Single-Molecule DNA Polymerase Dynamics at a Bacterial Replisome in Live Cells

Liao, Yi; Li, Yilai; Schroeder, Jeremy W.; Simmons, Lyle A.; Biteen, Julie S.

doi:10.1016/j.bpj.2016.11.006

Cited by 54 publications

(65 citation statements)

References 39 publications

(51 reference statements)

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“…In contrast to long-standing views that replisome architecture is static, single-molecule fluorescence experiments have documented dynamic exchange of components at physiologically relevant time scales in large multi-protein complexes across all domains of life (Geertsema et al, 2014;Liao et al). Our work provides the first direct evidence that Pols ε and δ are similarly exchanged from solution in a concentration-dependent manner during DNA synthesis.…”

Section: Discussionmentioning

confidence: 53%

Tunability of DNA polymerase stability during eukaryotic DNA replication

Lewis

Spenkelink

Schauer

et al. 2019

Preprint

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Structural and biochemical studies have revealed the basic principles of how the replisome duplicates genomic DNA, but little is known about its dynamics during DNA replication. We reconstitute the 34 proteins needed to form the S. cerevisiae replisome and show how changing local concentrations of the key DNA polymerases tunes the ability of the complex to efficiently recycle these proteins or to dynamically exchange them. Particularly, we demonstrate redundancy of the Pol α DNA polymerase activity in replication and show that Pol α primase and the lagging-strand Pol δ can be re-used within the replisome to support the synthesis of large numbers of Okazaki fragments. This unexpected malleability of the replisome might allow it to deal with barriers and resource challenges during replication of large genomes. Author contributions Conceptualization, J.S.L., L.M.S., G.D.S., M.E.O., A.M.v.O.; Formal analysis, J.S.L., L.M.S., S.H.M.; Funding acquisition, M.E.O., A.M.v.O.; Investigation, J.S.L., L.M.S., G.D.S., S.H.M., V.N., C.M., C.K.; Methodology, J.S.L., L.M.S., A.M.v.O.; Resources, G.D.S., O.Y., G.K.; Software, L.M.S.; Supervision, M.E.O., A.M.v.O.; Validation, J.S.L., L.M.S.; Visualization, J.S.L., L.M.S., A.M.v.O.; Writing -original draft, J.S.L., L.M.S., G.

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Section: Discussionmentioning

confidence: 53%

Tunability of DNA polymerase stability during eukaryotic DNA replication

Lewis

Spenkelink

Schauer

et al. 2019

Preprint

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“…Consequently, measurement of interactions that last longer than the photobleaching lifetime is impossible. Instead we imaged UvrA-YPet using an interval imaging strategy 10,22,27,28 (Fig. 2d) that elegantly deconvolutes the lifetime of the interaction of UvrA-YPet with DNA and the lifetime of the fluorescent probe.…”

Section: Interval Imaging Strategy To Measure Dna Binding Kineticsmentioning

confidence: 99%

Single-molecule live-cell imaging visualizes parallel pathways of prokaryotic nucleotide excision repair

Ghodke

Oijen

2019

Preprint

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In the model organism Escherichia coli, helix distorting lesions are recognized by the UvrAB damage surveillance complex in the global genomic nucleotide excision repair pathway (GGR). Alternately, RNA polymerases stalled or paused by lesions signal the presence of DNA damage in transcriptioncoupled nucleotide excision repair (TCR). Ultimately, damage recognition is mediated by UvrA, culminating in the loading of the damage verification enzyme UvrB. We set out to characterize the differences in the kinetics of damage recognition by UvrA complexes formed during GGR and TCR. We followed functional, fluorescently tagged UvrA molecules in live cells and measured their residence times in TCR-deficient or wild-type cells. We demonstrate that the lifetimes of UvrA in Mfd-dependent or Mfd-independent repair are similar in live cells, and are governed by UvrB. Here, we illustrate a non-perturbative, imaging-based approach to quantify the kinetic signatures of damage recognition enzymes participating in multiple pathways in cells. Across the various domains of life, the recognition and repair of bulky helix distorting lesions in chromosomal DNA is coordinated by nucleotide excision repair (NER) factors. Damage detection occurs in two stages: a dedicated set of damage surveillance enzymes (reviewed in ref. 1, 2, 3 ) (namely the prokaryotic UvrA, and the eukaryotic UV-DDB, XPC, XPA and homologs) constantly survey genomic DNA for lesions. Upon DNA damage recognition, these enzymes load specific factors (UvrB in prokaryotes, TFIIH and homologs in eukaryotes) that unwind the DNA and verify the location of the damage with nucleotide resolution (Fig. 1a) (reviewed in ref. 2, 3 ). Subsequently, specialized endonucleases (prokaryotic UvrC and homologs, and the eukaryotic XPF/XPG and homologs) are recruited to the site of the DNA, resulting in cleavage of the single-stranded DNA (ssDNA) patch containing the lesion (reviewed in ref. 2, 3 ).In all studied organisms, the recognition of DNA damage also occurs via the stalling of RNA polymerase at sites of lesions (reviewed in ref. 4 ). In this case, a transcription elongation complex that is unable to catalyse RNA primer extension manifests as an ultra-stable protein-DNA roadblock. Transcriptionrepair coupling factors such as the prokaryotic Mfd, and the eukaryotic homologs Rad26/CSB are dedicated factors that recognize these TECs and remodel them 5,6,7,8,9 . In prokaryotes, Mfd is recruited to the site of a failed TEC, and in turn it recruits the UvrA(B) protein (Fig. 1) 7,9,10,11 . Similarly, in eukaryotes, CSB is recruited to the site of a stalled RNAPII complex, and recruits the TFIIH complex 12 .Damage detection via elongating RNA polymerase is termed transcription-coupled repair (TCR), in contrast to the direct detection of lesions by the UvrAB damage sensor (global genomic repair, or GGR). Studies investigating the rate of repair during TCR vs. GGR, have reported an enhancement in the rate of removal of UV-induced lesions from the template strand in transcribed DNA compared to 14...

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“…Therefore, only up to three polymerases can be simultaneously bound to the clamp loader complex. Consistent with this stoichiometry, single‐molecule in vivo measurements have revealed three polymerases both in E. coli and B. subtilis . The additional polymerase may allow for alternating action of two polymerases on the lagging strand, providing alternative synchronization pathways.…”

Section: A Dynamic Interplay Drives Initiation Of Bacterial Dna Replimentioning

confidence: 63%

“…Nevertheless, there are still differing views on the polymerase stoichiometry and only two polymerases are used in many mechanistic studies . The large uncertainty in the in vivo estimates allows for the possibility of a mixture of two and three polymerase replisomes …”

Section: A Dynamic Interplay Drives Initiation Of Bacterial Dna Replimentioning

confidence: 99%

Noise in the Machine: Alternative Pathway Sampling is the Rule During DNA Replication

2017

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The astonishing efficiency and accuracy of DNA replication has long suggested that refined rules enforce a single highly reproducible sequence of molecular events during the process. This view was solidified by early demonstrations that DNA unwinding and synthesis are coupled within a stable molecular factory, known as the replisome, which consists of conserved components that each play unique and complementary roles. However, recent single-molecule observations of replisome dynamics have begun to challenge this view, revealing that replication may not be defined by a uniform sequence of events. Instead, multiple exchange pathways, pauses, and DNA loop types appear to dominate replisome function. These observations suggest we must rethink our fundamental assumptions and acknowledge that each replication cycle may involve sampling of alternative, sometimes parallel, pathways. Here, we review our current mechanistic understanding of DNA replication while highlighting findings that exemplify multi-pathway aspects of replisome function and considering the broader implications.

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Single-Molecule DNA Polymerase Dynamics at a Bacterial Replisome in Live Cells

Cited by 54 publications

References 39 publications

Tunability of DNA polymerase stability during eukaryotic DNA replication

Tunability of DNA polymerase stability during eukaryotic DNA replication

Single-molecule live-cell imaging visualizes parallel pathways of prokaryotic nucleotide excision repair

Noise in the Machine: Alternative Pathway Sampling is the Rule During DNA Replication

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