Quantifying the Assembly of Multicomponent Molecular Machines by Single-Molecule Total Internal Reflection Fluorescence Microscopy

Boehm, Elizabeth M.; Subramanyam, Shyamal; Ghoneim, Mohamed; Washington, M. Todd; Spies, Maria

doi:10.1016/bs.mie.2016.08.019

Cited by 19 publications

(28 citation statements)

References 84 publications

(144 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…For example, hybrid approaches, such as full ensemble methods that combine small-angle X-ray scattering data and molecular dynamics simulations, should be extremely useful in studying such systems [81]. Similarly, single-molecule total internal reflection fluorescence microscopy studies of the assembly and composition of multi-protein complexes should also prove useful [82]. Ultimately, these emerging technologies should provide important and exciting insights into these DNA damage bypass pathways and the maintenance of genome stability.…”

Section: Discussionmentioning

confidence: 99%

Control of DNA Damage Bypass by Ubiquitylation of PCNA

Ripley

Gildenberg

Washington

2020

Genes

View full text Add to dashboard Cite

DNA damage leads to genome instability by interfering with DNA replication. Cells possess several damage bypass pathways that mitigate the effects of DNA damage during replication. These pathways include translesion synthesis and template switching. These pathways are regulated largely through post-translational modifications of proliferating cell nuclear antigen (PCNA), an essential replication accessory factor. Mono-ubiquitylation of PCNA promotes translesion synthesis, and K63-linked poly-ubiquitylation promotes template switching. This article will discuss the mechanisms of how these post-translational modifications of PCNA control these bypass pathways from a structural and biochemical perspective. We will focus on the structure and function of the E3 ubiquitin ligases Rad18 and Rad5 that facilitate the mono-ubiquitylation and poly-ubiquitylation of PCNA, respectively. We conclude by reviewing alternative ideas about how these post-translational modifications of PCNA regulate the assembly of the multi-protein complexes that promote damage bypass pathways.

show abstract

Section: Discussionmentioning

confidence: 99%

Control of DNA Damage Bypass by Ubiquitylation of PCNA

Ripley

Gildenberg

Washington

2020

Genes

View full text Add to dashboard Cite

show abstract

“…2i, j). On shorter DNA lengths, both binding and dissociation phases are clearly observed for RPA-DBD-A MB543 ( providing quantifiable information on the formation and dissociation of the nucleoprotein complex 24 . Moreover, environmentally triggered fluorescence changes of the dye in single molecule trajectories can also report on the presence of conformational states in the dye-decorated protein 25 .…”

Section: Differential Effects Of Ssdna Length On Dbd Conformationsmentioning

confidence: 99%

Dynamics and Selective Remodeling of the DNA Binding Domains of RPA

Pokhrel

Caldwell

Corless

et al. 2018

Preprint

View full text Add to dashboard Cite

Replication protein A (RPA) coordinates important DNA metabolic events by stabilizing singlestrand DNA (ssDNA) intermediates, activating the DNA damage response, and handing off ssDNA to appropriate downstream players. Six DNA binding domains (DBDs) in RPA promote high affinity binding to ssDNA, but also allow RPA displacement by lower affinity proteins. We have made fluorescent versions of RPA and visualized the conformational dynamics of individual DBDs in the context of the full-length protein. We show that both DBD-A and DBD-D rapidly bind to and dissociate from ssDNA, while RPA as a whole remains bound to ssDNA.The recombination mediator protein Rad52 selectively modulates the dynamics of DBD-D. This demonstrates how RPA interacting proteins, with lower ssDNA binding affinity, can access the occluded ssDNA and remodel individual DBDs to replace RPA. Main Text:In every eukaryotic cell, RPA binds to transiently exposed ssDNA and serves as a hub protein to coordinate essential DNA metabolic processes including replication, recombination, repair, and telomere maintenance 1,2 . Cellular functions of RPA rely on its high affinity ssDNA binding, its ability to physically interact with over two dozen DNA processing enzymes, and to correctly position these enzymes on complex DNA structures. The precise mechanisms through which RPA functions in many contexts; and more importantly, how it differentiates between multiple DNA metabolic events (DNA replication, repair or recombination) is a long-standing puzzle 1,3 .RPA is heterotrimeric, flexible, and modular in structure. It is composed of three subunits:RPA70, RPA32 and RPA14 (Figs.1a, b). The subunits harbor six 3 oligonucleotide/oligosaccharide binding folds (OB-folds; labeled A through F). We refer to the DNA binding OB-folds as DNA binding domains (DBDs; Fig. 1a). RPA binds to ssDNA with high, sub-nanomolar affinity, but can be displaced by DNA binding proteins with much lower DNA binding affinity. Recent studies have suggested that the RPA-ssDNA complex is relatively dynamic 4,5,6, positing a selective dissociative mechanism where not all DBDs are stably bound to the DNA at the same time and microscopic dissociation of individual DNA binding domains occurs.In all existing models for RPA function, DBDs A and B are assigned as high affinity binding domains. Purified DBD-A, DBD-B and DBD-A/DBD-B constructs bind ssDNA with KD values 2 µM, 20 µM and 50 nM, respectively 7-9 . The trimerization core made up of DBD-C (RPA70), DBD-D (RPA32) and DBD-E (RPA14) is considered to have a weaker ssDNA binding affinity (Kd>5μM) 10 . Additionally, mutational analysis of individual aromatic residues that interact with the ssDNA in either DBD-C or DBD-D show minimal perturbations on ssDNA binding affinity 6 . Paradoxically, in the recently solved crystal structure of the RPA-ssDNA complex (Fig. 1b), the interactions of all four DBD's with ssDNA are similar with DBD-C having more contacts with ssDNA bases than DBD-A, DBD-B or DBD-D 11 . Thus, the exact nature of the contributions from...

show abstract

“…Analysis of the single-molecule data were carried out as outlined previously (75). Briefly, single-molecule trajectories were extracted using IDL software and selected for analysis based…”

Section: Analysis Of Single-molecule Tirf Imaging Datamentioning

confidence: 99%

The Tiam1 guanine nucleotide exchange factor is auto-inhibited by its pleckstrin homology coiled-coil extension domain

Xu¹,

Gakhar

Bain³

et al. 2017

Journal of Biological Chemistry

View full text Add to dashboard Cite

T-cell lymphoma invasion and metastasis 1 (Tiam1) is a Dbl-family guanine nucleotide exchange factor (GEF) that specifically activates the Rho-family GTPase Rac1 in response to upstream signals, thereby regulating cellular processes including cell adhesion and migration. Tiam1 contains multiple domains, including an N-terminal pleckstrin homology coiled-coiled extension (PH-CC-Ex) and catalytic Dbl homology and C-terminal pleckstrin homology (DH-PH) domain. Previous studies indicate that larger fragments of Tiam1, such as the region encompassing the N-terminal to C-terminal pleckstrin homology domains (PH-PH), are auto-inhibited. However, the domains in this region responsible for inhibition remain unknown. Here, we show that the PH-CC-Ex domain inhibits Tiam1 GEF activity by directly interacting with the catalytic DH-PH domain, preventing Rac1 binding and activation. Enzyme kinetics experiments suggested that Tiam1 is auto-inhibited through occlusion of the catalytic site rather than by allostery. Small angle X-ray scattering and ensemble modeling yielded models of the PH-PH fragment that indicate it is in equilibrium between "open" and "closed" conformational states. Finally, single-molecule experiments support a model in which conformational sampling between the open and closed states of Tiam1 contributes to Rac1 dissociation. Our results highlight the role of the PH-CC-Ex domain in Tiam1 GEF regulation and suggest a combinatorial model for GEF inhibition and activation of the Rac1 signaling pathway.

show abstract

Quantifying the Assembly of Multicomponent Molecular Machines by Single-Molecule Total Internal Reflection Fluorescence Microscopy

Cited by 19 publications

References 84 publications

Control of DNA Damage Bypass by Ubiquitylation of PCNA

Control of DNA Damage Bypass by Ubiquitylation of PCNA

Dynamics and Selective Remodeling of the DNA Binding Domains of RPA

The Tiam1 guanine nucleotide exchange factor is auto-inhibited by its pleckstrin homology coiled-coil extension domain

Contact Info

Product

Resources

About