Biology subjects

Loparo, J. J.

Publications and source records attributed to Loparo, J. J..

14 recordsLinked to original sources

bioRxiv2026

Core NHEJ Factors Maintain Synapsis Across Diverse DNA End Structures

DNA double strand breaks (DSBs) generated by genotoxic stress frequently contain chemically diverse termini that require end processing before ligation by non-homologous end joining (NHEJ). How end chemistry influences assembly and persistence of NHEJ synaptic complexes remains unclear. Using single-molecule imaging approaches in Xenopus egg extract, we measured formation and stability of the long-range (LRC) and short-range (SRC) complexes on nine DNA substrates spanning kinase-, polymerase- and nuclease-dependent processing requirements. LRC formation and lifetime were comparable across end types, and SRC formation and stability were largely insensitive to end chemistry. DNA hairpins, physiological intermediates of V(D)J recombination, were a notable exception that formed LRCs but inefficiently transitioned to SRCs. Depletion of cognate processing factors had modest effects on SRC formation and persistence, indicating that core NHEJ factors provide the principal structural support for synapsis across diverse termini. Although XLF was required for SRC formation on all substrates, reducing an XLF dimer from two intact tail/Ku-binding motifs to one selectively reduced productive SRC formation on ends requiring processing. Together, these results support a model in which partially redundant protein-protein interactions between core NHEJ factors maintain synapsis while end-specific processing reactions shape repair outcome.

biophysics↗

bioRxiv2026

Sequential division of labor between PAXX and XLF drives NHEJ synaptic complex stability and maturation

DNA double-strand breaks (DSBs) are primarily repaired by non-homologous end joining (NHEJ), which tethers and ligates DNA ends within a synaptic complex. PAXX is an NHEJ accessory factor related to XRCC4 and XLF, but its mechanistic role and genetic interaction with XLF are unclear. Using biochemical assays and single-molecule imaging within Xenopus laevis egg extracts and human cell assays, we show that PAXX bridges DNA ends by using its disordered tails to bind opposing Ku molecules. This PAXX tether substantially extends long-range synaptic complex (LRSC) lifetime and modestly stabilizes the short-range synaptic complex (SRSC). LRSC stabilization increases XLF residence and promotes the LRSC-to-SRSC transition. Using domain-swapped chimeras, we demonstrate that PAXXs tails provide synaptic stabilization, whereas XLFs heads interact with XRCC4 to drive the LRSC-to-SRSC transition: an XLF head-PAXX tail chimera recapitulates the functions of both proteins, revealing complementary, sequential roles for PAXX and XLF in NHEJ.

biophysics↗

bioRxiv2025

The NHEJ machinery is translocated off DNA ends toenable resection

Error-prone non-homologous end joining (NHEJ) and homologous recombination (HR) compete to repair DNA double strand breaks [1, 2, 3]. Given that NHEJ factors such as Ku and DNA-PKcs are rapidly recruited to DSBs and sterically occlude ends, they must be removed to enable 5' DNA end resection, the commitment step of HR. While resection is well characterized, how the NHEJ machinery is displaced from DNA ends has remained poorly understood. Here, we performed single-molecule imaging in Xenopus laevis egg extracts to monitor the removal of NHEJ factors from DNA ends while simultaneously tracking the progress of DNA end resection. We find that MRN and CtIP are essential to evict the core NHEJ factor Ku from DNA ends. Surprisingly, while Ku removal and resection of the 5' end are normally coordinated, these processes arise from distinct activities of the MRN complex and are differentially regulated by the action of cyclin dependent kinases and ATM. By directly imaging the position of Ku on DNA using smFRET, we found that Ku is translocated independently of DNA end resection in an ATP-dependent manner that requires both MRN and CtIP. In summary, our results provide a new mechanistic framework to understand how eviction of the NHEJ machinery is coordinated with DNA resection to initiate HR.

biophysics↗

bioRxiv2024

The hit-and-run of cell wall synthesis: LpoB transiently binds and activates PBP1b through a conserved allosteric switch.

The peptidoglycan (PG) cell wall is the primary protective layer of bacteria, making the process of PG synthesis a key antibiotic target. Class A penicillin-binding proteins (aPBPs) are a family of conserved and ubiquitous PG synthases that fortify and repair the PG matrix. In gram-negative bacteria, these enzymes are regulated by outer-membrane tethered lipoproteins. However, the molecular mechanism by which lipoproteins coordinate the spatial recruitment and enzymatic activation of aPBPs remains unclear. Here we use single-molecule FRET and single-particle tracking in E. coli to show that a prototypical lipoprotein activator LpoB triggers site-specific PG synthesis by PBP1b through conformational rearrangements. Once synthesis is initiated, LpoB affinity for PBP1b dramatically decreases and it dissociates from the synthesizing enzyme. Our results suggest that transient allosteric coupling between PBP1b and LpoB directs PG synthesis to areas of low peptidoglycan density, while simultaneously facilitating efficient lipoprotein redistribution to other sites in need of fortification.

microbiology↗

bioRxiv2024

MreC-MreD structure reveals a multifaceted interface that controls MreC conformation

The peptidoglycan (PG) cell wall is critical for bacterial growth and survival and is a primary antibiotic target. MreD is an essential accessory factor of the Rod complex, which carries out PG synthesis during elongation, yet little is known about how MreD facilitates this process. Here, we present the cryo-electron microscopy structure of Thermus thermophilus MreD in complex with another essential Rod complex component, MreC. The structure reveals that a periplasmic-facing pocket of MreD interacts with multiple membrane-proximal regions of MreC. We use single-molecule FRET to show that MreD controls the conformation of MreC through these contacts, inducing a state primed for Rod complex activation. Using E. coli as a model, we demonstrate that disrupting these interactions abolishes Rod complex activity in vivo. Our findings reveal the role of MreD in bacterial cell shape determination and highlight its potential as an antibiotic target.

microbiology↗

bioRxiv2024

A bipartite interaction with the processivity clamp potentiates Pol IV-mediated TLS

Processivity clamps mediate polymerase switching for translesion synthesis (TLS). All three E. coli TLS polymerases interact with the {beta}2 processivity clamp through a conserved clamp-binding motif (CBM), which is indispensable for TLS. Notably, Pol IV also makes a unique secondary contact with the clamp through non-CBM residues. However, the role of this "rim contact" in Pol IV-mediated TLS remains poorly understood. Here we show that the rim contact is critical for TLS past strong replication blocks. In in vitro reconstituted Pol IV-mediated TLS, ablating the rim contact compromises TLS past 3-methyl dA, a strong block, while barely affecting TLS past N2-furfuryl dG, a weak block. Similar observations are also made in E. coli cells bearing a single copy of these lesions in the genome. Within lesion-stalled replication forks, the rim interaction and ssDNA binding protein cooperatively poise Pol IV to better compete with Pol III for binding to a cleft through its CBM. We propose that this bipartite clamp interaction enables Pol IV to rapidly resolve lesion-stalled replication through TLS at the fork, which reduces damage induced mutagenesis.

biochemistry↗

bioRxiv2024

Identification of the main barriers to Ku accumulation in chromatin

Repair of DNA double strand breaks by the non-homologous end-joining pathway is initiated by the binding of Ku to DNA ends. Given its high affinity for ends, multiple Ku proteins load onto linear DNAs in vitro. However, in cells, Ku loading is limited to [~]1-2 molecules per DNA end. The mechanisms enforcing this limit are currently unknown. Here we show that the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), but not its protein kinase activity, is required to prevent excessive Ku entry into chromatin. Ku accumulation is further restricted by two mechanisms: a neddylation/FBXL12-dependent process which actively removes loaded Ku molecules throughout the cell cycle and a CtIP/ATM-dependent mechanism which operates in S-phase. Finally, we demonstrate that the misregulation of Ku loading leads to impaired transcription in the vicinity of DNA ends. Together our data shed light on the multiple layers of coordinated mechanisms operating to prevent Ku from invading chromatin and interfering with other DNA transactions. HighlightsO_LIDNA-PKcs structurally blocks Ku sliding into chromatin in human & Xenopus C_LIO_LIA neddylation/FBXL12-dependent mechanism limits Ku accumulation on chromatin C_LIO_LIIn S-phase, ATM/CtIP overcomes Ku accumulation C_LIO_LIIn absence of DNA-PKcs, transcription at the DNA end vicinity is inhibited C_LI eTOC blurbThe DNA end binding protein Ku can slide onto naked DNA but this is limited in cells. Using human cells and Xenopus egg extracts, DNA-PKcs is identified as the main structural barrier to Ku entry into chromatin, along with two active mechanisms which limit Ku accumulation in absence of DNA-PKcs. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=194 SRC="FIGDIR/small/574002v1_ufig1.gif" ALT="Figure 1"> View larger version (67K): org.highwire.dtl.DTLVardef@4ebc45org.highwire.dtl.DTLVardef@12a7805org.highwire.dtl.DTLVardef@12db0a8org.highwire.dtl.DTLVardef@a925cc_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2022

Allosteric activation of cell wall synthesis during bacterial growth

The peptidoglycan (PG) cell wall protects bacteria against osmotic lysis and determines cell shape, making this structure a key antibiotic target. Peptidoglycan is a polymer of glycan chains connected by peptide crosslinks, and its synthesis requires precise spatiotemporal coordination between glycan polymerization and crosslinking. However, the molecular mechanism by which these reactions are initiated and coupled is unclear. Here we use single-molecule FRET and cryo-EM to show that an essential PG synthase (RodA-PBP2) responsible for bacterial elongation undergoes dynamic exchange between closed and open states. Structural opening couples the activation of polymerization and crosslinking and is essential in vivo. Given the high conservation of this family of synthases, the opening motion that we uncovered likely represents a conserved regulatory mechanism that controls activation of PG synthesis during other cellular processes, including cell division.

biophysics↗

bioRxiv2022

H2A.Z deposition by SWR1C involves multiple ATP-dependent steps

The histone variant H2A.Z is a conserved feature of nucleosomes flanking protein-coding genes. Deposition of H2A.Z requires ATP-dependent replacement of nucleosomal H2A by a chromatin remodeler related to the multi-subunit enzyme, yeast SWR1C. How these enzymes use ATP to promote this nucleosome editing reaction remains unclear. Here we use single-molecule and ensemble methodologies to identify three ATP-dependent phases in the H2A.Z deposition reaction. Real-time analysis of single nucleosome remodeling events reveals an initial, priming step that occurs after ATP addition that likely involves transient DNA unwrapping from the nucleosome. Priming is followed by rapid loss of histone H2A, which is subsequently released from the H2A.Z nucleosomal product. Surprisingly, the rates of both priming and the release of the H2A/H2B dimer are sensitive to ATP concentration. This complex reaction pathway provides multiple opportunities to regulate the timely and accurate deposition of H2A.Z at key genomic locations.

biochemistry↗

bioRxiv2020

XLF acts as a flexible connector during non-homologous end joining

Non-homologous end joining (NHEJ) is the predominant pathway that repairs DNA double strand breaks in vertebrates. During NHEJ DNA ends are held together by a multi-protein synaptic complex until they are ligated. Here we investigate the role of the intrinsically disordered C-terminal tail of XLF, a critical factor in end synapsis. We demonstrate that the XLF tail along with the Ku binding motif (KBM) at the extreme C-terminus are required for end joining. While the underlying sequence of the tail can be varied, a minimal tail length is required for NHEJ. Single-molecule FRET experiments that observe end synapsis in real-time show that this defect is due to a failure to closely align DNA ends. Our data supports a model in which a single C-terminal tail tethers XLF to Ku while allowing XLF to form interactions with XRCC4 that enable synaptic complex formation.

biochemistry↗

bioRxiv2020

Compartmentalization of the replication fork by single-stranded DNA binding protein regulates translesion synthesis

DNA replication is mediated by the coordinated actions of multiple enzymes within replisomes. Processivity clamps tether many of these enzymes to DNA, allowing access to the primer/template junction. Many clamp-interacting proteins (CLIPs) are involved in genome maintenance pathways including translesion synthesis (TLS). Despite their abundance, DNA replication in bacteria is not perturbed by these CLIPs. Here we show that while the TLS polymerase Pol IV is largely excluded from moving replisomes, the remodeling of ssDNA binding protein (SSB) upon replisome stalling enriches Pol IV at replication forks. This enrichment is indispensable for Pol IV-mediated TLS on both the leading and lagging strands as it enables Pol IV-processivity clamp binding by overcoming the gatekeeping role of the Pol III epsilon subunit. As we have demonstrated for the Pol IV-SSB interaction, we propose that the binding of CLIPs to the processivity clamp must be preceded by interactions with factors that serve as localization markers for their site of action.

biochemistry↗

bioRxiv2019

A gatekeeping function of the replicative polymerase controls pathway choice in the resolution of lesion-stalled replisomes

DNA lesions stall the replisome and proper resolution of these obstructions is critical for genome stability. Replisomes can directly replicate past a lesion by error-prone translesion synthesis. Alternatively, replisomes can reprime DNA synthesis downstream of the lesion, creating a single-stranded DNA gap that is repaired primarily in an error-free, homology-directed manner. Here we demonstrate how structural changes within the bacterial replisome determine the resolution pathway of lesion-stalled replisomes. This pathway selection is controlled by a dynamic interaction between the proofreading subunit of the replicative polymerase and the processivity clamp, which sets a kinetic barrier to restrict access of TLS polymerases to the primer/template junction. Failure of TLS polymerases to overcome this barrier leads to repriming, which competes kinetically with TLS. Our results demonstrate that independent of its exonuclease activity, the proofreading subunit of the replisome acts as a gatekeeper and influences replication fidelity during the resolution of lesion-stalled replisomes.

biochemistry↗

bioRxiv2019

Non-homologous end joining minimizes errors by coordinating DNA processing with ligation

Genome stability requires efficient and faithful repair of DNA double-strand breaks (DSBs). The predominant DSB repair pathway in human cells is non-homologous end-joining (NHEJ), which directly ligates DNA ends1-5. Broken DNA ends at DSBs are chemically diverse, and many are not compatible for direct ligation by the NHEJ-associated DNA Ligase IV (Lig4). To solve this problem, NHEJ end-processing enzymes including polymerases and nucleases modify ends until they are ligatable. How cells regulate end processing to minimize unnecessary genomic alterations6 during repair of pathological DSBs remains unknown. Using a biochemical system that recapitulates key features of cellular NHEJ, we previously demonstrated that DNA ends are initially tethered at a distance, followed by Lig4-mediated formation of a "short-range synaptic complex" in which DNA ends are closely aligned for ligation7. Here, we show that a wide variety of end-processing activities all depend on formation of the short-range complex. Moreover, using real-time single molecule imaging, we find that end processing occurs within the short-range complex. Confining end processing to the Lig4-dependent short-range synaptic complex promotes immediate ligation of compatible ends and ensures that incompatible ends are ligated as soon as they become compatible, thereby minimizing end processing. Our results elucidate how NHEJ exploits end processing to achieve versatility while minimizing errors that cause genome instability.

molecular biology↗

bioRxiv2019

A flow extension tethered particle motion assay for single-molecule proteolysis

Regulated proteolysis of signaling proteins under mechanical tension enables cells to communicate with their environment in a variety of developmental and physiologic contexts. The role of force in inducing proteolytic sensitivity has been explored using magnetic tweezers at the single-molecule level with bead-tethered assays, but such efforts have been limited by challenges in ensuring that beads are not restrained by multiple tethers. Here, we describe a multiplexed assay for single-molecule proteolysis that overcomes the multiple-tether problem using a flow extension (FLEX) strategy on a microscope equipped with magnetic tweezers. Particle tracking and computational sorting of flow-induced displacements allows assignment of tethered substrates into singly-captured and multiply-tethered bins, with the fraction of fully mobile, single-tethered substrates depending inversely on the concentration of substrate loaded on the coverslip. Computational exclusion of multiply-tethered beads enables robust assessment of on-target proteolysis by the highly specific tobacco etch virus protease and the more promiscuous metalloprotease ADAM17. This method should be generally applicable to a wide range of proteases and readily extensible to robust evaluation of proteolytic sensitivity as a function of applied magnetic force.

biophysics↗