<b><i>Escherichia coli</i></b> Single-Stranded DNA-Binding Protein: NanoESI-MS Studies of Salt-Modulated Subunit Exchange and DNA Binding Transactions

Mason, Claire E; Jergic, Slobodan; Lo, Allen T.Y.; Wang, Yao; Dixon, Nicholas E.; Beck, Jennifer L.

doi:10.1007/s13361-012-0552-2

Cited by 38 publications

(56 citation statements)

References 65 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…These interactions are critical to cooperative binding of ssDNA as well as to SSB interactome function. In addition, and consistent with the work of others, the model proposes that the acidic tip is not involved in protein binding per se but is instead a regulatory element (Mason et al, 2013; Su et al, 2014). …”

Section: Introductionsupporting

confidence: 81%

“…Early biochemical experiments with C-terminally truncated E. coli SSB led to a model with the acidic tip bound to the OB-fold in the N-terminal domain (Curth et al, 1996; Kozlov et al, 2010; Mason et al, 2013; Williams et al, 1983). As ssDNA and C-peptide binding by the OB-fold are competitive, this model proposed that in solution, the C-termini of SSB are bound to the tetramer and are released upon ssDNA binding, making them available for interactions with target proteins.…”

Section: Introductionmentioning

confidence: 99%

“…Finally, it affects the stability of the SSB tetramer. In vitro in 10mM ammonium acetate, SSBΔC8 tetramers exchange subunits with 15 N-SSBΔC8 tetramers at a rate twice that of wild type (Mason et al, 2013). Collectively, these studies imply that the last eight residues of the C-terminus have a regulatory role in SSB function.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

The tale of SSB

Bianco

2017

Progress in Biophysics and Molecular Biology

116

View full text Add to dashboard Cite

The E.coli single stranded DNA binding protein (SSB) is essential to all aspects of DNA metabolism. Here, it has two seemingly disparate but equally important roles: it binds rapidly and cooperatively to single stranded DNA (ssDNA) and it binds to partner proteins that constitute the SSB interactome. These two roles are not disparate but are instead, intimately linked. A model is presented wherein the intrinsically disordered linker (IDL) is directly responsible for mediating protein-protein interactions. It does this by binding, via PXXP motifs, to the OB-fold (aka SH3 domain) of a nearby protein. When the nearby protein is another SSB tetramer, this leads to a highly efficient ssDNA binding reaction that rapidly and cooperatively covers and protects the exposed nucleic acid from degradation. Alternatively, when the nearby protein is a member of the SSB interactome, loading of the enzyme onto the DNA takes places.

show abstract

Section: Introductionsupporting

confidence: 81%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

The tale of SSB

Bianco

2017

Progress in Biophysics and Molecular Biology

116

View full text Add to dashboard Cite

show abstract

“…E. coli DNA replication proteins were produced as described previously: the β 2 sliding clamp (Oakley et al, 2003), SSB (Mason et al, 2013), the DnaB 6 (DnaC) 6 helicase–loader complex (Jergic et al, 2013), DnaG primase (Stamford et al, 1992), the Pol III τ 3 δδ’χψ clamp loader (Tanner et al, 2008) and Pol III αεθ core (Tanner et al, 2008). Highly purified E. coli Pol I and DNA ligase A were gifts of Yao Wang (Wang, 2015).…”

Section: Methodsmentioning

confidence: 99%

Single-molecule visualization of fast polymerase turnover in the bacterial replisome

Lewis

Spenkelink

Jergic

et al. 2017

eLife

Self Cite

117

153

View full text Add to dashboard Cite

The Escherichia coli DNA replication machinery has been used as a road map to uncover design rules that enable DNA duplication with high efficiency and fidelity. Although the enzymatic activities of the replicative DNA Pol III are well understood, its dynamics within the replisome are not. Here, we test the accepted view that the Pol III holoenzyme remains stably associated within the replisome. We use in vitro single-molecule assays with fluorescently labeled polymerases to demonstrate that the Pol III* complex (holoenzyme lacking the β2 sliding clamp), is rapidly exchanged during processive DNA replication. Nevertheless, the replisome is highly resistant to dilution in the absence of Pol III* in solution. We further show similar exchange in live cells containing labeled clamp loader and polymerase. These observations suggest a concentration-dependent exchange mechanism providing a balance between stability and plasticity, facilitating replacement of replisomal components dependent on their availability in the environment.DOI: http://dx.doi.org/10.7554/eLife.23932.001

show abstract

“…Previously published protocols were used to prepare gp4 (35), gp5/trx (36), gp2.5 (37), and E. coli SSB (38). Polymerase labeling reactions were performed according to the procedure described by Etson et al (39) with an excess of either Alexa Fluor 488 carboxylic acid succinimidyl ester (Invitrogen) or Cy5 NHS ester (Lumiprobe) for an hour at room temperature.…”

Section: Methodsmentioning

confidence: 99%

Single-molecule studies of polymerase dynamics and stoichiometry at the bacteriophage T7 replication machinery

Geertsema

Kulczyk

Richardson

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Replication of DNA plays a central role in transmitting hereditary information from cell to cell. To achieve reliable DNA replication, multiple proteins form a stable complex, known as the replisome, enabling them to act together in a highly coordinated fashion. Over the past decade, the roles of the various proteins within the replisome have been determined. Although many of their interactions have been characterized, it remains poorly understood how replication proteins enter and leave the replisome. In this study, we visualize fluorescently labeled bacteriophage T7 DNA polymerases within the replisome while we simultaneously observe the kinetics of the replication process. This combination of observables allows us to monitor both the activity and dynamics of individual polymerases during coordinated leading-and lagging-strand synthesis. Our data suggest that lagging-strand polymerases are exchanged at a frequency similar to that of Okazaki fragment synthesis and that two or more polymerases are present in the replisome during DNA replication. Our studies imply a highly dynamic picture of the replisome with lagging-strand DNA polymerases residing at the fork for the synthesis of only a few Okazaki fragments. Further, new lagging-strand polymerases are readily recruited from a pool of polymerases that are proximally bound to the replisome and continuously replenished from solution.polymerase exchange | single molecule | fluorescence microscopy T he organization of replisomes is highly conserved among various organisms (1), underlining the evolutionary importance of the replication machinery architecture. The bacteriophage T7 replication system offers an attractive model system to study the interplay between replication proteins because its replication machinery is relatively simple; a functional replisome can be reconstituted by just four purified proteins. Three of these proteins are encoded by the phage itself: helicase-primase (gp4), DNA polymerase (gp5), and single-stranded DNA (ssDNA) binding protein (gp2.5). A processivity factor for the gp5 polymerase, thioredoxin (trx), is provided by the host Escherichia coli.

show abstract

Escherichia coli Single-Stranded DNA-Binding Protein: NanoESI-MS Studies of Salt-Modulated Subunit Exchange and DNA Binding Transactions

Cited by 38 publications

References 65 publications

The tale of SSB

The tale of SSB

Single-molecule visualization of fast polymerase turnover in the bacterial replisome

Single-molecule studies of polymerase dynamics and stoichiometry at the bacteriophage T7 replication machinery

Contact Info

Product

Resources

About