Biology subjects

Mattice, J.

Publications and source records attributed to Mattice, J..

6 recordsLinked to original sources

bioRxiv2026

A Phosphorylation Switch Modulates Configurational Codes in the Oncofetal IGF2BP RNA Binding Paralogs

The insulin like growth factor 2 mRNA binding proteins (IGF2BP1-3) are oncofetal RNA regulators that control translation, stability, and localization of several transcripts, yet display paralog specific functions despite high structural similarity. Each paralog contains six RNA binding domains (two RRMs and four KH domains) linked by intrinsically disordered segments. mTORC2 phosphorylates IGF2BP1 and IGF2BP3 at a single conserved serine within the disordered linker between the RRM2 and KH1 domains, a modification required for proper regulation of mRNA translational fate. Pairing site specific phosphoserine incorporation with structural and biophysical interrogations, we show that this phosphorylation acts as a configurational switch that reorganizes long range arrangements of RNA binding domains and linkers without altering the secondary structure, and with only modest effects on RNA binding affinity. Critically, pSer driven rearrangements occur both in the RNA free state and upon RNA engagement, and the resulting architectures differ markedly between IGF2BP1 and IGF2BP3 despite >70% sequence identity. These paralog specific, phosphorylation dependent configurational landscapes likely underlie differences in mRNA recognition modes and functional outcomes. Our work identifies a post translational mechanism that tunes IGF2BP paralog dynamics across free and RNA bound states to program target mRNA selection, processing, and translational fate.

biochemistry↗

bioRxiv2023

C-terminus induced asymmetry within a Rad52 homodecamer dictates single-position Rad51 nucleation in homologous recombination

Homologous recombination (HR) is a pathway for the accurate repair of double-stranded DNA breaks. These breaks are resected to yield single-stranded DNA (ssDNA) that are coated by Replication Protein A (RPA). Saccharomyces cerevisiae Rad52 is a mediator protein that promotes HR by facilitating formation of Rad51 nucleoprotein filaments on RPA-coated ssDNA. Canonically, Rad52 has been described to function by displacing RPA to promote Rad51 binding. However, in vitro, Rad51 readily forms a filament by displacing RPA in the absence of Rad52. Yet, in vivo, Rad52 is essential for HR. Here, we resolve how Rad52 functions as a mediator using single-particle cryo-electron microscopy and biophysical approaches. We show that Rad52 functions as a homodecamer and catalyzes single-position nucleation of Rad51. The N-terminal half of Rad52 is a well-ordered ring, while the C-terminal half is disordered. An intrinsic asymmetry within Rad52 is observed, where one or a few of the C-terminal halves interact with the ordered N-terminal ring. Within the C-terminal half, we identify two conserved charged patches that harbor the Rad51 and RPA interacting motifs. Interactions between these two charged patches regulate a ssDNA binding. These features drive Rad51 binding to a single position on the Rad52 decameric ring. We propose a Rad52 catalyzed single-position nucleation model for the formation of pre-synaptic Rad51 filaments in HR.

biochemistry↗

bioRxiv2022

An Aurora B-RPA signaling axis secures chromosome segregation fidelity

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 phosphorylation markedly affects cell viability and causes defects in chromosome segregation with hypersensitivity to DNA damaging agents. Phosphorylation at Ser-384 remodels the protein interaction domains of RPA and facilitates formation of higher density filaments. 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 RPA-Aurora B signaling axis in mitosis that is essential for maintaining genomic integrity.

biochemistry↗

bioRxiv2022

Dynamic states of eIF6 and SDS variants modulate interactions with uL14 of the 60S ribosomal subunit.

Assembly of ribosomal subunits into active ribosomal complexes is integral to protein synthesis. Release of eIF6 from the 60S ribosomal subunit primes 60S to associate with the 40S subunit and engage in translation. The dynamics of eIF6 interaction with the uL14 (RPL23) interface of 60S and its regulation by somatic mutations acquired in Shwachman-Diamond Syndrome (SDS) is yet to be clearly understood. Here, by using a modified strategy to obtain high yields of recombinant human eIF6 we have uncovered the critical interface entailing 8 key residues in the C-tail of RPL23 that is essential for physical interactions between 60S and eIF6. Disruption of the complementary binding interface by conformational changes in eIF6 disease variants provide a mechanism for weakened interactions of variants with the 60S. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) analyses uncovered dynamic configurational rearrangements in eIF6 induced by binding to RPL23 and exposed an allosteric interface regulated by the C-tail of eIF6. Disrupting a key residue in the eIF6-60S binding interface markedly limits proliferation of cancer cells, which highlights the significance of therapeutically targeting this interface. Establishing these key interfaces thus provide a therapeutic framework for targeting eIF6 in cancers and SDS.

biochemistry↗

bioRxiv2022

Rtt105 configurationally staples RPA and blocks facilitated exchange and interactions with RPA-interacting proteins

Replication Protein A (RPA) binds to single-stranded DNA (ssDNA) and recruits over three dozen RPA-interacting proteins (RIPs) to coordinate multiple aspects of DNA metabolism including DNA replication, repair, and recombination. Rtt105 is a molecular chaperone that regulates nuclear localization of RPA. Whether and how Rtt105 regulates the activities of RPA is poorly understood. 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 (RIPs). 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 RPA facilitated exchange. Collectively, our data suggest that Rtt105 sequesters free RPA in the nucleus to prevent untimely RIP interaction, while stabilizing RPA-ssDNA filaments at DNA lesion sites.

biochemistry↗

bioRxiv2020

Hydrogen-deuterium exchange reveals a dynamic DNA binding map of Replication Protein A

Replication Protein A (RPA) binds to single-stranded DNA (ssDNA) and interacts with over three dozen enzymes and serves as a recruitment hub to coordinate most DNA metabolic processes including DNA replication, repair, and recombination. RPA binds ssDNA utilizing six oligosaccharide/oligonucleotide binding (OB) domains within a heterotrimeric complex of RPA70, RPA32 and RPA14 subunits. Based on their DNA binding affinities they are classified as high versus low-affinity DNA binding domains (DBDs). However, recent evidence suggests that the DNA-binding dynamics of DBDs better define their roles. Utilizing hydrogen-deuterium exchange mass spectrometry (HDX-MS) we assessed the contacts and dynamics of the individual domains of human RPA to determine the landscape of conformational changes upon binding to ssDNA. As expected, ssDNA interacts with the major DBDs (A, B, C, and D). However, DBD-A and DBD-B are dynamic and do not show robust DNA-dependent protection. DBD-C displays the most extensive changes in HDX, suggesting a major role in stabilizing RPA on ssDNA. DNA-dependent HDX kinetics are also captured for DBD-D and DBD-E. Slower allosteric changes transpire in the protein-protein interaction domains and the linker regions. We propose a dynamics-based DNA binding model for RPA utilizing a dynamic half and a less-dynamic half.

biochemistry↗