Biology subjects

Antony, E.

Publications and source records attributed to Antony, E..

30 records · Page 2Linked to original sources

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

Mechanistic Insight into AP-Endonuclease 1 Cleavage of Abasic Sites at Stalled Replication Forks

1.Many types of DNA damage stall replication fork progression, including abasic sites. AP-Endonuclease 1 (APE1) has been shown to cleave abasic sites in ssDNA substrates. Importantly, APE1 cleavage of ssDNA at a replication fork has significant biological implications by generating double strand breaks that could collapse the replication fork. Despite this, the molecular basis and efficiency of APE1 processing abasic sites at a replication fork remains elusive. Here, we investigate APE1 cleavage of several abasic substrates that mimic potential APE1 interactions at replication forks. We determine that APE1 has robust activity on these substrates, similar to dsDNA, and report rapid rates for cleavage and product release. X-ray crystal structures visualize the APE1 active site, highlighting that a similar mechanism is used to process ssDNA substrates as canonical APE1 activity on dsDNA. However, mutational analysis reveals R177 to be uniquely critical for the APE1 ssDNA cleavage mechanism. Additionally, we investigate the interplay between APE1 and Replication Protein A (RPA), the major ssDNA-binding protein at replication forks, revealing that APE1 can cleave an abasic site while RPA is still bound to the DNA substrate. Together, this work provides molecular level insights into abasic ssDNA processing by APE1, including the presence of RPA.

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↗

bioRxiv2021

A-type lamins are critical for the recruitment of RPA and RAD51 to stalled replication forks to maintain fork stability

Lamins provide a nuclear scaffold for compartmentalization of genome function that is important for genome integrity. The mechanisms whereby lamins regulate genome stability remain poorly understood. Here, we demonstrate a crucial role for A-type lamins preserving the integrity of the replication fork (RF) during replication stress (RS). We find that lamins bind to nascent DNA strands, especially during RS, and ensure the recruitment of fork protective factors RPA and RAD51. These ssDNA-binding proteins, considered the first and second responders to RS respectively, play crucial roles in the stabilization, remodeling and repair of the stalled fork to ensure proper restart and genome stability. Reduced recruitment of RPA and RAD51 upon lamins depletion elicits replication fork instability (RFI) depicted by MRE11 nuclease-mediated degradation of nascent DNA, RS-induced accumulation of DNA damage, and increased sensitivity to replication inhibitors. Importantly, in contrast to cells deficient in various homology recombination repair proteins, the RFI phenotype of lamins-depleted cells is not linked to RF reversal. This suggests that the point of entry of nucleases is not the reversed fork, but regions of ssDNA generated during RS that are not protected by RPA and RAD51. Consistently, RFI in lamins-depleted cells is rescued by forced elevation of the heterotrimeric RPA complex or RAD51. These data unveil a clear involvement of structural nuclear proteins in the protection of ssDNA from the action of nucleases during RS by warranting proper recruitment of ssDNA binding proteins RPA and RAD51 to stalled RFs. In support of this model, we show physical interaction between RPA and lamins. Our study also suggests that RS is a major source of genomic instability in laminopathies and in lamins-depleted tumors.

cancer biology↗

bioRxiv2021

Human HELB is a processive motor protein which catalyses RPA clearance from single-stranded DNA

Human HELB is a poorly-characterised helicase suggested to play both positive and negative regulatory roles in DNA replication and recombination. In this work, we used bulk and single molecule approaches to characterise the biochemical activities of HELB protein with a particular focus on its interactions with RPA and RPA-ssDNA filaments. HELB is a monomeric protein which binds tightly to ssDNA with a site size of [~]20 nucleotides. It couples ATP hydrolysis to translocation along ssDNA in the 5'-to-3' direction accompanied by the formation of DNA loops and with an efficiency of 1 ATP per base. HELB also displays classical helicase activity but this is very weak in the absence of an assisting force. HELB binds specifically to human RPA which enhances its ATPase and ssDNA translocase activities but inhibits DNA unwinding. Direct observation of HELB on RPA nucleoprotein filaments shows that translocating HELB concomitantly clears RPA from single-stranded DNA.

molecular biology↗

bioRxiv2021

Genetic Incorporation of Two Mutually Orthogonal Bioorthogonal Amino Acids That Enable Efficient Protein Dual-Labeling in Cells

The ability to site-specifically modify proteins at multiple sites in vivo will enable the study of protein function in its native environment with unprecedented levels of detail. Here, we present a versatile two-step strategy to meet this goal involving site-specific encoding of two distinct noncanonical amino acids bearing bioorthogonal handles into proteins in vivo followed by mutually orthogonal labeling. This general approach, that we call dual encoding and labeling (DEAL), allowed us to efficiently encoded tetrazine- and azide-bearing amino acids into a protein and demonstrate for the first time that the bioorthogonal labeling reactions with strained alkene and alkyne labels can function simultaneously and intracellularly with high yields when site-specifically encoded in a single protein. Using our DEAL system, we were able to perform topologically-defined protein-protein crosslinking, intramolecular stapling, and site-specific installation of fluorophores all inside living Escherichia coli cells, as well as study the DNA-binding properties of yeast Replication Protein A in vitro. By enabling the efficient dual modification of proteins in vivo, this DEAL approach provides a tool for the characterization and engineering of proteins in vivo.

synthetic biology↗

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↗

bioRxiv2020

Substrate sensing institutes sequential and asymmetric electron transfer in the nitrogenase-like DPOR Complex

Dark-operative protochlorophyllide oxidoreductase (DPOR) catalyzes the reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), a key penultimate step in the biosynthesis of bacteriochlorophyll. DPOR shares structural homology with nitrogenase and is made of electron donor (BchL) and electron acceptor (BchNB) component proteins. ATP driven assembly of the BchL and BchNB proteins drives electron transfer and Pchlide reduction. BchNB is composed of two subunits each of BchN and BchB arranged as an 2{beta}2 heterotetramer. Here, we describe extensive allosteric communication between the two identical active sites in BchNB that drives sequential and asymmetric electron transfer. Pchlide binding and electron transfer activities in one half of the BchNB tetramer allosterically regulates activities in the other half. Pchlide binding is sensed and recognized in trans by an Asp274 from the opposing half and is positioned in the active site to likely serve as the initial proton donor. An Asp274 to Ala substituted DPOR binds to two Pchlide molecules in the BchNB complex but is unable to conformationally poise one Pchlide molecule. Thus, stalling Pchlide reduction in both active sites. The [4Fe-4S] cluster of the BchNB protein is pre-reduced and donates the first electron to Pchlide, a mechanism similar to the deficit-spending model observed in nitrogenase. In half-reactive DPOR complexes, incapacitating proton donation in one half generates a stalled intermediate and Pchlide reduction in both halves is abolished. The results showcase long-range allosteric communication and sequential ET in the two symmetric halves. The findings shed light on the functional advantages imparted by the oligomeric architecture found in many electron transfer enzymes.

biochemistry↗

bioRxiv2019

The flexible N-terminus of BchL protects its cluster in oxygenic environments and autoinhibits activity

The dark-operative protochlorophyllide oxidoreductase (DPOR) enzyme contains two [4Fe-4S]- containing component proteins (BchL and BchNB) that assemble in an ATP-dependent fashion to coordinate electron transfer and reduction of protochlorophyllide to chlorophyllide. Photosynthesis generates an oxygenic environment that is non-optimal for [Fe-S] clusters and we here present an elegant evolutionarily conserved mechanism in BchL to protect its [4Fe-4S] cluster. We present a crystal structure of BchL in the nucleotide-free form with an ordered N-terminus that shields the [4Fe-4S] cluster at the docking interface between BchL and BchNB. Amino acid substitutions that perturb the shielding of the [4Fe-4S] cluster produce an unstable, but hyper-active enzyme complex, suggesting a role for the N-terminus in both auto-inhibition and enzyme stability. Upon ATP binding, a patch of amino acids, Asp-Phe-Asp ( DFD patch), situated at the mouth of the BchL ATP-binding pocket promotes inter-subunit cross stabilization of the two subunits and relieves the auto-inhibition by the N-terminus. A linked BchL dimer with one defective ATP-binding site does not support substrate reduction, illustrating that nucleotide binding to both subunits is a prerequisite for the inter-subunit cross stabilization. We propose that ATP-binding produces a conformational compaction of the BchL homodimer leading to a release of the flexible N-terminus from blocking the [4Fe-4S] cluster and promotes complex formation with BchNB to drive electron transfer. The auto-inhibitive feature and release mechanism appear unique to DPOR and is not found in the structurally similar nitrogenase.

biochemistry↗

bioRxiv2019

Elevated Expression of a Functional Suf Pathway in the E. coli BL21(DE3) Cell Line Enhances Recombinant Production of an Iron-Sulfur Cluster Containing Protein.

Structural and spectroscopic analysis of iron-sulfur [Fe-S] cluster-containing proteins is often limited by the occupancy and yield of recombinantly produced proteins. Here we report that Escherichia coli BL21(DE3), a strain routinely used to overexpress [Fe-S] cluster-containing proteins, has a nonfunctional Suf pathway, one of two E. coli [Fe-S] cluster biogenesis pathways. We confirmed that BL21(DE3) and commercially available derivatives carry a deletion that results in an inframe fusion of sufA and sufB genes within the sufABCDSE operon. We show that this fusion protein accumulates in cells but is - inactive in [Fe-S] biogenesis. Restoration of an intact Suf pathway combined with enhanced suf operon expression led to a remarkable (~3-fold) increase in the production of the [4Fe-4S] cluster-containing BchL protein, a key component of the dark-operative protochlorophyllide oxido-reductase complex. These results show that this engineered SufFeScient derivative of BL21(DE3) is suitable for enhanced large-scale synthesis of an [Fe-S] cluster-containing protein.\n\nIMPORTANCELarge quantities of recombinantly overexpressed iron-sulfur cluster-containing proteins are necessary for their in-depth biochemical characterization. Commercially available E. coli strain BL21(DE3) and its derivatives have a mutation that inactivates the function of one of the two native pathways (Suf pathway) responsible for cluster biogenesis. Correction of the mutation, combined with sequence changes that increase Suf expression can increase yield and cluster occupancy of [Fe-S] cluster-containing enzymes, facilitating the biochemical analysis of this fascinating group of proteins.

biochemistry↗

bioRxiv2018

Dynamics and Selective Remodeling of the DNA Binding Domains of RPA

Replication protein A (RPA) coordinates important DNA metabolic events by stabilizing single-strand DNA (ssDNA) intermediates, activating the DNA damage response, and handing off ssDNA to appropriate downstream players. Six DNA binding domains (DBDs) in RPA promote high affinity binding to ssDNA, but also allow RPA displacement by lower affinity proteins. We have made fluorescent versions of RPA and visualized the conformational dynamics of individual DBDs in the context of the full-length protein. We show that both DBD-A and DBD-D rapidly bind to and dissociate from ssDNA, while RPA as a whole remains bound to ssDNA. The recombination mediator protein Rad52 selectively modulates the dynamics of DBD-D. This demonstrates how RPA interacting proteins, with lower ssDNA binding affinity, can access the occluded ssDNA and remodel individual DBDs to replace RPA.\n\nOne Sentence SummaryThe choreography of binding and rearrangement of the individual domains of RPA during homologous recombination is revealed.

biochemistry↗