DNA combing reveals intrinsic temporal disorder in the replication of yeast chromosome VI.
Ontology highlight
ABSTRACT:
SUBMITTER: Czajkowsky DM
PROVIDER: S-EPMC2151843 | biostudies-literature | 2008 Jan
REPOSITORIES: biostudies-literature
Publications
DNA combing reveals intrinsic temporal disorder in the replication of yeast chromosome VI.
Czajkowsky Daniel M DM Liu Jie J Hamlin Joyce L JL Shao Zhifeng Z
Journal of molecular biology 20071023 1
It is generally believed that DNA replication in most eukaryotes proceeds according to a precise program in which there is a defined temporal order by which each chromosomal region is duplicated. However, the regularity of this program at the level of individual chromosomes, in terms of both the relative timing and the size of the DNA domain, has not been addressed. Here, the replication of chromosome VI from synchronized budding yeast was studied at a resolution of approximately 1 kb with DNA c ...[more]
Similar Datasets
Project description:The replication strategy of metazoan genomes is still unclear, mainly because definitive maps of replication origins are missing. High-throughput methods are based on population average and thus may exclusively identify efficient initiation sites, whereas inefficient origins go undetected. Single-molecule analyses of specific loci can detect both common and rare initiation events along the targeted regions. However, these usually concentrate on positioning individual events, which only gives an overview of the replication dynamics. Here, we computed the replication fork directionality (RFD) profiles of two large genes in different transcriptional states in chicken DT40 cells, namely untranscribed and transcribed DMD and CCSER1 expressed at WT levels or overexpressed, by aggregating hundreds of oriented replication tracks detected on individual DNA fibres stretched by molecular combing. These profiles reconstituted RFD domains composed of zones of initiation flanking a zone of termination originally observed in mammalian genomes and were highly consistent with independent population-averaging profiles generated by Okazaki fragment sequencing. Importantly, we demonstrate that inefficient origins do not appear as detectable RFD shifts, explaining why dispersed initiation has remained invisible to population-based assays. Our method can both generate quantitative profiles and identify discrete events, thereby constituting a comprehensive approach to study metazoan genome replication.
| S-EPMC8266662 | biostudies-literature
Project description:The replication strategy of metazoan genomes is still unclear, mainly because definitive maps of replication origins are missing. High-throughput methods are based on population average and thus may exclusively identify efficient initiation sites, whereas ineffective origins go undetected. Single-molecule analyses of specific loci can detect both common and rare initiation events along the targeted regions. However, these usually concentrate on positioning individual events, which only gives an overview of the replication dynamics. Here, we computed the replication fork directionality (RFD) profiles of two large genes in different transcriptional states in chicken DT40 cells, namely untranscribed and transcribed DMD and CCSER1 expressed at WT levels or overexpressed, by aggregating hundreds of oriented replication tracks detected on individual DNA fibres stretched by molecular combing. These profiles reconstituted RFD domains composed of zones of initiation flanking a zone of termination originally observed in mammalian genomes and were highly consistent with independent population-averaging profiles generated by Okazaki fragment sequencing (OK-seq). Importantly, we demonstrate that inefficient origins do not manifest as detectable RFD shifts, explaining why dispersed initiation has remained invisible to population-based assays. Our method can both generate quantitative profiles and identify discrete events, thereby constituting a comprehensive approach to study metazoan genome replication.
2021-03-16 | GSE164252 | GEO
Project description:Type of experiment: ChIP-chip (Chromatin immunoprecipitation on DNA chip) analyses of replication related, checkpoint related proteins and BrdU incorporated regions at 300bp resolution. Newly developed S.cerevisiae chromosome VI tiling array produced by affymetrix was used. Experimental factors:Distribution of various replication and checkpoint related proteins on chromosome VI during S-phase (with or w/o hydroxy urea). Distribution of BrdU incorporated regions on chromosome VI during S-phase with hydroxy urea. Wild type, tof1 deletion mutant, mrc1 deletion mutant, rad9 deletion mutant, sml1 deletion mutant, and sml1 mec1 tel1 triple deletion mutant were also investigated. The results of 74 hybridization files presented in the paper are shown. The type of reference used for the hybridizations, if any: For the analysis of FLAG tagged protein, we usually used Cdc45-3XHA as an internal reference. For the analysis of HA tagged protein, POL1-3XFLAG was usually analyzed simultaneously as a reference. Hybridization design: Comparison of ChIP fraction with SUP fraction for protein binding profile. For the analyses of BrdU incorporated regions, anti BrdU antibody bound fraction was compared with SUP (Sup of purified DNA from cells at G1 phase) fraction. During a course of these experiments, we found that S-phase Sup fractions were essentially the same among different genetic backgrounds and did not affect the binding profiles. Based on this observation we use standardized Sups for all experiments. No locus was scored as significantly enriched in the control ChIP experiment using HU treated untagged cells. Quality control steps taken: Confirmation of ChIP fraction by Western blotting. Confirmation by conventional ChIP PCR methods for selected locus. Or Duplication. The origin of the biological sample: Budding Yeast (Saccharomyces cerevisiae) Manipulation of biological samples and protocols used:For the preparation of HU-arrested cells, we synchronized yeast cells with alpha-factor (2mM) and then released cells into 200mM HU for 60 min. at 23°C. For the preparation of S-phase cells, cells were released into S-phase without HU and at 16°C collected at the time indicated. These cells were fixed by 1% Formaldehyde and then used for ChIP of protein of interest. For the analyses of BrdU incorporation, the same fraction of cells used for ChIP were fixed by ice cold buffer containing 0.1% Azide, and then used for detection of BrdU incorporated regions. Protocol for preparing the hybridization extract:We disrupted 1.5X10^8 cells by MULTI-BEADS SHOCKER (MB400U, YASUI KIKAI, Osaka), which was able to keep cells precisely at lower than 6°C during disruption by glass beads. This enabled us to retrieve more than 60% of tagged proteins to soluble fraction routinely. Chromosomal DNA was shared into the average size of 400-600bp by sonication. Anti-HA monoclonal antibody HA.11 (16B12) (CRP Inc., Denver, PA) and anti-FLAG monoclonal antibody M2 (Sigma-Aldrich Co., St Louis, MO) were used for chromatin immuno-precipitation. Immunoprecipitated chromosomal DNA was subsequently purified following the protocol of Youngâs lab (http://inside.wi.mit.edu/young/pub/locationanalysis.html). Purified DNA was random primed and amplified by the protocol of Brownâs lab (http://www.microarrays.org). 2ug of amplified DNA was digested with DNaseI to a mean size of 100bp and used for labeling as previously described by Winzeler et al. (Science. 281, 1194-1197, 1998). For the analysis of BrdU incorporated regions, total DNA from 3X10^8 cells was purified by Qiagen Genomic-tip system (P/N10223, QIAGEN, Germany). DNA was sheared to 300bp by sonication, denatured, and mixed with 2ug of anti-mouse IgG1 dynabeads (P/N110.12, Dynal AS, Norway) bound anti-BrdU monoclonal antibody (2B1D5F5H4E2, MBL, Japan). Immunoprecipitation and purification of antibody bound DNA fraction was carried out following the protocol of Cimbora et al. (Mol. Cel. Biol. 20, 5581-5591, 2000). Purified DNA was random primed and amplified by the Brownâs lab protocol. 2ug of amplified DNA was digested with DNaseI to a mean size of 100bp and used for labeling. Labeling protocol:DNA was end-labelled with Biotin-N6ddATP by Terminal Transferase as previously described by Winzeler et al. (Science. 281, 1194-1197, 1998) The protocol and conditions used during hybridization, blocking and washing: Hybridization, blocking and washing steps were carried out as previously described by Winzeler et al. (Science. 281, 1194-1197, 1998). Each sample was hybridized to the array in 150ul containing 6XSSPE, 0.005% TritonX-100, 15ug fragmented denatured salmon sperm DNA (Gibco-BRL), and 1nmole of a 3â-biotin control oligonucleotide (oligo B2 probes) that hybridized to the border features on the array. Samples were heated to 100°C for 10 min., and then cooled on ice. Samples were hybridized for 16h at 42°C in a hybridization oven (GeneChip hybri oven 320, Affymetrix, CA). Washing and staining protocol (Mini_euk1 ver2) provided by Affymetrix was performed automatically on a fluidics station (GeneChip fluidics station 400, Affymetrix, CA). Measurement data and specifications:Each array was scanned by HP GeneArray Scanner (Affymetrix, CA) at an emission wavelength of 560nm at 7.5uM resolution. Grids were aligned to the scanned images using the know feature of the array. Primary data analyses were carried out using the Affymetrix Microarray Suite Ver.5.0 software to obtain hybridization intensity, fold change value, change p-value, and detection p-value for each locus. For the discrimination of positive and negative signals for the binding, we compared ChIP fraction with Sup (supernatant) fraction by using three criteria as follows. First, the reliability of strength of signal was judged by detection p-value of each locus (p-value lower than 0.025 was considered as significant). Secondly, reliability of binding ratio was judged by change p-value (p-value lower than 0.025 was considered as significant). Thirdly, clusters consisted of at least three contiguous loci which filled above two criteria were selected as significantly enriched locus, because it is apparent that a single site of protein-DNA interaction will result in immuno-precipitation of DNA fragments that hybridized not only to the locus of the actual binding site but also to its neighbors. This third criterion is very unique, make the data highly reliable and can be only applicable to the chip data obtained by our high-resolution tiling array. In the case of BrdU experiment, loci c6-462 to c6-479 (Ty element) and c6-591 to c6-601 (YFR016c) were excluded from calculation, because those loci gave high background in IP fraction even without BrdU treatment. The software to present the result of discriminant analyses (makeps) is available at our web site. Keywords = DNA replication checkpoint binding profile Keywords: other
2010-06-05 | E-GEOD-486 | biostudies-arrayexpress
Project description:This series is a complementary data set for the manuscript entitled "Nup-PI: The Nucleopore-Promoter Interactions of Genes in Yeast." (Mol. Cell, 2006). Experimental Background: Genomic interaction sites of nuclear proteins were mapped by the in vivo ChEC technique described in Schmid et al. (2004) Mol. Cell 16, 147-157. Genome-wide probes that are suitable for hybridization of microarrays were prepared. In brief, MboI restriction fragments that were internally cleaved by the MN moiety of the fusion proteins were amplified and labelled. The procedure is explained in detail in the manuscript. "Nup-PI: The Nucleopore-Promoter Interactions of Genes in Yeast." (Schmid et al., Mol. Cell 2006). Experiments: Genome-wide probes were prepared from control, uncleaved chromatin, as well as chromatin cleaved by H2b-MN and Nup2-MN. All samples were from raffinose grown cells induced for 1 hour by galactose in logarithmic growth phase. Keywords: Genome-wide ChEC analysis, Mapping of Nuclear Pore Proteins
2005-12-12 | GSE3756 | GEO
Project description:Type of experiment: ChIP-chip (Chromatin immunoprecipitation on DNA chip) analyses of replication related, checkpoint related proteins and BrdU incorporated regions at 300bp resolution. Newly developed S.cerevisiae chromosome VI tiling array produced by affymetrix was used. Experimental factors:Distribution of various replication and checkpoint related proteins on chromosome VI during S-phase (with or w/o hydroxy urea). Distribution of BrdU incorporated regions on chromosome VI during S-phase with hydroxy urea. Wild type, tof1 deletion mutant, mrc1 deletion mutant, rad9 deletion mutant, sml1 deletion mutant, and sml1 mec1 tel1 triple deletion mutant were also investigated. The results of 74 hybridization files presented in the paper are shown. The type of reference used for the hybridizations, if any: For the analysis of FLAG tagged protein, we usually used Cdc45-3XHA as an internal reference. For the analysis of HA tagged protein, POL1-3XFLAG was usually analyzed simultaneously as a reference. Hybridization design: Comparison of ChIP fraction with SUP fraction for protein binding profile. For the analyses of BrdU incorporated regions, anti BrdU antibody bound fraction was compared with SUP (Sup of purified DNA from cells at G1 phase) fraction. During a course of these experiments, we found that S-phase Sup fractions were essentially the same among different genetic backgrounds and did not affect the binding profiles. Based on this observation we use standardized Sups for all experiments. No locus was scored as significantly enriched in the control ChIP experiment using HU treated untagged cells. Quality control steps taken: Confirmation of ChIP fraction by Western blotting. Confirmation by conventional ChIP PCR methods for selected locus. Or Duplication. The origin of the biological sample: Budding Yeast (Saccharomyces cerevisiae) Manipulation of biological samples and protocols used:For the preparation of HU-arrested cells, we synchronized yeast cells with alpha-factor (2mM) and then released cells into 200mM HU for 60 min. at 23°C. For the preparation of S-phase cells, cells were released into S-phase without HU and at 16°C collected at the time indicated. These cells were fixed by 1% Formaldehyde and then used for ChIP of protein of interest. For the analyses of BrdU incorporation, the same fraction of cells used for ChIP were fixed by ice cold buffer containing 0.1% Azide, and then used for detection of BrdU incorporated regions. Protocol for preparing the hybridization extract:We disrupted 1.5X10^8 cells by MULTI-BEADS SHOCKER (MB400U, YASUI KIKAI, Osaka), which was able to keep cells precisely at lower than 6°C during disruption by glass beads. This enabled us to retrieve more than 60% of tagged proteins to soluble fraction routinely. Chromosomal DNA was shared into the average size of 400-600bp by sonication. Anti-HA monoclonal antibody HA.11 (16B12) (CRP Inc., Denver, PA) and anti-FLAG monoclonal antibody M2 (Sigma-Aldrich Co., St Louis, MO) were used for chromatin immuno-precipitation. Immunoprecipitated chromosomal DNA was subsequently purified following the protocol of Young’s lab (http://inside.wi.mit.edu/young/pub/locationanalysis.html). Purified DNA was random primed and amplified by the protocol of Brown’s lab (http://www.microarrays.org). 2ug of amplified DNA was digested with DNaseI to a mean size of 100bp and used for labeling as previously described by Winzeler et al. (Science. 281, 1194-1197, 1998). For the analysis of BrdU incorporated regions, total DNA from 3X10^8 cells was purified by Qiagen Genomic-tip system (P/N10223, QIAGEN, Germany). DNA was sheared to 300bp by sonication, denatured, and mixed with 2ug of anti-mouse IgG1 dynabeads (P/N110.12, Dynal AS, Norway) bound anti-BrdU monoclonal antibody (2B1D5F5H4E2, MBL, Japan). Immunoprecipitation and purification of antibody bound DNA fraction was carried out following the protocol of Cimbora et al. (Mol. Cel. Biol. 20, 5581-5591, 2000). Purified DNA was random primed and amplified by the Brown’s lab protocol. 2ug of amplified DNA was digested with DNaseI to a mean size of 100bp and used for labeling. Labeling protocol:DNA was end-labelled with Biotin-N6ddATP by Terminal Transferase as previously described by Winzeler et al. (Science. 281, 1194-1197, 1998) The protocol and conditions used during hybridization, blocking and washing: Hybridization, blocking and washing steps were carried out as previously described by Winzeler et al. (Science. 281, 1194-1197, 1998). Each sample was hybridized to the array in 150ul containing 6XSSPE, 0.005% TritonX-100, 15ug fragmented denatured salmon sperm DNA (Gibco-BRL), and 1nmole of a 3’-biotin control oligonucleotide (oligo B2 probes) that hybridized to the border features on the array. Samples were heated to 100°C for 10 min., and then cooled on ice. Samples were hybridized for 16h at 42°C in a hybridization oven (GeneChip hybri oven 320, Affymetrix, CA). Washing and staining protocol (Mini_euk1 ver2) provided by Affymetrix was performed automatically on a fluidics station (GeneChip fluidics station 400, Affymetrix, CA). Measurement data and specifications:Each array was scanned by HP GeneArray Scanner (Affymetrix, CA) at an emission wavelength of 560nm at 7.5uM resolution. Grids were aligned to the scanned images using the know feature of the array. Primary data analyses were carried out using the Affymetrix Microarray Suite Ver.5.0 software to obtain hybridization intensity, fold change value, change p-value, and detection p-value for each locus. For the discrimination of positive and negative signals for the binding, we compared ChIP fraction with Sup (supernatant) fraction by using three criteria as follows. First, the reliability of strength of signal was judged by detection p-value of each locus (p-value lower than 0.025 was considered as significant). Secondly, reliability of binding ratio was judged by change p-value (p-value lower than 0.025 was considered as significant). Thirdly, clusters consisted of at least three contiguous loci which filled above two criteria were selected as significantly enriched locus, because it is apparent that a single site of protein-DNA interaction will result in immuno-precipitation of DNA fragments that hybridized not only to the locus of the actual binding site but also to its neighbors. This third criterion is very unique, make the data highly reliable and can be only applicable to the chip data obtained by our high-resolution tiling array. In the case of BrdU experiment, loci c6-462 to c6-479 (Ty element) and c6-591 to c6-601 (YFR016c) were excluded from calculation, because those loci gave high background in IP fraction even without BrdU treatment. The software to present the result of discriminant analyses (makeps) is available at our web site. Keywords = DNA replication checkpoint binding profile Keywords: other
2003-08-27 | GSE486 | GEO
Project description:This series is a complementary data set for the manuscript entitled "Nup-PI: The Nucleopore-Promoter Interactions of Genes in Yeast." (Mol. Cell, 2006). Experimental Background: Genomic interaction sites of nuclear proteins were mapped by the in vivo ChEC technique described in Schmid et al. (2004) Mol. Cell 16, 147-157. Genome-wide probes that are suitable for hybridization of microarrays were prepared. In brief, MboI restriction fragments that were internally cleaved by the MN moiety of the fusion proteins were amplified and labelled. The procedure is explained in detail in the manuscript. "Nup-PI: The Nucleopore-Promoter Interactions of Genes in Yeast." (Schmid et al., Mol. Cell 2006). Experiments: Genome-wide probes were prepared from control, uncleaved chromatin, as well as chromatin cleaved by H2b-MN and Nup2-MN. All samples were from raffinose grown cells induced for 1 hour by galactose in logarithmic growth phase. Keywords: Genome-wide ChEC analysis, Mapping of Nuclear Pore Proteins 3 independent genome-wide probes were prepared for each Sample. That is, the ChEC experiments as well as preparation of genome-wide probes and hybridization of microarrays were carried independently (on different days). RMA values were calculated from the original .CEL files of each array and Average and standard deviation calculated.
2010-06-05 | E-GEOD-3756 | biostudies-arrayexpress
Project description:DNA molecular combing-based replication fork directionality profiling
| PRJNA689720 | ENA
Project description:Temporal regulation of origin activation is widely thought to explain the pattern of early- and late-replicating domains in the Saccharomyces cerevisiae genome. Recently, single-molecule analysis of replication suggested that stochastic processes acting on origins with different probabilities of activation could generate the observed kinetics of replication without requiring an underlying temporal order. To distinguish between these possibilities, we examined a clb5Delta strain, where origin firing is largely limited to the first half of S phase, to ask whether all origins nonspecifically show decreased firing (as expected for disordered firing) or if only some origins ("late" origins) are affected. Approximately half the origins in the mutant genome show delayed replication while the remainder replicate largely on time. The delayed regions can encompass hundreds of kilobases and generally correspond to regions that replicate late in wild-type cells. Kinetic analysis of replication in wild-type cells reveals broad windows of origin firing for both early and late origins. Our results are consistent with a temporal model in which origins can show some heterogeneity in both time and probability of origin firing, but clustering of temporally like origins nevertheless yields a genome that is organized into blocks showing different replication times.
| S-EPMC2600925 | biostudies-literature
Project description:A surveillance mechanism, the S phase checkpoint, blocks progression into mitosis in response to DNA damage and replication stress. Segregation of damaged or incompletely replicated chromosomes results in genomic instability. In humans, the S phase checkpoint has been shown to constitute an anti-cancer barrier. Inhibition of mitotic cyclin dependent kinase (M-CDK) activity by Wee1 kinases is critical to block mitosis in some organisms. However, such mechanism is dispensable in the response to genotoxic stress in the model eukaryotic organism Saccharomyces cerevisiae. We show here that the Wee1 ortholog Swe1 does indeed inhibit M-CDK activity and chromosome segregation in response to genotoxic insults. Swe1 dispensability in budding yeast is the result of a redundant control of M-CDK activity by the checkpoint kinase Rad53. In addition, our results indicate that Swe1 is an effector of the checkpoint central kinase Mec1. When checkpoint control on M-CDK and on Pds1/securin stabilization are abrogated, cells undergo aberrant chromosome segregation.
| S-EPMC4558037 | biostudies-literature
Project description:Background: Generalized anxiety disorder (GAD) and panic disorder (PD) are the two severe subtypes of anxiety disorders (ADs), which are similar in clinical manifestation, pathogenesis, and treatment. Earlier studies have taken a whole-brain perspective on GAD and PD in the assumption that intrinsic fluctuations are static throughout the entire scan. However, it has recently been suggested that the dynamic alternations in functional connectivity (FC) may reflect the changes in macroscopic neural activity patterns underlying the critical aspects of cognition and behavior, and thus may act as biomarkers of disease. Methods: In this study, the resting-state functional MRI (fMRI) data were collected from 26 patients with GAD, 22 patients with PD, and 26 healthy controls (HCs). We investigated dynamic functional connectivity (DFC) by using the group spatial independent component analysis, a sliding window approach, and the k-means clustering methods. For group comparisons, the temporal properties of DFC states were analyzed statistically. Results: The dynamic analysis demonstrated two discrete connectivity "States" across the entire group, namely, a more segregated State I and a strongly integrated State II. Compared with HCs, patients with both GAD and PD spent more time in the weakly within-network State I, while performing fewer transitions and dwelling shorter in the integrated State II. Additionally, the analysis of DFC strength showed that connections associated with ADs were identified including the regions that belonged to default mode (DM), executive control (EC), and salience (SA) networks, especially the connections between SA and DM networks. However, no significant difference was found between the GAD and PD groups in temporal features and connection strength. Conclusions: More common but less specific alterations were detected in the GAD and PD groups, which implied that they might have similar state-dependent neurophysiological mechanisms and, in addition, could hopefully help us better understand their abnormal affective and cognitive performances in the clinic.
| S-EPMC8319536 | biostudies-literature