An Aurora B-RPA signaling axis secures chromosome segregation fidelity

Roshan, Poonam; Kuppa, Sahiti; Mattice, Jenna; Kaushik, Vikas; Chadda, Rahul; Pokhrel, Nilisha; Tumala, Brunda; Biswas, Aparna; Bothner, Brian; Antony, Edwin; Origanti, Sofia

doi:10.1038/s41467-023-38711-2

Cited by 4 publications

(7 citation statements)

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“…We have used such FRET experiments to monitor assembly and disassembly of RPA on longer DNA substrates. 41,42 Such experiments would be powerful to investigate the role of post-translational modifications or mutations in RPA and to assess the role of RPA-interacting proteins in remodeling RPA.…”

Section: Resultsmentioning

confidence: 99%

Fluorescent human RPA to track assembly dynamics on DNA

Kaushik,

Chadda,

Kuppa

et al. 2023

Preprint

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DNA metabolic processes including replication, repair, recombination, and telomere maintenance occur on single-stranded DNA (ssDNA). In each of these complex processes, dozens of proteins function together on the ssDNA template. However, when double-stranded DNA is unwound, the transiently open ssDNA is protected and coated by the high affinity heterotrimeric ssDNA binding Replication Protein A (RPA). Almost all downstream DNA processes must first remodel/remove RPA or function alongside to access the ssDNA occluded under RPA. Formation of RPA-ssDNA complexes trigger the DNA damage checkpoint response and is a key step in activating most DNA repair and recombination pathways. Thus, in addition to protecting the exposed ssDNA, RPA functions as a gatekeeper to define functional specificity in DNA maintenance and genomic integrity. RPA achieves functional dexterity through a multi-domain architecture utilizing several DNA binding and protein-interaction domains connected by flexible linkers. This flexible and modular architecture enables RPA to adopt a myriad of configurations tailored for specific DNA metabolic roles. To experimentally capture the dynamics of the domains of RPA upon binding to ssDNA and interacting proteins we here describe the generation of active site-specific fluorescent versions of human RPA (RPA) using 4-azido-L-phenylalanine (4AZP) incorporation and click chemistry. This approach can also be applied to site-specific modifications of other multi-domain proteins. Fluorescence-enhancement through non-canonical amino acids (FEncAA) and Förster Resonance Energy Transfer (FRET) assays for measuring dynamics of RPA on DNA are also described.HighlightsRPA is an essential protein for most DNA metabolic processes including replication, repair, and recombination.RPA is a ssDNA binding protein made of six domains situated across RPA70, RPA32 and RPA14 subunits. Four high affinity DNA binding domains engage the DNA.Site-specific fluorescent probes were incorporated into two domains of RPA and report on ssDNA binding dynamics.Bulk-level kinetic and single-molecule assays are described to monitor the binding and remodeling of individual RPA domains on ssDNA.

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Section: Resultsmentioning

confidence: 99%

Fluorescent human RPA to track assembly dynamics on DNA

Kaushik,

Chadda,

Kuppa

et al. 2023

Preprint

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“…There are two important features that need to be considered: The first is the intrinsic dynamic interactions between the DBDs and ssDNA, and the second is the configurational rearrangements of the domains with respect to each other and the ssDNA. We, and others, have shown that the domains possess different dynamic properties, with DBD-A and DBD-B being more dynamic on ssDNA compared to DBD-C and DBD-D (19,(22)(23)(24). Using C-trap experiments, we recently showed that the rates of diffusion for RPA on long stretches of ssDNA are regulated by the trimerization core (41).…”

Section: Post-translational Modification Of Human Rpa By Aurora Kinas...mentioning

confidence: 99%

“…Thus, incoming proteins can access the buried ssDNA through transient dissociation of one or more DBDs. The second mode of ssDNA access might be provided through post-translational modifications of the DBDs or the disordered linkers (22,24). Using hydrogen-deuterium exchange mass spectrometry, we recently showed that the domains of RPA are not splayed apart but are tightly organized along with the protein-interaction domains and this configuration is altered upon phosphorylation by Aurora kinase B (22).…”

Section: Post-translational Modification Of Human Rpa By Aurora Kinas...mentioning

confidence: 99%

“…Aurora kinase B phosphorylates RPA at a single Ser-384 position in the large RPA70 subunit (22). Using genetic code expansion (33,34) we generated site-specific phospho-serine (pSer) modified RPA at Ser-384 (RPA-pSer 384 ).…”

Section: Post-translational Modification Of Human Rpa By Aurora Kinas...mentioning

confidence: 99%

“…OB-F and wh coordinate protein-protein interactions and are called protein-interaction domains (PIDs). Dynamic rearrangements of the DBDs and PIDs are observed upon binding to DNA and in response to post-translational modifications (22,23). Each DBD exhibits a moderate affinity for ssDNA binding.…”

Section: Introductionmentioning

confidence: 99%

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Wrapping of single-stranded DNA by Replication Protein A and modulation through phosphorylation

Chadda,

Kaushik,

Ahmad

et al. 2024

Preprint

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Single-stranded DNA (ssDNA) intermediates, which emerge during DNA metabolic processes are shielded by Replication Protein A (RPA). RPA binds to ssDNA and acts as a gatekeeper, directing the ssDNA towards downstream DNA metabolic pathways with exceptional specificity. Understanding the mechanistic basis for such RPA-dependent specificity requires a comprehensive understanding of the structural conformation of ssDNA when bound to RPA. Previous studies suggested a stretching of ssDNA by RPA. However, structural investigations uncovered a partial wrapping of ssDNA around RPA. Therefore, to reconcile the models, in this study, we measured the end-to-end distances of free ssDNA and RPA-ssDNA complexes using single-molecule FRET and Double Electron-Electron Resonance (DEER) spectroscopy and found only a small systematic increase in the end-to-end distance of ssDNA upon RPA binding. This change does not align with a linear stretching model but rather supports partial wrapping of ssDNA around the contour of DNA binding domains of RPA. Furthermore, we reveal how phosphorylation at the key Ser-384 site in the RPA70 subunit provides access to the wrapped ssDNA by remodeling the DNA-binding domains. These findings establish a precise structural model for RPA-bound ssDNA, providing valuable insights into how RPA facilitates the remodeling of ssDNA for subsequent downstream processes.

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