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Jerard Hurwitz - One of the best experts on this subject based on the ideXlab platform.
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atomic force microscopic analysis of the binding of the schizosaccharomyces pombe origin recognition complex and the sporc4 protein with origin dna
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Maria Gaczynska, Pawel A Osmulski, Yun Jiang, Joon Kyu Lee, Vladimir P Bermudez, Jerard HurwitzAbstract:In eukaryotes, the initiation of DNA replication requires the interaction between origin Sequences and the origin recognition complex (ORC), which is highly conserved. In this report, atomic force microscopy (AFM) was used to examine the binding of Schizosaccharomyces pombe (sp) ORC and the spOrc4 protein with the sp Autonomously Replicating Sequence 1 (ars1). AFM imaging revealed that spORC binding to ars1 occurred solely through spOrc4p and depended on the N-terminal AT-hook domains present in spOrc4p. At high molar ratios of spORC (or spOrc4p alone) to DNA (6:1), all of the input ars1 was bound in a one protein complex to one plasmid manner. Restriction digestion and AFM analysis of protein–DNA fragments revealed the presence of two binding sites in ars1. One site mapped to a region centered at nucleotide 838 of ars1 previously detected by DNase I protection that was reported to be essential for the Autonomously Replicating Sequence activity of ars1. The second site mapped to a previously uncharacterized region centered at nucleotide 1148. AFM showed that the length of the DNA fragment complexed with either spORC or spOrc4p was shortened by ≈140 bp, suggesting the wrapping of two turns of the DNA around the spOrc4p alone as well as the spOrc4p in spORC. We also show that treatment of the spORC (spOrc4p)–ars1 complex with topoisomerase I induced a negative shift in the topoisomer distribution. These findings suggest that the binding of spORC to origin DNA alters the structure of the DNA. Thus, in the case of spORC, due to its unusual spOrc4p, at least two factors are likely to influence ars1 activation. These include the selective binding of the complex to A- and T-rich regions and the alteration of the DNA structure due to its wrapping around spOrc4p.
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the schizosaccharomyces pombe origin recognition complex interacts with multiple at rich regions of the replication origin dna by means of the at hook domains of the sporc4 protein
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Kyeongyeop Moon, Yun Jiang, Jerard HurwitzAbstract:The interaction between an origin Sequence and the origin recognition complex (ORC), which is highly conserved in eukaryotes, is critical for the initiation of DNA replication. In this report, we have examined the interaction between the Schizosaccharomyces pombe (sp) Autonomously Replicating Sequence 1 (ars1) and the spORC. For this purpose, we have purified the spORC containing all six subunits, a six-subunit complex containing the N-terminal-deleted spOrc4 subunit (spORCΔN-Orc4), and the spOrc4 subunit by using the baculovirus expression system. Wild-type spORC showed Sequence-specific binding to ars1, and the spOrc4 protein alone showed the same DNA-binding properties as wild-type spORC. In contrast, the spORCΔN-Orc4 and the ΔN-spOrc4p alone did not bind significantly to ars1. These findings indicate that the N-terminal domain of the spOrc4 protein that contains multiple AT-hook motifs is essential for the ars1-binding activity. DNA-binding competition assays with fragments of ars1 and DNase I footprinting studies with full-length ars1 revealed that the spORC interacted with several AT-rich Sequence regions of ars1. These DNA-binding properties of spORC correlate with the previously determined Sequence requirements of the S. pombe ars1. These studies indicate that because of its unique Orc4 subunit, S. pombe uses a mechanism to recognize its origins different from that used by Saccharomyces cerevisiae.
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identification purification and molecular cloning of Autonomously Replicating Sequence binding protein 1 from fission yeast schizosaccharomyces pombe
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Yota Murakami, Joel A Huberman, Jerard HurwitzAbstract:Abstract Autonomously Replicating Sequence (ARS) elements of the fission yeast Schizosaccharomyces pombe contain multiple imperfect copies of the consensus Sequence reported by Maundrell et al. [Maundrell K., Hutchison, A. & Shall, S. (1988) EMBO J. 7, 2203-2209]. When cell free extracts of S. pombe were incubated with a dimer or tetramer of an oligonucleotide containing the ARS consensus Sequence, several complexes were detected using a gel mobility-shift assay. The proteins forming these complexes also bind ars3002, which is the most active origin in the ura4 region of chromosome III of S. pombe. One protein, partly responsible for the binding activity observed with crude extracts, was purified to near homogeneity. It is a 60-kDa protein and was named ARS-binding protein 1 (Abp1). Abp1 preferentially binds to multiple sites in ARS 3002 and to the DNA polymer poly[d(A.T)]. The cloning and Sequence of the gene coding for Abp1 revealed that it encodes a protein of 59.8 kDa (522 amino acids). Abp1 has significant homology (25% identity, 50% similarity) to the N-terminal region (approximately 300 amino acids) of the human and mouse centromere DNA-binding protein CENP-B. Because centromeres of S. pombe contain a high density of ARS elements, Abp1 may play a role connecting DNA replication and chromosome segregation.
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identification purification and molecular cloning of Autonomously Replicating Sequence binding protein 1 from fission yeast schizosaccharomyces pombe dna binding proteinycenp b
1996Co-Authors: Yota Murakami, Joel A Huberman, Jerard HurwitzAbstract:Autonomously Replicating Sequence (ARS) elements of the fission yeast Schizosaccharomyces pombe con- tain multiple imperfect copies of the consensus Sequence reported by Maundrell et al. (Maundrell K., Hutchison, A. & Shall, S. (1988) EMBO J. 7, 2203-2209). When cell free extracts of S. pombe were incubated with a dimer or tetramer of an oligonucleotide containing the ARS consensus Sequence, several complexes were detected using a gel mobility-shift assay. The proteins forming these complexes also bind ars3002, which is the most active origin in the ura4 region of chromosome III of S. pombe. One protein, partly responsible for the binding activity observed with crude extracts, was purified to near homogeneity. It is a 60-kDa protein and was named ARS-binding protein 1 (Abp1). Abp1 preferentially binds to multiple sites in ARS 3002 and to the DNA polymer poly(d(AzT)). The cloning and Sequence of the gene coding for Abp1 revealed that it encodes a protein of 59.8 kDa (522 amino acids). Abp1 has significant homology (25% identity, 50% similarity) to the N-terminal region ('300 amino acids) of the human and mouse centromere DNA-binding protein CENP-B. Because centromeres of S. pombe contain a high density of ARS elements, Abp1 may play a role connecting DNA repli- cation and chromosome segregation. In the budding yeast Saccharomyces cerevisiae, ''replicators'' have been identified as short stretches of DNA ('100 bp), called Autonomously Replicating Sequence (ARS) elements, that support the autonomous replication of plasmids in cells. A subset of ARS elements was shown to colocalize with origins on chromosomes (1). Fine structure analyses of the ARS1 and ARS307 origins revealed the presence of several short func- tional elements, including an essential A element that contains the 11-bp ARS consensus Sequence and other important B elements (2-4). A multisubunit origin recognition complex (ORC) that binds to the A and B1 elements in the presence of ATP has been identified as a candidate for an ''initiator protein,'' which facilitates formation of an initiation complex at replication origins (5). In contrast, little is known about the Sequence requirements for replication origins in animal cells due to the lack of reliable ARS assays and difficulty in the direct genetic manipulations of chromosomes. However, available data suggest that animal replicators may be less specific or function differently than those of budding yeast (6). Because the fission yeast Schizosaccharomyces pombe is thought to be similar in some respects to higher eukaryotes than S. cerevisiae, a study of DNA replication in S. pombe may give us a better appreciation of this process in higher eukary- otic cells. ARS elements have been identified in S. pombe and were shown to colocalize with origins on chromosomes (7-9). The ARS elements of S. pombe are larger than those of S. cerevisiae (.500 bp vs. '100 bp) and are thought to contain multiple redundant Sequence elements distributed over a longer region (9, 10), supporting the idea that S. pombe origins may be more related to mammalian origins than those of S. cerevisiae. We have begun to identify origin-binding protein(s) from fission yeast using ars3002, which is the most active origin in the ura4 origin region (containing three distinct origins) (7). Several binding activities have been detected that interact with ars3002, and one of these proteins has been purified to homogeneity. This 60-kDa protein, named Abp1, binds to multiple sites in ars3002. The gene for Abp1¶ has been cloned, and the deduced amino acid Sequence reveals that the protein has homology to the human centromere DNA-binding protein CENP-B.
Carol S Newlon - One of the best experts on this subject based on the ideXlab platform.
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analysis of chromosome iii replicators reveals an unusual structure for the ars318 silencer origin and a conserved wtw Sequence within the origin recognition complex binding site
Molecular and Cellular Biology, 2008Co-Authors: Fujung Chang, James F Theis, Carol S Newlon, Jeremy A Miller, Conrad A Nieduszynski, Michael WeinreichAbstract:Saccharomyces cerevisiae chromosome III encodes 11 Autonomously Replicating Sequence (ARS) elements that function as chromosomal replicators. The essential 11-bp ARS consensus Sequence (ACS) that binds the origin recognition complex (ORC) has been experimentally defined for most of these replicators but not for ARS318 (HMR-I), which is one of the HMR silencers. In this study, we performed a comprehensive linker scan analysis of ARS318. Unexpectedly, this replicator depends on a 9/11-bp match to the ACS that positions the ORC binding site only 6 bp away from an Abf1p binding site. Although a largely inactive replicator on the chromosome, ARS318 becomes active if the nearby HMR-E silencer is deleted. We also performed a multiple Sequence alignment of confirmed replicators on chromosomes III, VI, and VII. This analysis revealed a highly conserved WTW motif 17 to 19 bp from the ACS that is functionally important and is apparent in the 228 phylogenetically conserved ARS elements among the six sensu stricto Saccharomyces species.
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linear derivatives of saccharomyces cerevisiae chromosome iii can be maintained in the absence of Autonomously Replicating Sequence elements
Molecular and Cellular Biology, 2007Co-Authors: Ann Dershowitz, Marylynn Snyder, Mohammed Sbia, Joan Skurnick, Loke Y Ong, Carol S NewlonAbstract:Replication origins in Saccharomyces cerevisiae are spaced at intervals of approximately 40 kb. However, both measurements of replication fork rate and studies of hypomorphic alleles of genes encoding replication initiation proteins suggest the question of whether replication origins are more closely spaced than should be required. We approached this question by systematically deleting replicators from chromosome III. The first significant increase in loss rate detected for the 315-kb full-length chromosome occurred only after all five efficient chromosomal replicators in the left two-thirds of the chromosome (ARS305, ARS306, ARS307, ARS309, and ARS310) had been deleted. The removal of the inefficient replicator ARS308 from this originless region caused little or no additional increase in loss rate. Chromosome fragmentations that removed the normally inactive replicators on the left end of the chromosome or the replicators distal to ARS310 on the right arm showed that both groups of replicators contribute significantly to the maintenance of the originless chromosome. Surprisingly, a 142-kb derivative of chromosome III, lacking all Sequences that function as Autonomously Replicating Sequence elements in plasmids, replicated and segregated properly 97% of the time. Both the replication initiation protein ORC and telomeres or a linear topology were required for the maintenance of chromosome fragments lacking replicators.
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two compound replication origins in saccharomyces cerevisiae contain redundant origin recognition complex binding sites
Molecular and Cellular Biology, 2001Co-Authors: James F Theis, Carol S NewlonAbstract:While many of the proteins involved in the initiation of DNA replication are conserved between yeasts and metazoans, the structure of the replication origins themselves has appeared to be different. As typified by ARS1, replication origins in Saccharomyces cerevisiae are <150 bp long and have a simple modular structure, consisting of a single binding site for the origin recognition complex, the replication initiator protein, and one or more accessory Sequences. DNA replication initiates from a discrete site. While the important Sequences are currently less well defined, metazoan origins appear to be different. These origins are large and appear to be composed of multiple, redundant elements, and replication initiates throughout zones as large as 55 kb. In this report, we characterize two S. cerevisiae replication origins, ARS101 and ARS310, which differ from the paradigm. These origins contain multiple, redundant binding sites for the origin recognition complex. Each binding site must be altered to abolish origin function, while the alteration of a single binding site is sufficient to inactivate ARS1. This redundant structure may be similar to that seen in metazoan origins. The replication of eukaryotic chromosomes initiates at multiple origins during each S phase. These DNA replication origins are best understood in the budding yeast Saccharomyces cerevisiae, in which they were initially recognized by their ability to promote the autonomous replication of plasmids. For this reason, they are referred to as Autonomously Replicating Sequence (ARS) elements (29, 55). The paradigm S. cerevisiae replication origin is ARS1. It has a modular structure that spans about 120 bp and includes a small essential region, domain A, and three small accessory Sequences, B1, B2, and B3, mutations in which reduce but do not abolish activity (40). Domain A, which encompasses the essential match to the 11-bp ARS consensus Sequence (ACS), is the core of the binding site for the S. cerevisiae replication initiator protein, the origin recognition complex (ORC). The six-subunit ORC complex also contacts and protects DNA in the B1 element, and some mutations in B1 compromise ORC binding in vitro (3, 37, 50, 52). The B3 element contains a binding site for the transcriptional activator-repressor Abf1p, which can be replaced by the binding sites for the transcriptional regulators Rap1p and Gal4p (40). The precise role of the B2 element has not been defined, although at least one of its functions is likely to be unwinding the DNA duplex to allow entry of the replication machinery (38, 41).
Shiladitya Dassarma - One of the best experts on this subject based on the ideXlab platform.
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multiple replication origins of halobacterium sp strain nrc 1 properties of the conserved orc7 dependent oric1
Journal of Bacteriology, 2009Co-Authors: James A. Coker, Brian R Berquist, Hua Xiang, Priya Dassarma, Melinda Capes, Tammitia Wallace, Karen Mcgarrity, Rachael Gessler, Roman Tatusov, Shiladitya DassarmaAbstract:The eukaryote-like DNA replication system of the model haloarchaeon Halobacterium NRC-1 is encoded within a circular chromosome and two large megaplasmids or minichromosomes, pNRC100 and pNRC200. We previously showed by genetic analysis that 2 (orc2 and orc10) of the 10 genes coding for Orc-Cdc6 replication initiator proteins were essential, while a third (orc7), located near a highly conserved Autonomously Replicating Sequence, oriC1, was nonessential for cell viability. Here we used whole-genome marker frequency analysis (MFA) and found multiple peaks, indicative of multiple replication origins. The largest chromosomal peaks were located proximal to orc7 (oriC1) and orc10 (oriC2), and the largest peaks on the extrachromosomal elements were near orc9 (oriP1) in both pNRC100 and -200 and near orc4 (oriP2) in pNRC200. MFA of deletion strains containing different combinations of chromosomal orc genes showed that replication initiation at oriC1 requires orc7 but not orc6 and orc8. The initiation sites at oriC1 were determined by replication initiation point analysis and found to map divergently within and near an AT-rich element flanked by likely Orc binding sites. The oriC1 region, Orc binding sites, and orc7 gene orthologs were conserved in all Sequenced haloarchaea. Serial deletion of orc genes resulted in the construction of a minimal strain containing not only orc2 and orc10 but also orc9. Our results suggest that replication in this model system is intriguing and more complex than previously thought. We discuss these results from the perspective of the replication strategy and evolution of haloarchaeal genomes.
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an archaeal chromosomal Autonomously Replicating Sequence element from an extreme halophile halobacterium sp strain nrc 1
Journal of Bacteriology, 2003Co-Authors: Brian R Berquist, Shiladitya DassarmaAbstract:We report on the identification and first cloning of an Autonomously Replicating Sequence element from the chromosome of an archaeon, the extreme halophile Halobacterium strain NRC-1. The putative replication origin was identified by association with the orc7 gene and replication ability in the host strain, demonstrated by cloning into a nonReplicating plasmid. Deletion analysis showed that Sequences located up to 750 bp upstream of the orc7 gene translational start, plus the orc7 gene and 50 bp downstream, are sufficient to endow the plasmid with replication ability, as judged by expression of a plasmid-encoded mevinolin resistance selectable marker and plasmid recovery after transformation. Sequences located proximal to the two other chromosomally carried haloarchaeal orc genes (orc6 and orc8) are not able to promote efficient autonomous replication. Located within the 750-bp region upstream of orc7 is a nearly perfect inverted repeat of 31 bp, which flanks an extremely AT-rich (44%) stretch of 189 bp. The replication ability of the plasmid was lost when one copy of the inverted repeat was deleted. Additionally, the inverted repeat structure near orc7 homologs in the genomic Sequences of two other halophiles, Haloarcula marismortui and Haloferax volcanii, is highly conserved. Our results indicate that, in halophilic archaea, a chromosomal origin of replication is physically linked to orc7 homologs and that this element is sufficient to promote autonomous replication. We discuss the finding of a functional haloarchaeal origin in relation to the large number of orc1-cdc6 homologs identified in the genomes of all haloarchaea to date.
Karl Friehs - One of the best experts on this subject based on the ideXlab platform.
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a mitochondrial Autonomously Replicating Sequence from pichia pastoris for uniform high level recombinant protein production
Frontiers in Microbiology, 2017Co-Authors: Jan Philipp Schwarzhans, Tobias Luttermann, Daniel Wibberg, Anika Winkler, Wolfgang Hubner, Thomas R Huser, Jorn Kalinowski, Karl FriehsAbstract:Pichia pastoris is a non-conventional methylotrophic yeast that is widely used for recombinant protein production, typically by stably integrating the target gene into the genome as part of an expression cassette. However, the comparatively high clonal variability associated with this approach usually necessitates a time intense screening step in order to find strains with the desired productivity. Some of the factors causing this clonal variability can be overcome using episomal vectors containing an Autonomously Replicating Sequence (ARS). Here, we report on the discovery, characterization, and application of a fragment of mitochondrial DNA from P. pastoris for use as an ARS. First encountered as an off-target event in an experiment aiming for genomic integration, the newly created circular plasmid named “pMito” consists of the expression cassette and a fragment of mitochondrial DNA. Multiple matches to known ARS consensus Sequence motifs, but no exact match to known chromosomal ARS from P. pastoris were detected on the fragment, indicating the presence of a novel ARS element. Different variants of pMito were successfully used for transformation and their productivity characteristics were assayed. All analyzed clones displayed a highly uniform expression level, exceeding by up to fourfold that of a reference with a single copy integrated in its genome. Expressed GFP could be localized exclusively to the cytoplasm via super-resolution fluorescence microscopy, indicating that pMito is present in the nucleus. While expression levels were homogenous among pMito clones, an apparent upper limit of expression was visible that could not be explained based on the gene dosage. Further investigation is necessary to fully understand the bottle-neck hindering this and other ARS vectors in P. pastoris from reaching their full capability. Lastly, we could demonstrate that the mitochondrial ARS from P. pastoris is also suitable for episomal vector transformation in Saccharomyces cerevisiae, widening the potential for biotechnological application. pMito displayed strong potential to reduce clonal variability in experiments targeting recombinant protein production. These findings also showcase the as of yet largely untapped potential of mitochondrial ARS from different yeasts for biotechnological applications.
James F Theis - One of the best experts on this subject based on the ideXlab platform.
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analysis of chromosome iii replicators reveals an unusual structure for the ars318 silencer origin and a conserved wtw Sequence within the origin recognition complex binding site
Molecular and Cellular Biology, 2008Co-Authors: Fujung Chang, James F Theis, Carol S Newlon, Jeremy A Miller, Conrad A Nieduszynski, Michael WeinreichAbstract:Saccharomyces cerevisiae chromosome III encodes 11 Autonomously Replicating Sequence (ARS) elements that function as chromosomal replicators. The essential 11-bp ARS consensus Sequence (ACS) that binds the origin recognition complex (ORC) has been experimentally defined for most of these replicators but not for ARS318 (HMR-I), which is one of the HMR silencers. In this study, we performed a comprehensive linker scan analysis of ARS318. Unexpectedly, this replicator depends on a 9/11-bp match to the ACS that positions the ORC binding site only 6 bp away from an Abf1p binding site. Although a largely inactive replicator on the chromosome, ARS318 becomes active if the nearby HMR-E silencer is deleted. We also performed a multiple Sequence alignment of confirmed replicators on chromosomes III, VI, and VII. This analysis revealed a highly conserved WTW motif 17 to 19 bp from the ACS that is functionally important and is apparent in the 228 phylogenetically conserved ARS elements among the six sensu stricto Saccharomyces species.
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two compound replication origins in saccharomyces cerevisiae contain redundant origin recognition complex binding sites
Molecular and Cellular Biology, 2001Co-Authors: James F Theis, Carol S NewlonAbstract:While many of the proteins involved in the initiation of DNA replication are conserved between yeasts and metazoans, the structure of the replication origins themselves has appeared to be different. As typified by ARS1, replication origins in Saccharomyces cerevisiae are <150 bp long and have a simple modular structure, consisting of a single binding site for the origin recognition complex, the replication initiator protein, and one or more accessory Sequences. DNA replication initiates from a discrete site. While the important Sequences are currently less well defined, metazoan origins appear to be different. These origins are large and appear to be composed of multiple, redundant elements, and replication initiates throughout zones as large as 55 kb. In this report, we characterize two S. cerevisiae replication origins, ARS101 and ARS310, which differ from the paradigm. These origins contain multiple, redundant binding sites for the origin recognition complex. Each binding site must be altered to abolish origin function, while the alteration of a single binding site is sufficient to inactivate ARS1. This redundant structure may be similar to that seen in metazoan origins. The replication of eukaryotic chromosomes initiates at multiple origins during each S phase. These DNA replication origins are best understood in the budding yeast Saccharomyces cerevisiae, in which they were initially recognized by their ability to promote the autonomous replication of plasmids. For this reason, they are referred to as Autonomously Replicating Sequence (ARS) elements (29, 55). The paradigm S. cerevisiae replication origin is ARS1. It has a modular structure that spans about 120 bp and includes a small essential region, domain A, and three small accessory Sequences, B1, B2, and B3, mutations in which reduce but do not abolish activity (40). Domain A, which encompasses the essential match to the 11-bp ARS consensus Sequence (ACS), is the core of the binding site for the S. cerevisiae replication initiator protein, the origin recognition complex (ORC). The six-subunit ORC complex also contacts and protects DNA in the B1 element, and some mutations in B1 compromise ORC binding in vitro (3, 37, 50, 52). The B3 element contains a binding site for the transcriptional activator-repressor Abf1p, which can be replaced by the binding sites for the transcriptional regulators Rap1p and Gal4p (40). The precise role of the B2 element has not been defined, although at least one of its functions is likely to be unwinding the DNA duplex to allow entry of the replication machinery (38, 41).
