Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Sequence-dependent pausing of single lambda exonuclease molecules.

ABSTRACT: Lambda exonuclease processively degrades one strand of duplex DNA, moving 5'-to-3' in an ATP-independent fashion. When examined at the single-molecule level, the speeds of digestion were nearly constant at 4 nanometers per second (12 nucleotides per second), interspersed with pauses of variable duration. Long pauses, occurring at stereotypical locations, were strand-specific and sequence-dependent. Pause duration and probability varied widely. The strongest pause, GGCGAT TCT, was identified by gel electrophoresis. Correlating single-molecule dwell positions with sequence independently identified the motif GGCGA. This sequence is found in the left lambda cohesive end, where exonuclease inhibition may contribute to the reduced recombination efficiency at that end.

SUBMITTER: Perkins TT

PROVIDER: S-EPMC1539570 | biostudies-literature | 2003 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Sequence-dependent pausing of single lambda exonuclease molecules.

Perkins Thomas T TT Dalal Ravindra V RV Mitsis Paul G PG Block Steven M SM

Science (New York, N.Y.) 20030828 5641

Lambda exonuclease processively degrades one strand of duplex DNA, moving 5'-to-3' in an ATP-independent fashion. When examined at the single-molecule level, the speeds of digestion were nearly constant at 4 nanometers per second (12 nucleotides per second), interspersed with pauses of variable duration. Long pauses, occurring at stereotypical locations, were strand-specific and sequence-dependent. Pause duration and probability varied widely. The strongest pause, GGCGAT TCT, was identified by g ...[more]

PMID: 12947034

Similar Datasets

Sequence-resolved detection of pausing by single RNA polymerase molecules.

Project description:Transcriptional pausing by RNA polymerase (RNAP) plays an important role in the regulation of gene expression. Defined, sequence-specific pause sites have been identified biochemically. Single-molecule studies have also shown that bacterial RNAP pauses frequently during transcriptional elongation, but the relationship of these "ubiquitous" pauses to the underlying DNA sequence has been uncertain. We employed an ultrastable optical-trapping assay to follow the motion of individual molecules of RNAP transcribing templates engineered with repeated sequences carrying imbedded, sequence-specific pause sites of known regulatory function. Both the known and ubiquitous pauses appeared at reproducible locations, identified with base-pair accuracy. Ubiquitous pauses were associated with DNA sequences that show similarities to regulatory pause sequences. Data obtained for the lifetimes and efficiencies of pauses support a model where the transition to pausing branches off of the normal elongation pathway and is mediated by a common elemental state, which corresponds to the ubiquitous pause.

| S-EPMC1483142 | biostudies-literature

Nascent strand with BrdU or lambda exonuclease

Project description:Nascent strand were purified with either the BrdU or the lambda exonuclease methods from culture human basophilic erythroblasts and sequenced/ Two methods were compared to isolated the Nascent strand, lambda exonuclease digestion or immunoprecipitation after pulse labelling with BrdU The following files were generated by combining round 1 and 2 sample files; FNY01_2_2_Ery_NS_BrdU_macs_summits.bed FNY01_2_2_Ery_NS_BrdU_macs_peaks.bed FNY01_2_2_Ery_NS_lexo_macs_peaks.bed FNY01_2_2_Ery_NS_lexo_macs_summits.bed

2014-05-01 | E-GEOD-50976 | biostudies-arrayexpress

Nascent strand with BrdU or lambda exonuclease

Project description:Nascent strand were purified with either the BrdU or the lambda exonuclease methods from culture human basophilic erythroblasts and sequenced/

2014-05-01 | GSE50976 | GEO

A sequence-dependent exonuclease activity from Tetrahymena thermophila.

Project description:BackgroundTelomere function requires a highly conserved G rich 3'- overhang. This structure is formed by 5'-resection of the C-rich telomere strand. However, while many nucleases have been suggested to play a role in processing, it is not yet clear which nucleases carry out this 5'-resection.ResultsWe used biochemical purification to identify a sequence-dependent exonuclease activity in Tetrahymena thermophila cell extracts. The nuclease activity showed specificity for 5'-ends containing AA or AC sequences, unlike Exo1, which showed sequence-independent cleavage. The Tetrahymena nuclease was active on both phosphorylated and unphosphorylated substrates whereas Exo1 requires a 5'-phosphate for cleavage.ConclusionsThe specificities of the enzyme indicate that this novel Tetrahymena exonuclease is distinct from Exo1 and has properties required for 3'-overhang formations at telomeres.

| S-EPMC2998447 | biostudies-literature

The enzymatic basis of processivity in lambda exonuclease.

Project description:Lambda exonuclease is a highly processive 5'-->3' exonuclease that degrades double-stranded (ds)DNA. The single-stranded DNA produced by lambda exonuclease is utilized by homologous pairing proteins to carry out homologous recombination. The extensive studies of lambda biology, lambda exonuclease enzymology and the availability of the X-ray crystallographic structure of lambda exonuclease make it a suitable model to dissect the mechanisms of processivity. lambda Exonuclease is a toroidal homotrimeric molecule and this quaternary structure is a recurring theme in proteins engaged in processive reactions in nucleic acid metabolism. We have identified residues in lambda exonuclease involved in recognizing the 5'-phosphate at the ends of broken dsDNA. The preference of lambda exonuclease for a phosphate moiety at 5' dsDNA ends has been established in previous studies; our results indicate that the low activity in the absence of the 5'-phosphate is due to the formation of inert enzyme-substrate complexes. By examining a lambda exonuclease mutant impaired in 5'-phosphate recognition, the significance of catalytic efficiency in modulating the processivity of lambda exonuclease has been elucidated. We propose a model in which processivity of lambda exonuclease is expressed as the net result of competition between pathways that either induce forward translocation or promote reverse translocation and dissociation.

| S-EPMC152868 | biostudies-literature

Downstream sequence-dependent RNA cleavage and pausing by RNA polymerase I.

Project description:The sequence of the DNA template has long been thought to influence the rate of transcription by DNA-dependent RNA polymerases, but the influence of DNA sequence on transcription elongation properties of eukaryotic RNA polymerase I (Pol I) from Saccharomyces cerevisiae has not been defined. In this study, we observe changes in dinucleotide production, transcription elongation complex stability, and Pol I pausing in vitro in response to downstream DNA. In vitro studies demonstrate that AT-rich downstream DNA enhances pausing by Pol I and inhibits Pol I nucleolytic cleavage activity. Analysis of Pol I native elongating transcript sequencing data in Saccharomyces cerevisiae suggests that these downstream sequence elements influence Pol I in vivo Native elongating transcript sequencing studies reveal that Pol I occupancy increases as downstream AT content increases and decreases as downstream GC content increases. Collectively, these data demonstrate that the downstream DNA sequence directly impacts the kinetics of transcription elongation prior to the sequence entering the active site of Pol I both in vivo and in vitro.

| S-EPMC6996881 | biostudies-literature

Half a century of bacteriophage lambda recombinase: In vitro studies of lambda exonuclease and Red-beta annealase.

Project description:DNA recombination, replication, and repair are intrinsically interconnected processes. From viruses to humans, they are ubiquitous and essential to all life on Earth. Single-strand annealing homologous DNA recombination is a major mechanism for the repair of double-stranded DNA breaks. An exonuclease and an annealase work in tandem, forming a complex known as a two-component recombinase. Red? annealase and ?-exonuclease from phage lambda form the archetypal two-component recombinase complex. In this short review article, we highlight some of the in vitro studies that have led to our current understanding of the lambda recombinase system. We synthesize insights from more than half a century of research, summarizing the state of our current understanding. From this foundation, we identify the gaps in our knowledge and cast an eye forward to consider what the next 50?years of research may uncover.

| S-EPMC7496540 | biostudies-literature

Homology sensing via non-linear amplification of sequence-dependent pausing by RecQ helicase.

Project description:RecQ helicases promote genomic stability through their unique ability to suppress illegitimate recombination and resolve recombination intermediates. These DNA structure-specific activities of RecQ helicases are mediated by the helicase-and-RNAseD like C-terminal (HRDC) domain, via unknown mechanisms. Here, employing single-molecule magnetic tweezers and rapid kinetic approaches we establish that the HRDC domain stabilizes intrinsic, sequence-dependent, pauses of the core helicase (lacking the HRDC) in a DNA geometry-dependent manner. We elucidate the core unwinding mechanism in which the unwinding rate depends on the stability of the duplex DNA leading to transient sequence-dependent pauses. We further demonstrate a non-linear amplification of these transient pauses by the controlled binding of the HRDC domain. The resulting DNA sequence- and geometry-dependent pausing may underlie a homology sensing mechanism that allows rapid disruption of unstable (illegitimate) and stabilization of stable (legitimate) DNA strand invasions, which suggests an intrinsic mechanism of recombination quality control by RecQ helicases.

| S-EPMC6773442 | biostudies-literature

Calcium-dependent folding of single calmodulin molecules.

Project description:Calmodulin is the primary calcium binding protein in living cells. Its function and structure depend strongly on calcium concentration. We used single molecule force spectroscopy by optical tweezers to study the folding of calmodulin in the physiologically relevant range. We find that full-length calmodulin switches from a rich and complex folding behavior at high calcium to a simple folding pathway at apo conditions. Using truncation mutants, we studied the individual domains separately. Folding and stability of the individual domains differ significantly at low calcium concentrations. With increasing calcium, the folding rate constants increase while unfolding rate constants decrease. The complete kinetic as well as energetic behavior of both domains could be modeled using a calcium-dependent three-pathway model. We find that the dominant folding pathway at high calcium concentrations proceeds via a transition state capable of binding one calcium ion. The folding of calmodulin seems to be designed to occur fast robustly over a large range of calcium concentrations and hence energetic stabilities.

| S-EPMC3497792 | biostudies-literature

A universal transcription pause sequence is an element of initiation factor ?70-dependent pausing.

Project description:The Escherichia coli ?70 initiation factor is required for a post-initiation, promoter-proximal pause essential for regulation of lambdoid phage late gene expression; potentially, ?70 acts at other sites during transcription elongation as well. The pause is induced by ?70 binding to a repeat of the promoter -10 sequence. After ?70 binding, further RNA synthesis occurs as DNA is drawn (or 'scrunched') into the enzyme complex, presumably exactly as occurs during initial synthesis from the promoter; this synthesis then pauses at a defined site several nucleotides downstream from the active center position when ?70 first engages the -10 sequence repeat. We show that the actual pause site in the stabilized, scrunched complex is the 'elemental pause sequence' recognized from its frequent occurrence in the E. coli genome. ?70 binding and the elemental pause sequence together, but neither alone, produce a substantial transcription pause.

| S-EPMC5001585 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data