Single-Molecule Analysis of Replication Protein A–DNA Interactions

Bain, Fletcher E; Fischer, Laura A.; Chen, Ran; Wold, Marc S.

doi:10.1016/bs.mie.2017.11.016

Cited by 17 publications

(11 citation statements)

References 18 publications

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“…In all forms of life, coating and protecting exposed ssDNA from nucleases is essential (Oliveira, 2021). In Eukarya, RPA has been shown to be an abundant multi-domain heterotrimeric protein complex, which is essential to all DNA processing events in DNA replication, recombination and repair (Bain et al, 2018; Brosey et al, 2013; Dueva and Iliakis, 2020). Here, we explored the quaternary structure of PabRPA and the conformational changes that occur upon DNA-binding.…”

Section: Discussionmentioning

confidence: 99%

“…In all forms of life, single-stranded DNA-binding proteins (SSBs) are essential components of the DNA replication machinery (Bain et al, 2018; Oliveira, 2021; Taib et al, 2021). They play vital roles in nearly all aspects of DNA metabolism by protecting exposed single-stranded DNA (ssDNA) and acting as platforms onto which DNA-processing enzymes can assemble (Brosey et al, 2013; Marceau, 2012).…”

Section: Introductionmentioning

confidence: 99%

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DNA-binding mechanism and evolution of Replication Protein A

Madru

Martínez‐Carranza

Laurent

et al. 2022

Preprint

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Replication Protein A (RPA) is a heterotrimeric single stranded DNA-binding protein with essential roles in DNA replication, recombination and repair, in both eukaryotic and archaeal cells. By using an integrative approach that combines three crystal structures, four cryo-EM structures in complex with single-stranded DNA (ssDNA) of different lengths, we extensively characterized RPA from Pyrococcus abyssi in different states. These structures show two essential features conserved in eukaryotes: a trimeric core and a module that promotes cooperative binding to ssDNA, as well as a newly identified archaeal-specific domain. These structures reveal for the first time how ssDNA is handed over from one RPA complex to the other, and uncover an unanticipated mechanism of self-association on ssDNA tracts. This work constitutes a significant step forward in the molecular understanding of the structure and DNA-binding mechanism of RPA, with far-reaching implications for the evolution of this primordial replication factor in Archaea and Eukarya.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

DNA-binding mechanism and evolution of Replication Protein A

Madru

Martínez‐Carranza

Laurent

et al. 2022

Preprint

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“…al . for details; (Bain et al 2018)). The Cy3 fluorescence was excited using 532nm (Coherent, Compass 215M-50) laser, and Cy5 fluorescence was excited using 641nm (Coherent, Cube 1150205 / AD) lasers.…”

Section: Methodsmentioning

confidence: 99%

Generation of site-specifically labelled fluorescent human XPA to investigate DNA binding dynamics during nucleotide excision repair

Kuppa,

Corless,

Caldwell

et al. 2023

Preprint

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Nucleotide excision repair (NER) promotes genomic integrity by correcting bulky DNA adducts damage caused by external factors such as ultraviolet light. Defects in NER enzymes are associated with pathological conditions such as Xeroderma Pigmentosum, trichothiodystrophy, and Cockayne syndrome. A critical step in NER is the binding of the Xeroderma Pigmentosum group A protein (XPA) to the DNA adduct. To better capture the dynamics of XPA interactions with DNA during NER we have utilized the fluorescence enhancement through non-canonical amino acids (FEncAA) approach. 4-azido-L-phenylalanine (4AZP) was incorporated at Arg-153 in human XPA and conjugated to Cy3 using strain-promoted azide-alkyne cycloaddition. The resulting fluorescent human XPA protein (hXPACy3) shows no loss in DNA binding activity and generates a robust change in fluorescence upon binding to DNA. Here we describe methods to generate hXPACy3and detail experimental conditions required to stably maintain the protein during biochemical and biophysical studies.

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“…Yet, how RPA diffuses on long ssDNA and the underlying mechanistic basis are poorly understood. Single molecule measurements are ideally suited for highly dynamic proteins such as RPA (28,(38)(39)(40)(41)(42)(43)(44). Single molecule FRET (smFRET) (45) has been used to study RPA-ssDNA interactions and revealed the dynamic nature of RPA on ssDNA where each DBD can undergo microscopic association and dissociation while RPA is bound to the ssDNA (28,37,39,40,46) and RNA (40).…”

Section: Introductionmentioning

confidence: 99%

Rapid long-distance migration of RPA on single stranded DNA occurs through intersegmental transfer utilizing multivalent interactions

Pangeni,

Biswas,

Kaushik

et al. 2023

Preprint

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Replication Protein A (RPA) is a single stranded DNA (ssDNA) binding protein that coordinates diverse DNA metabolic processes including DNA replication, repair, and recombination. RPA is a heterotrimeric protein with six functional oligosaccharide/oligonucleotide (OB) domains and flexible linkers. Flexibility enables RPA to adopt multiple configurations and is thought to modulate its function. Here, using single molecule confocal fluorescence microscopy combined with optical tweezers and coarse-grained molecular dynamics simulations, we investigated the diffusional migration of single RPA molecules on ssDNA under tension. The diffusion coefficientDis the highest (20,000 nucleotides2/s) at 3 pN tension and in 100 mM KCl and markedly decreases when tension or salt concentration increases. We attribute the tension effect to intersegmental transfer which is hindered by DNA stretching and the salt effect to an increase in binding site size and interaction energy of RPA-ssDNA. Our integrative study allowed us to estimate the size and frequency of intersegmental transfer events that occur through transient bridging of distant sites on DNA by multiple binding sites on RPA. Interestingly, deletion of RPA trimeric core still allowed significant ssDNA binding although the reduced contact area made RPA 15-fold more mobile. Finally, we characterized the effect of RPA crowding on RPA migration. These findings reveal how the high affinity RPA-ssDNA interactions are remodeled to yield access, a key step in several DNA metabolic processes.SignificanceReplication Protein A (RPA) binds to the exposed single stranded DNA (ssDNA) during DNA metabolism. RPA dynamics are essential to reposition RPA on ssDNA and recruit downstream proteins at the bound site. Here in this work, we perform a detailed biophysical study on dynamics of yeast RPA on ssDNA. We show that RPA can diffuse on ssDNA and is affected by tension and salt. Our observations are best explained by the intersegmental transfer model where RPA can transiently bridge two distant DNA segments for its migration over long distances. We further dissect the contributions of the trimerization core of RPA and other adjacent RPA molecules on RPA migration. This study provides detailed experimental and computational insights into RPA dynamics on ssDNA.

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Single-Molecule Analysis of Replication Protein A–DNA Interactions

Cited by 17 publications

References 18 publications

DNA-binding mechanism and evolution of Replication Protein A

DNA-binding mechanism and evolution of Replication Protein A

Generation of site-specifically labelled fluorescent human XPA to investigate DNA binding dynamics during nucleotide excision repair

Rapid long-distance migration of RPA on single stranded DNA occurs through intersegmental transfer utilizing multivalent interactions

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