Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Single-molecule analysis of the Escherichia coli replisome and use of clamps to bypass replication barriers.

ABSTRACT: The process of chromosome duplication faces many obstacles. One way to circumvent blocks is to hop over them by placing a new clamp on a downstream primer. This resembles lagging strand synthesis, where the tight grip of polymerase to the clamp and DNA must be overcome upon completing each Okazaki fragment so it can transfer to new primed sites. This review focuses on recent single-molecule studies showing that Escherichia coli Pol III can hop from one clamp to another without leaving the replication fork. This capability provides a means to circumvent obstacles like transcription or DNA lesions without fork collapse.

SUBMITTER: Georgescu RE

PROVIDER: S-EPMC2919775 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Single-molecule analysis of the Escherichia coli replisome and use of clamps to bypass replication barriers.

Georgescu Roxana E RE Yao Nina Y NY O'Donnell Mike M

FEBS letters 20100411 12

The process of chromosome duplication faces many obstacles. One way to circumvent blocks is to hop over them by placing a new clamp on a downstream primer. This resembles lagging strand synthesis, where the tight grip of polymerase to the clamp and DNA must be overcome upon completing each Okazaki fragment so it can transfer to new primed sites. This review focuses on recent single-molecule studies showing that Escherichia coli Pol III can hop from one clamp to another without leaving the replic ...[more]

PMID: 20388515

Similar Datasets

Replisome dynamics and use of DNA trombone loops to bypass replication blocks.

Project description:Replisomes are dynamic multiprotein machines capable of simultaneously replicating both strands of the DNA duplex. This review focuses on the structure and function of the E. coli replisome, many features of which generalize to other bacteria and eukaryotic cells. For example, the bacterial replisome utilizes clamps and clamp loaders to coordinate the actions required of the trombone model of lagging strand synthesis made famous by Bruce Alberts. All cells contain clamps and clamp loaders and this review summarizes their structure and function. Clamp loaders are pentameric spirals that bind DNA in a structure specific fashion and thread it through the ring shaped clamp. The recent structure of the E. coli beta clamp in complex with primed DNA has implications for how multiple polymerases function on sliding clamps and how the primed DNA template is exchanged between them. Recent studies reveal a remarkable fluidity in replisome function that enables it to bypass template lesions on either DNA strand. During these processes the polymerases within the replisome functionally uncouple from one another. Mechanistic processes that underlie these actions may involve DNA looping, similar to the trombone loops that mediate the lagging strand Okazaki fragment synthesis cycle.

| S-EPMC4011192 | biostudies-literature

Single-molecule studies of fork dynamics in Escherichia coli DNA replication.

Project description:We present single-molecule studies of the Escherichia coli replication machinery. We visualize individual E. coli DNA polymerase III (Pol III) holoenzymes engaging in primer extension and leading-strand synthesis. When coupled to the replicative helicase DnaB, Pol III mediates leading-strand synthesis with a processivity of 10.5 kilobases (kb), eight-fold higher than that by Pol III alone. Addition of the primase DnaG causes a three-fold reduction in the processivity of leading-strand synthesis, an effect dependent upon the DnaB-DnaG protein-protein interaction rather than primase activity. A single-molecule analysis of the replication kinetics with varying DnaG concentrations indicates that a cooperative binding of two or three DnaG monomers to DnaB halts synthesis. Modulation of DnaB helicase activity through the interaction with DnaG suggests a mechanism that prevents leading-strand synthesis from outpacing lagging-strand synthesis during slow primer synthesis on the lagging strand.

| S-EPMC2651573 | biostudies-literature

Single-molecule visualization of stalled replication-fork rescue by the Escherichia coli Rep helicase.

Project description:Genome duplication occurs while the template DNA is bound by numerous DNA-binding proteins. Each of these proteins act as potential roadblocks to the replication fork and can have deleterious effects on cells. In Escherichia coli, these roadblocks are displaced by the accessory helicase Rep, a DNA translocase and helicase that interacts with the replisome. The mechanistic details underlying the coordination with replication and roadblock removal by Rep remain poorly understood. Through real-time fluorescence imaging of the DNA produced by individual E. coli replisomes and the simultaneous visualization of fluorescently-labeled Rep, we show that Rep continually surveils elongating replisomes. We found that this association of Rep with the replisome is stochastic and occurs independently of whether the fork is stalled or not. Further, we visualize the efficient rescue of stalled replication forks by directly imaging individual Rep molecules as they remove a model protein roadblock, dCas9, from the template DNA. Using roadblocks of varying DNA-binding stabilities, we conclude that continuation of synthesis is the rate-limiting step of stalled replication rescue.

| S-EPMC10123100 | biostudies-literature

Rescuing Replication from Barriers: Mechanistic Insights from Single-Molecule Studies.

Project description:To prevent replication failure due to fork barriers, several mechanisms have evolved to restart arrested forks independent of the origin of replication. Our understanding of these mechanisms that underlie replication reactivation has been aided through unique dynamic perspectives offered by single-molecule techniques. These techniques, such as optical tweezers, magnetic tweezers, and fluorescence-based methods, allow researchers to monitor the unwinding of DNA by helicase, nucleotide incorporation during polymerase synthesis, and replication fork progression in real time. In addition, they offer the ability to distinguish DNA intermediates after obstacles to replication at high spatial and temporal resolutions, providing new insights into the replication reactivation mechanisms. These and other highlights of single-molecule techniques and remarkable studies on the recovery of the replication fork from barriers will be discussed in this review.

| S-EPMC6497992 | biostudies-other

Probing DNA clamps with single-molecule force spectroscopy.

Project description:Detailed mechanisms of DNA clamps in prokaryotic and eukaryotic systems were investigated by probing their mechanics with single-molecule force spectroscopy. Specifically, the mechanical forces required for the Escherichia coli and Saccharomyces cerevisiae clamp opening were measured at the single-molecule level by optical tweezers. Steered molecular dynamics simulations further examined the forces involved in DNA clamp opening from the perspective of the interface binding energies associated with the clamp opening processes. In combination with additional molecular dynamics simulations, we identified the contact networks between the clamp subunits that contribute significantly to the interface stability of the S.cerevisiae and E. coli clamps. These studies provide a vivid picture of the mechanics and energy landscape of clamp opening and reveal how the prokaryotic and eukaryotic clamps function through different mechanisms.

| S-EPMC3763527 | biostudies-literature

Bypass of complex co-directional replication-transcription collisions by replisome skipping.

Project description:Collisions between the replisome and RNA polymerases [RNAP(s)] are the main obstacle to DNA replication. These collisions can occur either head-on or co-directionally with respect to the direction of translocation of both complexes. Whereas head-on collisions require additional factors to be resolved, co-directional collisions are thought to be overcome by the replisome itself using the mRNA transcript as a primer. We show that mRNA takeover is utilized primarily after collisions with single RNAP complexes with short transcripts. Bypass of more complex transcription complexes requires the synthesis of a new primer downstream of the RNAP for the replisome to resume leading-strand synthesis. In both cases, bypass proceeds with displacement of the RNAP. Rep, Mfd, UvrD and RNase H can process the RNAP block and facilitate replisome bypass by promoting the formation of continuous leading strands. Bypass of co-directional RNAP(s) and/or R-loops is determined largely by the length of the obstacle that the replisome needs to traverse: R-loops are about equally as potent obstacles as RNAP arrays if they occupy the same length of the DNA template.

| S-EPMC8464059 | biostudies-literature

Single-molecule mapping of replisome progression

Project description:Replicon-seq is a method to study the progression of sister replisomes during DNA replication. This method relies excision of the full-length of replicons by the fusion of MNase to MCM4 and sequencing via Nanopore technology.

2022-01-26 | GSE194025 | GEO

Single-molecule analysis reveals that the lagging strand increases replisome processivity but slows replication fork progression.

Project description:Single-molecule techniques are developed to examine mechanistic features of individual E. coli replisomes during synthesis of long DNA molecules. We find that single replisomes exhibit constant rates of fork movement, but the rates of different replisomes vary over a surprisingly wide range. Interestingly, lagging strand synthesis decreases the rate of the leading strand, suggesting that lagging strand operations exert a drag on replication fork progression. The opposite is true for processivity. The lagging strand significantly increases the processivity of the replisome, possibly reflecting the increased grip to DNA provided by 2 DNA polymerases anchored to sliding clamps on both the leading and lagging strands.

| S-EPMC2726342 | biostudies-literature

MutL sliding clamps coordinate exonuclease-independent Escherichia coli mismatch repair.

Project description:A shared paradigm of mismatch repair (MMR) across biology depicts extensive exonuclease-driven strand-specific excision that begins at a distant single-stranded DNA (ssDNA) break and proceeds back past the mismatched nucleotides. Historical reconstitution studies concluded that Escherichia coli (Ec) MMR employed EcMutS, EcMutL, EcMutH, EcUvrD, EcSSB and one of four ssDNA exonucleases to accomplish excision. Recent single-molecule images demonstrated that EcMutS and EcMutL formed cascading sliding clamps on a mismatched DNA that together assisted EcMutH in introducing ssDNA breaks at distant newly replicated GATC sites. Here we visualize the complete strand-specific excision process and find that long-lived EcMutL sliding clamps capture EcUvrD helicase near the ssDNA break, significantly increasing its unwinding processivity. EcSSB modulates the EcMutL-EcUvrD unwinding dynamics, which is rarely accompanied by extensive ssDNA exonuclease digestion. Together these observations are consistent with an exonuclease-independent MMR strand excision mechanism that relies on EcMutL-EcUvrD helicase-driven displacement of ssDNA segments between adjacent EcMutH-GATC incisions.

| S-EPMC6876574 | biostudies-literature

The Escherichia coli replisome is inherently DNA damage tolerant.

Project description:The Escherichia coli DNA replication machinery must frequently overcome template lesions under normal growth conditions. Yet, the outcome of a collision between the replisome and a leading-strand template lesion remains poorly understood. Here, we demonstrate that a single, site-specific, cyclobutane pyrimidine dimer leading-strand template lesion provides only a transient block to fork progression in vitro. The replisome remains stably associated with the fork after collision with the lesion. Leading-strand synthesis is then reinitiated downstream of the damage in a reaction that is dependent on the primase, DnaG, but independent of any of the known replication-restart proteins. These observations reveal that the replisome can tolerate leading-strand template lesions without dissociating by synthesizing the leading strand discontinuously.

| S-EPMC3593629 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

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