Replication Protein A (RPA) and the coordination of DNA metabolism
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Select Recent Publications
Roshan et. al. Mechanism of RPA phosphocode priming and tuning by Cdk1/Wee1 signaling circuit. Nature Communications. 2026 Chadda et. al. Partial wrapping of single-stranded DNA by Replication Protein A and modulation through phosphorylation. Nucleic Acids Research. 2024 Roshan et. al. An Aurora B-RPA signaling axis secures chromosome segregation fidelity. Nature Communications. 2023 |
DNA metabolic processes including replication, repair, recombination, and telomere maintenance are enacted through single-stranded DNA (ssDNA) intermediates. In each of these complex processes, dozens of proteins function together on the ssDNA. 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 processes must first remodel/remove RPA, or function alongside, to access the ssDNA occluded by 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. The precise mechanisms of how RPA imparts functional specificity is poorly resolved. Our long-term goals are to answer the following questions:
- RPA physically interacts with over three dozen DNA processing enzymes. How are these interactions determined, regulated, and prioritized?
- RPA binds to ssDNA with high affinity. How do DNA metabolic enzymes that bind to ssDNA with hundred-fold lower affinities remove RPA?
- RPA plays a role in positioning the recruited enzymes (with appropriate polarity) onto the DNA. What are the structural, kinetic, and thermodynamic properties that regulate this process?
- How are the DNA and protein interaction activities of RPA tuned by post translational modifications such as phosphorylation?
Homologous recombination and maintenance of genome integrity and stability
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Deveryshetty et. al. Rad52 sorts and stacks Rad51 at the DNA junction to promote homologous recombination. Nature Communications. 2025 Deveryshetty et. al. C-terminus induced asymmetry within a Rad52 homodecamer dictates single-position Rad51 nucleation in homologous recombination. Nature Communications. 2023 Kuppa et. al. Rtt105 configurationally staples RPA and blocks facilitated exchange and interactions with RPA-interacting proteins. Nature Communications. 2022 |
ouble-stranded DNA breaks (DSBs) are drivers of genomic instability and associated cancers. On average, ~10 to 50 DSBs occur per cell per day and thus pose the highest risk to genomic integrity. Homologous Recombination (HR), single strand annealing (SSA), and RNA-templated DNA repair (RDR) are key biological processes that protect the cell from DSBs. The fundamental problem with a DSB is that physical continuity is broken within a chromosome. Thus, for accurate repair, the undamaged sister allele is used as a template to copy genetic information. Homology directed DSB repair transpires through either Rad51-dependent HR or Rad51-independent SSA pathways. For both processes, the DSB must first be processed (resected) to generate 3′ single strand DNA (ssDNA) tails which are coated by the ssDNA binding Replication Protein A (RPA). Binding of RPA triggers activation of the ATR kinase and associated repair signaling mechanisms. In HR, mediator proteins such as Rad52 and BRCA2 promote loading of the Rad51 protein (recombinase) onto RPA-coated ssDNA. The Rad51-coated 3′-ssDNA filament invades the undamaged sister chromatid and base pairs with the complementary region, which then is used as a template to replicate the missing information. In certain cases, when the 3′-ssDNA overhangs contain highly homologous direct repeats, or during replication stress in common fragile sites, SSA is triggered. Here, Rad52 promotes the direct annealing of the direct repeats between the two 3′-ssDNA overhangs. The non-annealed 3′-flaps are excised, and the resulting structures are subsequently processed by DNA repair synthesis. While HR is an accurate process for DSB repair, SSA is relatively error prone. Finally, RNA can serve as a template for DSB repair where R-loop intermediates (RNA-DNA hybrids) can be used as substrate to copy the missing information. While decades of efforts have identified the proteins involved in DSB repair, how they assemble together to enact the complex steps, and their regulation, remains poorly resolved. Utilizing biophysical, mass-spectrometry, single-molecule fluorescence, and cryoEM approaches we seek to address the following questions:
- How do mediator proteins (Rad52 and BRCA2) promote Rad51 filament formation during HR?
- How do Rad51-paralogs regulate Rad51 filament formation and stability?
- How do anti-HR mediators remove Rad51 filaments?
Mechanisms of long-range electron transfer in oxidoreductases
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Select Recent Publications
Kashyap et. al. Cryo-EM structure of a methanogen nitrogenase-PII protein supercomplex. BioRxiv. 2025 Kashyap et. al. Cryo-EM captures the coordination of asymmetric electron transfer through a bi-copper cluster in DPOR. Nature Communications. 2025 Corless et. al. The flexible N-terminus of BchL autoinhibits activity through interaction with its [4Fe-4S] and relieved upon ATP binding. Journal of Biological Chemistry. 2020 |
Redox chemistry is characterized by the addition/removal of electrons and is a characteristic of most reactions that occur in nature and in living organisms. Processes such as nitrogen fixation and photosynthesis are essential to sustaining all life on earth. Our research focus are on nitrogenase and nitrogenase-like enzymes that catalyze ATP-dependent multi-electron substrate reduction reactions. The architecture of these enzymes and how they mediate long-range electron transfer (ET) over a series of sophisticated metal centers have served as blueprints for the design of bio-inspired catalysts. Nitrogenase fixes atmospheric dinitrogen in the biological nitrogen cycle, and two nitrogenase-like enzymes - DPOR (Dark-operative Protochlorophyllide Oxidoreductase) and COR (Chlorophyllide a Oxidoreductase) catalyze key reduction reactions in the ‘dark’ chlorophyll biosynthetic pathway and are essential for photosynthesis. In addition to their significance in life-sustaining fundamental biological processes, DPOR, COR, and nitrogenase serve as excellent model systems to decipher complex long-range electron transfer processes in biology. These enzymes are made of homodimeric electron donor and heterotetrameric electron acceptor component proteins that transiently assemble in the presence of ATP to perform electron transfer. The heterotetrameric acceptor component possesses two identical active sites. We uncovered that the two halves in nitrogenase and DPOR function in an asymmetric manner with catalytic events in one half allosterically controlling the other. Perturbing activities in one half stalls substrate reduction in the complex. The functional significance of this asymmetry and the mechanistic basis of allosteric control are not understood. We seek to decipher the following:
- What is the role for structural and functional allostery in these enzymes?
- We discovered a new di-Cu center in the DPOR enzyme. What is the role of this metal center in enzyme function and redox catalysis?
- We discovered that nitrogenase in Methanosarcina acetivorans is a supercomplex. How does this higher order architecture regulate N2 fixation in these archaea?
- How do the component proteins assemble and how is electron transfer coupled to ATP binding and hydrolysis?
- How are the substrate and product differentiated, and how is allostery propagated within these large enzyme complexes?
mRNA recognition by the oncofetal IMP family of RNA binding proteins
RNA binding proteins recognize hundreds of mRNA targets and regulate their expression through splicing, end processing, nuclear export, translational control, and altering mRNA stability. Most such proteins lack enzymatic activity but serve as a scaffold for the recruitment of other proteins onto the mRNA. The assembly of such complexes, on the appropriate mRNA target, confer specificity to accurate and rapid gene expression demands within the cell. A small number of RNA binding proteins control the expression of thousands of mRNAs, and our long-term goal is to decipher how these proteins gain specificity for a defined mRNA target. In other words, how do these proteins pick out one mRNA from thousands of potential mRNA substrates? We address this question in the IMP family of mRNA binding proteins. Insulin-like growth factor 2 mRNA binding proteins (IMPs) is a family of three paralogs (IMP1, IMP2 and IMP3) and functions in normal development, stem cell maintenance, and when deregulated leads to cancers. These proteins use six high affinity RNA binding domains tethered by flexible linkers, thus making them challenging to investigate through traditional biochemical approaches. Our work aims to:
- Obtain structural and mechanistic knowledge of how these enzymes function.
- Probe how each member contributes to embryonic development.
- Why do mutations and re-expression lead to tumor development.
- Develop small molecule inhibitors for chemotherapy.