Biology subjects

Antony, E.

Publications and source records attributed to Antony, E..

At least 19 recordsLinked to original sources

bioRxiv2026

A Binuclear Cluster Supports Functional Asymmetry in Dark-Operative Protochlorophyllide Reductase

Many photosynthetic bacteria rely on the nitrogenase-like dark-operative protochlorophyllide oxidoreductase (DPOR) to sustain (bacterio)chlorophyll synthesis in the dark. DPOR couples ATP hydrolysis to electron transfer from a reducing component (L-protein) to a catalytic component (NB-protein) that reduces protochlorophyllide. NB-protein is an 2{beta}2 heterotetramer, and each heterodimeric half contains an active site and a [Fe4S4] cluster. Although the two halves are identical, they react sequentially rather than in parallel. How the two halves communicate has remained unknown. Here we show, using Cu K-edge x-ray absorption spectroscopy and cryo-EM, that the NB-protein tetramer interface holds a binuclear, sulfur-bridged [Cu2S2] cluster. Each Cu is reduced and three-coordinate, with short Cu-S and Cu-Cu distances (2.26 and 2.62 &Aring). Mutagenesis excludes the nearest cysteine as the bridging ligand, supporting inorganic sulfide as the most likely sulfur donor. To our knowledge, this is the first sulfide-bridged dicopper cluster identified in a protein. The cluster is conserved in three purple non-sulfur bacteria and connects the BchB C-terminal helices that contact opposing active sites. Loss of Cu abolishes DPOR activity and asymmetric L-protein binding without disturbing overall fold. We propose that the cluster relays substrate-binding information between active sites, suggesting a metal-mediated mechanism for cooperativity in nitrogenase-like enzymes.

biochemistry↗

bioRxiv2026

A novel mechanism of ubiquitin-charging a bivalent E2:RING E3 complex

Protein ubiquitination results from a cascade of enzyme interactions that transfer ubiquitin from one covalent bond to another. First, an E1 is activated by attaching ubiquitin to itself through a thioester bond. Next, in E2 charging, activated E1 interacts with an E2 and transfers ubiquitin to a thioester bond on the E2. Finally, an E3 mediates the transfer of ubiquitin from a charged E2 to an isopeptide bond on a protein target. RING E3s, the largest E3 family in eukaryotes, function as scaffolds rather than enzymes and facilitate direct transfer of ubiquitin from a charged E2 to a protein target by simultaneously engaging both. All RING E3s engage an E2 at an interface that overlaps with the E1-binding site and several form stable, bivalent complexes with an E2 through additional binding surfaces. How E2 charging proceeds within these complexes remains unclear.Here, we utilize human Rad6(Rad18)2 as a model bivalent E2:RING E3 complex to delineate the interplay of protein{middle dot}protein interactions among the primary human E1 (Uba1), an E2 (Rad6), and a RING E3 (Rad18) during E2 charging. Collectively, the results reveal a novel mechanism that is not directed by ubiquitin thioester affinity switches. Rather, interactions of Rad18 with Rad6 slow the chemistry step and all preceding steps of the Uba1 catalytic cycle via competitive inhibition but accelerate release of charged Rad6 from Uba1, ultimately stimulating Rad6 charging overall. To the best of our knowledge, this represents the first example of a RING E3 stimulating Uba1-dependent charging of an E2.

biochemistry↗

bioRxiv2026

Dissecting the role of PCNA and Pif1 in replication of individual DNA molecules by DNA polymerase δ.

PCNA loading and stabilization at primer-template junctions is crucial for processive DNA synthesis by replicative polymerases. It is also essential for strand displacement synthesis during Break Induced Replication (BIR). In this study, we employed a single molecule approach to directly visualize the role of PCNA, DNA polymerase {delta} and the Pif1 DNA helicase in these pathways. We first dissected the preferential loading of PCNA by RFC to 3-junctions of gaps, nicks and flapped substrates. Moreover, we found that Pol {delta} and Pif1 both stabilize loaded PCNA and reduce its diffusion coefficient along DNA. We further show that the interaction of PCNA with Pol {delta} is essential for gap-filling synthesis. On a 5-flapped substrate, we show that both Pol {delta} and Pif1 are required for strand displacement synthesis, while PCNA is not; although, it stimulates the process.

biochemistry↗

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↗

bioRxiv2025

Delineation of a novel assembly intermediate in retroviral integration pathway

Retroviral integration is mediated by viral integrase (IN) which synapses two viral long terminal repeat (LTR) DNA ends and produces a series of nucleoprotein complexes known as intasomes. While structural studies of mature intasomes have illuminated key aspects of their architecture and provided insights into the integration reaction, the sequence of events driving IN oligomerization and engagement of the viral DNA pairing remains unclear. Here, using complementary biochemical and biophysical approaches, including ensemble and single molecule FRET, we reveal that integration progresses through a key transient intermediate that leads to the mature intasome. We demonstrate that Rous Sarcoma Virus (RSV) intasome assembly pathway proceeds through a tetrameric intermediate where two IN dimers engage a single DNA end. This complex subsequently oligomerizes to form mature functional octameric intasome in which two DNA ends are juxtaposed for concerted integration. These findings provide mechanistic insights into the stepwise pathway of retroviral integration and define a previously uncharacterized intermediate critical for intasome maturation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=97 SRC="FIGDIR/small/675266v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@57a82aorg.highwire.dtl.DTLVardef@e17110org.highwire.dtl.DTLVardef@c5f4c4org.highwire.dtl.DTLVardef@4886d2_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

Cryo-EM structure of a methanogen nitrogenase-PII protein supercomplex

Nitrogenases are metalloenzymes that catalyze the reduction of atmospheric dinitrogen to ammonia, sustaining the global nitrogen cycle. While bacterial nitrogenase has been extensively characterized, the architecture and regulation of archaeal nitrogenases remain unknown despite longstanding evidence of nitrogen fixation in methanogens. Here we report a 3.2 [A] cryo-electron microscopy structure of a native nitrogenase-PII protein supercomplex from Methanosarcina acetivorans. The structure reveals an unprecedented assembly of three NifDK heterotetramers bridged by six NifI1,2 heterotrimeric PII complexes, which sterically block NifH association and lock the enzyme in an inactive state. The PII complexes display asymmetric binding of ADP and 2-oxoglutarate, coupling nitrogenase inhibition directly to cellular energy and nitrogen status. Addition of 2-oxoglutarate and ATP releases the NifI complexes, stimulating a threefold increase in NifDK activity in vitro. This higher-order architecture uncovers a previously unrecognized regulatory strategy in methanogens, in which PII proteins drive nitrogenase oligomerization to control activity. The discovery that nitrogenase activity may be modulated through direct assembly into higher-order structures opens new avenues for exploring nitrogenase evolution, regulation, and biotechnological applications. One sentence summaryDiscovery of a nitrogenase-PII protein supercomplex in methanogens, uncovering a metabolite-gated assembly mechanism for nitrogenase inhibition.

biochemistry↗

bioRxiv2025

PCNA encircling primer/template junctions is eliminated by exchange of RPA for Rad51: Implications for the interplay between human DNA damage tolerance pathways

The DNA genome is constantly exposed to agents, such as ultraviolet radiation (UVR), that can alter or eliminate its coding properties through covalent modifications of the template bases. Many of these damaging modifications (i.e., lesions) persist into S-phase of the cell cycle where they may stall the canonical DNA replication machinery. In humans, these stalling events are circumvented by one of at least three interconnected DNA damage tolerance (DDT) pathways; translesion DNA synthesis (TLS), Template Switching (TS), and Homology-dependent Recombination (HDR). Currently, the functional interplay between these pathways is unclear, leaving wide gaps in our fundamental understanding of human DDT. To gain insights, we focus on the activation mechanisms of the DDT pathways. PCNA sliding clamps encircling primer/template (P/T) junctions of stalled replication sites are central to the activation of both TLS and TS whereas exchange of RPA for Rad51 filaments on the single strand DNA (ssDNA) sequences of stalled replication sites is central to HDR activation. Utilizing direct, ensemble FRET approaches developed by our lab, we independently monitor and directly compare PCNA occupancy and RPA/Rad51 exchange at P/T junctions under a variety of conditions that mimic in vivo scenarios. Collectively, the results reveal that assembly of stable Rad51 filaments at P/T junctions via RPA/Rad51 exchange causes complete and irreversible unloading of the resident PCNA, both in the presence and absence of an abundant PCNA-binding protein complex. Further investigations decipher the mechanism of RPA/Rad51 exchange-dependent unloading of PCNA. Collectively, these studies provide critical insights into the interplay between human DDT pathways and direction for future studies.

biophysics↗

bioRxiv2025

Mechanistic insights into direct DNA and RNA strand transfer and dynamic protein exchange of SSB and RPA

Single-stranded DNA-binding proteins (SSBs) are essential for genome stability, facilitating replication, repair, and recombination by binding ssDNA, recruiting other proteins, and dynamically relocating in response to cellular demands. Using single-molecule fluorescence resonance energy transfer (smFRET) assays, we elucidated the mechanisms underlying direct strand transfer from one locale to another, protein exchange, and RNA interactions at high resolution. Both bacterial SSB and eukaryotic replication protein A (RPA) exhibited direct strand transfer to competing ssDNA, with rates strongly influenced by ssDNA length. Strand transfer proceeded through multiple failed attempts before a successful transfer, forming a ternary intermediate complex with transient interactions, supporting a direct transfer mechanism. Both proteins efficiently exchanged DNA-bound counterparts with freely diffusing molecules, while hetero-protein exchange revealed that SSB and RPA could replace each other on ssDNA in a length-dependent manner, indicating that protein exchange does not require specific protein-protein interactions. Additionally, both proteins bound RNA and underwent strand transfer to competing RNA, with RPA demonstrating faster RNA transfer kinetics. Competitive binding assays confirmed a strong preference for DNA over RNA. These findings provide critical insights into the dynamic behavior of SSB and RPA in nucleic acid interactions, advancing our understanding of their essential roles in genome stability, regulating RNA metabolism, and orchestrating nucleic acid processes.

molecular biology↗

bioRxiv2025

Mechanism of RPA phosphocode priming and tuning by Cdk1/Wee1 signaling circuit

Replication protein A (RPA) is a heterotrimeric single-strand DNA binding protein that is integral to DNA metabolism. Segregation of RPA functions in response to DNA damage is fine-tuned by hyperphosphorylation of the RPA32 subunit that is dependent on Cyclin-dependent kinase (Cdk)-mediated priming phosphorylation at the Ser-23 and Ser-29 sites. However, the mechanism of priming-driven hyperphosphorylation of RPA remains unresolved. Furthermore, the modulation of cell cycle progression by the RPA-Cdk axis is not clearly understood. Here, we uncover that the RPA70 subunit is also phosphorylated by Cdk1 at Thr-191. This modification is crucial for the G2 to M phase transition. This function is enacted through reciprocal regulation of Cdk1 activity through a feedback circuit espoused by stabilization of Wee1 kinase. The Thr-191 phosphosite on RPA70 is also crucial for priming hyperphosphorylation of RPA32 in response to DNA damage. Structurally, phosphorylation by Cdk1 primes RPA by reconfiguring the domains to release the N-terminus of RPA32 and the two protein-interaction domains that markedly enhances the efficiency of multisite phosphorylation by other kinases. Our findings establish a unique phosphocode-dependent feedback mechanism between RPA and RPA-regulating kinases that is fine-tuned to enact bipartite functions in cell cycle progression and DNA damage response.

biochemistry↗

bioRxiv2024

Rad52 sorts and stacks Rad51 at the DNA junction to promote homologous recombination

Homologous recombination (HR) repairs double-stranded DNA breaks (DSBs). The DSBs are resected to yield single-stranded DNA (ssDNA) that are coated by Replication Protein A (RPA). Rad51 is a recombinase and catalyzes strand invasion and the search for homology. However, it binds to ssDNA with lower affinity than RPA. Thus, mediator proteins such as Rad52/BRCA2 are required to promote Rad51 binding to RPA-coated ssDNA, but the underlying mechanisms remain poorly understood. Saccharomyces cerevisiae Rad52 interacts with Rad51 through two distinct binding modes. We here uncover that the Rad51-binding site in the disordered C-terminus of Rad52 (mode-1) sorts polydisperse Rad51 into discrete monomers. The second Rad51 binding site resides in the ordered N-terminal ring of Rad52 (mode-2), but this interaction occurs at only one position on the ring. In single molecule confocal fluorescence microscopy combined with optical tweezer analysis, we directly visualize filament formation using fluorescent-Rad51. Rad52 catalyzes Rad51 loading onto RPA-coated ssDNA, with a distinct preference for junctions, but no filament growth is observed. Deletion of the C-terminus of Rad52 results in loss of Rad51 sorting and abrogates Rad51 binding to RPA-coated DNA. While BRCA2 and Rad52 are structurally unrelated, many of these functional features are conserved. We describe a concerted Sort & Stack mechanism for mediator proteins in promoting HR.

biochemistry↗

bioRxiv2024

Cryo-EM captures the coordination of long-range allostery and asymmetric electron transfer by a bi-copper cluster in the nitrogenase-like DPOR complex

Enzymes that catalyze long-range electron transfer reactions are often structurally evolved to possess two symmetrical halves. The functional advantages and mechanistic principles for such architecture remain a mystery. Using Cryo-EM we capture snapshots of the nitrogenase-like Dark-operative Protochlorophyllide Oxidoreductase (DPOR) enzyme during substrate recognition and turnover. The structures reveal that asymmetry is enforced upon substrate binding and leads to an allosteric inhibition of protein-protein interactions and electron transfer in one half. Residues that form a conduit for electron transfer are aligned in one half while misaligned in the other. An ATP-turnover coupled switch is triggered once electron transfer is accomplished in one half and relayed through a bi-copper cluster at the oligomeric interface, leading to activation of enzymatic events in the other. The findings provide a mechanistic blueprint for regulation of asymmetric long-range electron transfer. One-Sentence SummaryA bi-copper cluster coordinates electron transfer for substrate reduction in the nitrogenase-like DPOR enzyme and the structures reveal how allostery and asymmetry are enacted over 100[A] and utilized for sequential electron transfer.

biochemistry↗

bioRxiv2024

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

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.

biochemistry↗

bioRxiv2023

Rapid long-distance migration of RPA on single stranded DNA occurs through intersegmental transfer utilizing multivalent interactions

Replication Protein A (RPA) is a single stranded DNA (ssDNA) binding protein that coordinates diverse DNA metabolic processes including DNA replication, repair, and recombination. RPA is a heterotrimeric protein with six functional oligosaccharide/oligonucleotide (OB) domains and flexible linkers. Flexibility enables RPA to adopt multiple configurations and is thought to modulate its function. Here, using single molecule confocal fluorescence microscopy combined with optical tweezers and coarse-grained molecular dynamics simulations, we investigated the diffusional migration of single RPA molecules on ssDNA under tension. The diffusion coefficient D is the highest (20,000 nucleotides2/s) at 3 pN tension and in 100 mM KCl and markedly decreases when tension or salt concentration increases. We attribute the tension effect to intersegmental transfer which is hindered by DNA stretching and the salt effect to an increase in binding site size and interaction energy of RPA-ssDNA. Our integrative study allowed us to estimate the size and frequency of intersegmental transfer events that occur through transient bridging of distant sites on DNA by multiple binding sites on RPA. Interestingly, deletion of RPA trimeric core still allowed significant ssDNA binding although the reduced contact area made RPA 15-fold more mobile. Finally, we characterized the effect of RPA crowding on RPA migration. These findings reveal how the high affinity RPA-ssDNA interactions are remodeled to yield access, a key step in several DNA metabolic processes. SignificanceReplication Protein A (RPA) binds to the exposed single stranded DNA (ssDNA) during DNA metabolism. RPA dynamics are essential to reposition RPA on ssDNA and recruit downstream proteins at the bound site. Here in this work, we perform a detailed biophysical study on dynamics of yeast RPA on ssDNA. We show that RPA can diffuse on ssDNA and is affected by tension and salt. Our observations are best explained by the intersegmental transfer model where RPA can transiently bridge two distant DNA segments for its migration over long distances. We further dissect the contributions of the trimerization core of RPA and other adjacent RPA molecules on RPA migration. This study provides detailed experimental and computational insights into RPA dynamics on ssDNA.

biophysics↗

bioRxiv2023

Fluorescent human RPA to track assembly dynamics on DNA

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 Forster Resonance Energy Transfer (FRET) assays for measuring dynamics of RPA on DNA are also described. HighlightsO_LIRPA is an essential protein for most DNA metabolic processes including replication, repair, and recombination. C_LIO_LIRPA 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. C_LIO_LISite-specific fluorescent probes were incorporated into two domains of RPA and report on ssDNA binding dynamics. C_LIO_LIBulk-level kinetic and single-molecule assays are described to monitor the binding and remodeling of individual RPA domains on ssDNA. C_LI

biochemistry↗

bioRxiv2023

Generation of site-specifically labelled fluorescent human XPA to investigate DNA binding dynamics during nucleotide excision repair

Nucleotide excision repair (NER) promotes genomic integrity by correcting bulky DNA adducts damage caused by external factors such as ultraviolet light. Defects in NER enzymes are associated with pathological conditions such as Xeroderma Pigmentosum, trichothiodystrophy, and Cockayne syndrome. A critical step in NER is the binding of the Xeroderma Pigmentosum group A protein (XPA) to the DNA adduct. To better capture the dynamics of XPA interactions with DNA during NER we have utilized the fluorescence enhancement through non-canonical amino acids (FEncAA) approach. 4-azido-L-phenylalanine (4AZP) was incorporated at Arg-153 in human XPA and conjugated to Cy3 using strain-promoted azide-alkyne cycloaddition. The resulting fluorescent human XPA protein (hXPACy3) shows no loss in DNA binding activity and generates a robust change in fluorescence upon binding to DNA. Here we describe methods to generate hXPACy3 and detail experimental conditions required to stably maintain the protein during biochemical and biophysical studies.

biochemistry↗

bioRxiv2023

Human hnRNPA1 reorganizes telomere-bound Replication Protein A

Human replication protein A (RPA) is a heterotrimeric ssDNA binding protein responsible for many aspects of cellular DNA metabolism. Dynamic interactions of the four RPA DNA binding domains (DBDs) with DNA control replacement of RPA by downstream proteins in various cellular metabolic pathways. RPA plays several important functions at telomeres where it binds to and melts telomeric G-quadruplexes, non-canonical DNA structures formed at the G-rich telomeric ssDNA overhangs. Here, we combine single-molecule total internal reflection fluorescence microscopy (smTIRFM) and mass photometry (MP) with biophysical and biochemical analyses to demonstrate that heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) specifically remodels RPA bound to telomeric ssDNA by dampening the RPA configurational dynamics and forming a ternary complex. Uniquely, among hnRNPA1 target RNAs, telomeric repeat-containing RNA (TERRA) is selectively capable of releasing hnRNPA1 from the RPA-telomeric DNA complex. We speculate that this telomere specific RPA-DNA-hnRNPA1 complex is an important structure in telomere protection. One Sentence SummaryAt the single-stranded ends of human telomeres, the heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) binds to and modulates conformational dynamics of the ssDNA binding protein RPA forming a ternary complex which is controlled by telomeric repeat-containing RNA (TERRA).

biophysics↗

bioRxiv2023

Interplay of macromolecular interactions during assembly of human DNA polymerase δ holoenzymes and initiation of DNA synthesis

In humans, DNA polymerase {delta} (Pol {delta}) holoenzymes, comprised of Pol {delta} and the processivity sliding clamp, proliferating cell nuclear antigen (PCNA), carry out DNA synthesis during lagging strand DNA replication, initiation of leading strand DNA replication, and the major DNA damage repair and tolerance pathways. Pol {delta} holoenzymes are assembled at primer/template (P/T) junctions and initiate DNA synthesis in a coordinated process involving the major single strand DNA-binding protein complex, replication protein A (RPA), the processivity sliding clamp loader, replication factor C (RFC), PCNA, and Pol {delta}. Each of these factors interact uniquely with a P/T junction and most directly engage one another. Currently, the interplay between these macromolecular interactions is largely unknown. In the present study, novel Forster Resonance Energy Transfer (FRET) assays reveal that dynamic interactions of RPA with a P/T junction during assembly of a Pol {delta} holoenzyme and initiation of DNA synthesis maintain RPA at a P/T junction and accommodate RFC, PCNA, and Pol {delta}, maximizing the efficiency of each process. Collectively, these studies significantly advance our understanding of human DNA replication and DNA repair.

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↗