Browsing by Author "Mattice, Jenna R."

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An Aurora B-RPA signaling axis secures chromosome segregation fidelity
(Springer Science and Business Media LLC, 2023-05) Roshan, Poonam; Kuppa, Sahiti; Mattice, Jenna R.; Kaushik, Vikas; Chadda, Rahul; Pokhrel, Nilisha; Tumala, Brunda R.; Biswas, Aparna; Bothner, Brian; Antony, Edwin; Origanti, Sofia
Errors in chromosome segregation underlie genomic instability associated with cancers. Resolution of replication and recombination intermediates and protection of vulnerable single-stranded DNA (ssDNA) intermediates during mitotic progression requires the ssDNA binding protein Replication Protein A (RPA). However, the mechanisms that regulate RPA specifically during unperturbed mitotic progression are poorly resolved. RPA is a heterotrimer composed of RPA70, RPA32 and RPA14 subunits and is predominantly regulated through hyperphosphorylation of RPA32 in response to DNA damage. Here, we have uncovered a mitosis-specific regulation of RPA by Aurora B kinase. Aurora B phosphorylates Ser-384 in the DNA binding domain B of the large RPA70 subunit and highlights a mode of regulation distinct from RPA32. Disruption of Ser-384 phosphorylation in RPA70 leads to defects in chromosome segregation with loss of viability and a feedback modulation of Aurora B activity. Phosphorylation at Ser-384 remodels the protein interaction domains of RPA. Furthermore, phosphorylation impairs RPA binding to DSS1 that likely suppresses homologous recombination during mitosis by preventing recruitment of DSS1-BRCA2 to exposed ssDNA. We showcase a critical Aurora B-RPA signaling axis in mitosis that is essential for maintaining genomic integrity.
The flexible N-terminus of BchL autoinhibits activity through interaction with its [4Fe-4S] cluster and released upon ATP binding
(Elsevier BV, 2021-01) Corless, Elliot I.; Muhammad Saad Imran, Syed; Watkins, Maxwell B.; Bacik, John-Paul; Mattice, Jenna R.; Patterson, Angela; Danyal, Karamatullah; Soffe, Mark; Kitelinger, Robert; Seefeldt, Lance C.; Origanti, Sofia; Bennett, Brian; Bothner, Brian; Ando, Nozomi; Antony, Edwin
A key step in bacteriochlorophyll biosynthesis is the reduction of protochlorophyllide to chlorophyllide, catalyzed by dark-operative protochlorophyllide oxidoreductase (DPOR). DPOR contains two [4Fe-4S]-containing component proteins (BchL and BchNB) that assemble upon ATP binding to BchL to coordinate electron transfer and protochlorophyllide reduction. But the precise nature of the ATP-induced conformational changes are poorly understood. We present a crystal structure of BchL in the nucleotide-free form where a conserved, flexible region in the N-terminus masks the [4Fe-4S] cluster at the docking interface between BchL and BchNB. Amino acid substitutions in this region produce a hyper-active enzyme complex, suggesting a role for the N-terminus in auto-inhibition. Hydrogen deuterium exchange mass spectrometry shows that ATP-binding to BchL produces specific conformational changes leading to release of the flexible N-terminus from the docking interface. The release also promotes changes within the local environment surrounding the [4Fe-4S] cluster and promotes BchL complex formation with BchNB. A key patch of amino acids, Asp-Phe-Asp (the ‘DFD patch’), situated at the mouth of the BchL ATP-binding pocket promotes inter-subunit cross stabilization of the two subunits. A linked BchL dimer with one defective ATP-binding site does not support protochlorophyllide reduction, illustrating nucleotide binding to both subunits as a prerequisite for the inter-subunit cross stabilization. The masking of the [4Fe-4S] cluster by the flexible N-terminal region and the associated inhibition of activity is a novel mechanism of regulation in metalloproteins. Such mechanisms are possibly an adaptation to the anaerobic nature of eubacterial cells with poor tolerance for oxygen.
Rtt105 regulates RPA function by configurationally stapling the flexible domains
(Springer Science and Business Media LLC, 2022-09) Kuppa, Sahiti; Deveryshetty, Jaigeeth; Chadda, Rahul; Mattice, Jenna R.; Pokhrel, Nilisha; Kaushik, Vikas; Patterson, Angela; Dhingra, Nalini; Pangeni, Sushil; Sadauskas, Marisa K.; Shiekh, Sajad; Balci, Hamza; Ha, Taekjip; Zhao, Xiaolan; Bothner, Brian; Antony, Edwin
Replication Protein A (RPA) is a heterotrimeric complex that binds to single-stranded DNA (ssDNA) and recruits over three dozen RPA-interacting proteins to coordinate multiple aspects of DNA metabolism including DNA replication, repair, and recombination. Rtt105 is a molecular chaperone that regulates nuclear localization of RPA. Here, we show that Rtt105 binds to multiple DNA binding and protein-interaction domains of RPA and configurationally staples the complex. In the absence of ssDNA, Rtt105 inhibits RPA binding to Rad52, thus preventing spurious binding to RPA-interacting proteins. When ssDNA is available, Rtt105 promotes formation of high-density RPA nucleoprotein filaments and dissociates during this process. Free Rtt105 further stabilizes the RPA-ssDNA filaments by inhibiting the facilitated exchange activity of RPA. Collectively, our data suggest that Rtt105 sequesters free RPA in the nucleus to prevent untimely binding to RPA-interacting proteins, while stabilizing RPA-ssDNA filaments at DNA lesion sites.
Yeast Rad52 is a homodecamer and possesses BRCA2-like bipartite Rad51 binding modes
(Springer Science and Business Media LLC, 2023-10) Deveryshetty, Jaigeeth; Chadda, Rahul; Mattice, Jenna R.; Karunakaran, Simrithaa; Rau, Michael J.; Basore, Katherine; Pokhrel, Nilisha; Englander, Noah; Fitzpatrick, James A. J.; Bothner, Brian; Antony, Edwin
Homologous recombination (HR) is an essential double-stranded DNA break repair pathway. In HR, Rad52 facilitates the formation of Rad51 nucleoprotein filaments on RPA-coated ssDNA. Here, we decipher how Rad52 functions using single-particle cryo-electron microscopy and biophysical approaches. We report that Rad52 is a homodecameric ring and each subunit possesses an ordered N-terminal and disordered C-terminal half. An intrinsic structural asymmetry is observed where a few of the C-terminal halves interact with the ordered ring. We describe two conserved charged patches in the C-terminal half that harbor Rad51 and RPA interacting motifs. Interactions between these patches regulate ssDNA binding. Surprisingly, Rad51 interacts with Rad52 at two different bindings sites: one within the positive patch in the disordered C-terminus and the other in the ordered ring. We propose that these features drive Rad51 nucleation onto a single position on the DNA to promote formation of uniform pre-synaptic Rad51 filaments in HR.