model in which the PcrA helicase is targeted to the pT181 origin through a protein-protein interaction with RepC and facilitates the movement of the replisome by initiating unwinding from the RepC-generated nick.
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Plasmid Rolling‐Circle Replication: recent developments
Molecular microbiology, 2002Co-Authors: Saleem A. KhanAbstract:It is now well established that a large majority of small, multicopy plasmids of Gram-positive bacteria use the Rolling-Circle (RC) mechanism for their Replication. Furthermore, the host range of RC plasmids now includes Gram-negative organisms as well as archaea. RC plasmids can be broadly classified into at least five families, individual members of which are spread among widely different bacteria. There is significant homology in the basic replicons of plasmids belonging to a particular family, and there is compelling evidence that such plasmids have evolved from common ancestors. Major advances have recently been made in our understanding of plasmid RC Replication, including the characterization of the biochemical activities of the plasmid initiator proteins and their interaction with the double-strand origin, the domain structure of the initiator proteins and the molecular basis for the function of single-strand origins in plasmid lagging strand synthesis. Over the past several years, there has been a ‘renaissance’ in studies on RC Replication as a result of the discovery that many plasmids replicate by this mechanism, and studies in the next few years are likely to reveal new and novel mechanisms used by RC plasmids for their regulated Replication.
Gang Chen - One of the best experts on this subject based on the ideXlab platform.
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How RNA catalyzes cyclization
Nature Chemical Biology, 2015Co-Authors: Zhensheng Zhong, Gang ChenAbstract:The long-awaited crystal structure of the Varkud satellite (VS) ribozyme dimer provides atomic-level insights into how the VS ribozyme folds and catalyzes RNA circularization during Rolling Circle Replication, as well as revealing convergent evolution used by RNAs to catalyze an S_N2 reaction.
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Ribozymes: How RNA catalyzes cyclization
Nature chemical biology, 2015Co-Authors: Zhensheng Zhong, Gang ChenAbstract:The long-awaited crystal structure of the Varkud satellite (VS) ribozyme dimer provides atomic-level insights into how the VS ribozyme folds and catalyzes RNA circularization during Rolling Circle Replication, as well as revealing convergent evolution used by RNAs to catalyze an SN2 reaction.
Zhensheng Zhong - One of the best experts on this subject based on the ideXlab platform.
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How RNA catalyzes cyclization
Nature Chemical Biology, 2015Co-Authors: Zhensheng Zhong, Gang ChenAbstract:The long-awaited crystal structure of the Varkud satellite (VS) ribozyme dimer provides atomic-level insights into how the VS ribozyme folds and catalyzes RNA circularization during Rolling Circle Replication, as well as revealing convergent evolution used by RNAs to catalyze an S_N2 reaction.
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Ribozymes: How RNA catalyzes cyclization
Nature chemical biology, 2015Co-Authors: Zhensheng Zhong, Gang ChenAbstract:The long-awaited crystal structure of the Varkud satellite (VS) ribozyme dimer provides atomic-level insights into how the VS ribozyme folds and catalyzes RNA circularization during Rolling Circle Replication, as well as revealing convergent evolution used by RNAs to catalyze an SN2 reaction.
Takashi Horiuchi - One of the best experts on this subject based on the ideXlab platform.
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Complex repeat structure promotes hyper-amplification and amplicon evolution through Rolling-Circle Replication
Nucleic acids research, 2018Co-Authors: Takaaki Watanabe, Hisashi Tanaka, Takashi HoriuchiAbstract:Inverted repeats (IRs) are abundant in genomes and frequently serve as substrates for chromosomal aberrations, including gene amplification. In the early stage of amplification, repeated cycles of chromosome breakage and rearrangement, called breakage-fusion-bridge (BFB), generate a large inverted structure, which evolves into highly-amplified, complex end products. However, it remains to be determined how IRs mediate chromosome rearrangements and promote subsequent hyper-amplification and amplicon evolutions. To dissect the complex processes, we constructed repetitive structures in a yeast chromosome and selected amplified cells using genetic markers with limited expression. The genomic architecture was associated with Replication stress and produced extra-/intra-chromosomal amplification. Genetic analysis revealed structure-specific endonucleases, Mus81 and Rad27, and post-Replication DNA repair protein, Rad18, suppress the amplification processes. Following BFB cycles, the intra-chromosomal products undergo intensive rearrangements, such as frequent inversions and deletions, indicative of Rolling-Circle Replication. This study presents an integrated view linking BFB cycles to hyper-amplification driven by Rolling-Circle Replication.
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Gene amplification system based on double Rolling-Circle Replication as a model for oncogene-type amplification
Nucleic acids research, 2011Co-Authors: Takaaki Watanabe, Hideyuki Tanabe, Takashi HoriuchiAbstract:Gene amplification contributes to a variety of biological phenomena, including malignant progression and drug resistance. However, details of the molecular mechanisms remain to be determined. Here, we have developed a gene amplification system in yeast and mammalian cells that is based on double Rolling-Circle Replication (DRCR). Cre-lox system is used to efficiently induce DRCR utilizing a recombinational process coupled with Replication. This system shows distinctive features seen in amplification of oncogenes and drug-resistance genes: (i) intra- and extrachromosomal amplification, (ii) intensive chromosome rearrangement and (iii) scattered-type amplification resembling those seen in cancer cells. This system can serve as a model for amplification of oncogenes and drug-resistance genes, and improve amplification systems used for making pharmaceutical proteins in mammalian cells.
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Double Rolling Circle Replication (DRCR) is recombinogenic
Genes to cells : devoted to molecular & cellular mechanisms, 2011Co-Authors: Haruko Okamoto, Takaaki Watanabe, Takashi HoriuchiAbstract:Homologous recombination plays a critical role in maintaining genetic diversity as well as genome stability. Interesting examples implying hyper-recombination are found in nature. In chloroplast DNA (cpDNA) and the herpes simplex virus 1 (HSV-1) genome, DNA sequences flanked by inverted repeats undergo inversion very frequently, suggesting hyper-recombinational events. However, mechanisms responsible for these events remain unknown. We previously observed very frequent inversion in a designed amplification system based on double Rolling Circle Replication (DRCR). Here, utilizing the yeast 2-μm plasmid and an amplification system, we show that DRCR is closely related to hyper-recombinational events. Inverted repeats or direct repeats inserted into these systems frequently caused inversion or deletion/duplication, respectively, in a DRCR-dependent manner. Based on these observations, we suggest that DRCR might be also involved in naturally occurring chromosome rearrangement associated with gene amplification and the Replication of cpDNA and HSV genomes. We propose a model in which DRCR markedly stimulates homologous recombination.
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a novel gene amplification system in yeast based on double Rolling Circle Replication
The EMBO Journal, 2005Co-Authors: Takaaki Watanabe, Takashi HoriuchiAbstract:Gene amplification is involved in various biological phenomena such as cancer development and drug resistance. However, the mechanism is largely unknown because of the complexity of the amplification process. We describe a gene amplification system in Saccharomyces cerevisiae that is based on double Rolling‐Circle Replication utilizing break‐induced Replication. This system produced three types of amplification products. Type‐1 products contain 5–7 inverted copies of the amplification marker, leu2d . Type‐2 products contain 13 to ≈100 copies of leu2d (up to ≈730 kb increase) with a novel arrangement present as randomly oriented sequences flanked by inverted leu2d copies. Type‐3 products are acentric multicopy minichromosomes carrying leu2d . Structures of type‐2 and ‐3 products resemble those of homogeneously staining region and double minutes of higher eukaryotes, respectively. Interestingly, products analogous to these were generated at low frequency without deliberate DNA cleavage. These features strongly suggest that the processes described here may contribute to natural gene amplification in higher eukaryotes.
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A novel gene amplification system in yeast based on double Rolling-Circle Replication
The EMBO journal, 2004Co-Authors: Takaaki Watanabe, Takashi HoriuchiAbstract:Gene amplification is involved in various biological phenomena such as cancer development and drug resistance. However, the mechanism is largely unknown because of the complexity of the amplification process. We describe a gene amplification system in Saccharomyces cerevisiae that is based on double Rolling-Circle Replication utilizing break-induced Replication. This system produced three types of amplification products. Type-1 products contain 5-7 inverted copies of the amplification marker, leu2d. Type-2 products contain 13 to approximately 100 copies of leu2d (up to approximately 730 kb increase) with a novel arrangement present as randomly oriented sequences flanked by inverted leu2d copies. Type-3 products are acentric multicopy minichromosomes carrying leu2d. Structures of type-2 and -3 products resemble those of homogeneously staining region and double minutes of higher eukaryotes, respectively. Interestingly, products analogous to these were generated at low frequency without deliberate DNA cleavage. These features strongly suggest that the processes described here may contribute to natural gene amplification in higher eukaryotes.
Christopher Thomas - One of the best experts on this subject based on the ideXlab platform.
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78 Structural studies of Rolling Circle Replication initiator proteins
Journal of Biomolecular Structure & Dynamics, 2013Co-Authors: Stephen B. Carr, Simon E. V. Phillips, Lauren B. Mecia, Alice J. Stelfox, Christopher ThomasAbstract:Plasmids of the pT181 family replicate by a Rolling-Circle mechanism. The process is initiated by a plasmid-encoded Rep initiator protein, which has sequence-specific DNA nicking and religation activity. The plasmid Replication origin is nicked by Rep, which binds covalently to one DNA strand via an active site tyrosine, initiating Rolling Circle Replication and religating the strand at the end of the cycle. Rep proteins also associate with PcrA helicase to form a highly processive complex. We have determined the structure of the Rep protein from cryptic plasmid pSTK1 of Geobacillus stearothermophilus (Gst), and several variants of RepD from Staphylococcus aureus (Sau), representing the first structural information on this class of initiators. Cloning and expression of the designated 269 aa Rep product from pSTK1 failed to yield soluble, active protein. However, expression from an arbitrary point upstream yielded an elongated product capable of relaxing plasmid substrates encoding an inverted repeat seque...
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Structural Studies of Rolling Circle Replication Initiator Proteins
Biophysical Journal, 2013Co-Authors: Simon E. V. Phillips, Stephen B. Carr, Lauren B. Mecia, Alice J. Stelfox, Christopher ThomasAbstract:pT181 family plasmids replicate by a Rolling-Circle mechanism. This is initiated by a plasmid-encoded Rep initiator protein, which has sequence-specific DNA nicking and religation activity. The Replication origin is nicked by Rep, which binds covalently to one DNA strand via an active site tyrosine, initiating Rolling Circle Replication and religating the strand at the end of the cycle. Rep proteins also associate with PcrA helicase to form a highly processive complex. We have determined the structure of the Rep protein from cryptic plasmid pSTK1 of Geobacillus stearothermophilus (Gst), and several variants of RepD from Staphylococcus aureus (Sau), representing the first structural information on this class of initiators.Cloning and expression of a construct derived from the from pSTK1 Rep yielded a product that relaxed plasmid substrates encoding an inverted repeat sequence from pSTK1, which resembles the Replication origin of the pT181 family, and activated the cognate Gst PcrA helicase. The crystal structure for the 31 kDa fragment of Gst Rep has been solved at 2.3 A, showing a novel, ring-shaped dimer with a 20A diameter pore. The inner surface is formed by an 18-stranded β-sheet, while the outer surface is decorated with 18 α-helices. The protein has a novel fold, but the extended sheet exhibits similarities to that in TATA-binding protein (TBP). The active site Tyr179 residues, one from each subunit, lie 26 A apart across the pore, with a nearby catalytic magnesium ion co-ordinated by three carboxylate side-chains.Crystal structures for the Sau Rep variants RepDE, RepDN and RepDC have been solved by molecular repacement using the Gst Rep as a model, and show similar structural features. The implications for the mechanism of Rolling Circle Replication will be discussed in the light of extensive functional data available for Sau RepD.
