Biology subjects

Luger, K.

Publications and source records attributed to Luger, K..

At least 19 recordsLinked to original sources

bioRxiv2026

PARP1 Exhibits an Enzymatically Inactive Chromatin Binding Mode

Poly (ADP-ribose) Polymerase 1 (PARP1) is an abundant nuclear enzyme that dynamically engages chromatin in diverse cellular scenarios. In the context of DNA repair, PARP1 becomes enzymatically activated and subsequently attaches ADP-ribose units onto various proteins, including histones, to signal and coordinate the DNA damage response. In the absence of DNA damage, PARP1 modulates chromatin structure by directly binding to nucleosomes, however, the molecular basis of this interaction is unknown. Here, we define a distinct, enzymatically inactive mode of PARP1 chromatin binding, in which the Zn1, Zn2, Zn3, and BRCT domains cooperatively bind nucleosomal linker DNA and drive compaction of undamaged chromatin. This binding mode does not trigger catalytic activation and therefore is insensitive to PARP inhibitors (PARPi). Together, our results support a model in which PARP1 associates with the genome in an inactive state to compact chromatin and to surveil for DNA lesions. SummaryPARP1 engages undamaged chromatin in a distinct binding mode that results in chromatin compaction but does not lead to enzymatic activation.

biochemistry↗

bioRxiv2026

Structure of the human HIRA histone chaperone with a nucleosome suggests a stepwise nucleosome assembly mechanism

The restoration of chromatin in the wake of a DNA or RNA polymerase is essential to maintain the integrity of eukaryotic genomes. Human HIRA is a 1.8-megadalton, three-subunit histone chaperone that mediates all replication-independent deposition of the histone variant H3.3 at active chromatin regions1-6. Disruption of HIRA perturbs active-chromatin organization and has wide-ranging consequences for development, cellular senescence, and genome integrity7-11. Despite its central biological role in reassembling nucleosomes post-transcription, the structure of native human HIRA and the mechanism by which it organizes histones and DNA during nucleosome assembly remain unknown. In particular, the function of the largest HIRA subunit CABIN1 is enigmatic. Here, we show that HIRA is not simply a passive histone hand-off factor but remains engaged across multiple stages of nucleosome assembly, including a close interaction with the nucleosome. Cryo-EM structures reveal that HIRA forms an extended arch-like structure that binds the nucleosome primarily through extensive CABIN1 contacts with histones, histone tails, nucleosomal DNA, and linker DNA, during the final stage of nucleosome assembly. Together, our results suggest a testable mechanism for HIRA-mediated nucleosome assembly and product release and provide the basis for elucidating the molecular details of this fundamental biological process.

biochemistry↗

bioRxiv2026

TFAM organizes DNA into compact higher order structures

TFAM (Transcription Factor A, Mitochondrial) is an essential human protein that plays two key roles in mitochondrial DNA (mtDNA) homeostasis. TFAM acts as a transcription factor that specifically binds to promoter regions, but it is also solely responsible for organizing mtDNA into nucleoids by nonspecifically covering the entire genome. Many studies have addressed TFAM in transcription regulation, but its role as a genome organizing entity is not well characterized. The current understanding of how TFAM compacts DNA into nucleoids is based on crystal structures of a TFAM monomer bound to short fragments of DNA (22-28 bp). However, this does not adequately reflect the biological role of TFAM in organizing the nucleoid where multiple TFAM molecules oligomerize on the 16.5 kb genome to form the nucleoid. Here, we present a biochemical and structural analysis of TFAM oligomerization on longer DNA. Our results show that TFAM compacts longer segments of DNA into higher order complexes that are homogenous yet exhibit continuous conformational dynamics. Significance statementMutations or damage to mitochondrial DNA (mtDNA) severely impairs cellular respiration and is implicated in many human diseases and aging. Transcription Factor A, Mitochondrial (TFAM) is an essential protein that is solely responsible for packaging mtDNA into nucleoids thereby shielding it from DNA damage. Despite its importance, the mechanism by which this is accomplished is poorly understood. Here, we use biochemistry to show that TFAM oligomerizes on DNA to form compact, homogenous higher order structures in solution. We also examined these complexes at a low resolution using cryo-EM, suggesting an organizational unit of mtDNA. This work reveals there may be a regular organization to mitochondrial nucleoids, providing the basis for further understanding mtDNA compaction by TFAM.

biophysics↗

bioRxiv2025

Histone diversity in the archaeal domain of life

Archaea represent a distinct domain of life that is genetically and biochemically unique from bacteria and eukaryotes. Two-thirds of all archaea encode histones, proteins that are ubiquitously used to structure chromatin in eukaryotes. Archaeal histone sequences are much less conserved than their eukaryotic counterparts, yet insight into how they structure DNA is limited to only a few species that fail to represent the diversity of the archaeal domain. Archaea have adapted to the most diverse and extreme environments on our planet, requiring protection of the genome against a multitude of external pressures. Here, we use bioinformatics, structure prediction, and molecular dynamics simulations to survey the diversity of histone-like sequences in all available archaeal genomes and to understand how they might interact with DNA. We have identified five distinct types of histones which are combined in seven different strategies, involving either single histones, multiple histones of the same type, or combinations of several types of histones in one genome. We show that some strategies correlate with environmental pressures, and some are phylogenetically restricted. Despite highly divergent amino acid sequences, structure predictions and simulations suggest similar histone DNA binding modes for most classes. Our work provides a guide to efficiently survey diverse strategies for histone-based DNA organization in archaea using biophysical and structural approaches, for a complete view of the rich diversity of histone strategies in the archaeal domain in a targeted manner.

microbiology↗

bioRxiv2025

Melbournevirus encodes a shorter H2B-H2A doublet histone variant that forms structurally distinct nucleosome structures.

Unique among viruses, some giant viruses utilize histones to organize their genomes into nucleosomes. Melbournevirus encodes a distinct H2B-H2A histone doublet variant in addition to the canonical H4-H3 and H2B-H2A doublets. This viral histone variant has a truncated H2B portion and its amino acid sequence deviates from that of the main viral H2B-H2A throughout the entire coding region. It is less abundant than the main H2B-H2A doublet, is essential for melbournevirus fitness, and is conserved in all Marseilleviridae. The cryo-EM structure of a nucleosome-like particle reconstituted with this H2B-H2A variant and viral H4-H3 reveals that only 90 base pairs of DNA are stably bound, significantly less than in eukaryotic nucleosomes and viral nucleosomes made with the main fused viral histone doublets. The reduced ability to bind DNA can be attributed to structural differences between variant and main H2B-H2A. Variant melbournevirus nucleosomes are less stable, possibly aiding rapid genome unpacking to initiate gene expression. Our results highlight the remarkable propensity of giant viruses to appropriate the utility of histones for their specialized purposes.

molecular biology↗

bioRxiv2024

SMARCAD1 Regulates R-Loops at Active Replication Forks Linked to Cancer Mutation Hotspots

DNA replication often encounters obstacles like the stalled transcription machinery and R-loops. While ribonucleases and DNA-RNA helicases can resolve these structures, the role of chromatin remodelers remains understudied. Through a series of in vitro and in vivo experiments, we show that the chromatin remodeler SMARCAD1, which associates with active replication forks, is crucial for resolving nearby R-loops to maintain fork stability. SMARCAD1 directly binds R-loops via its ATPase domain and associates with the replisome through its N-terminus region. Both interactions are critical for resolving R-loops within cells. Genome-wide assays reveal that cells expressing mutant SMARCAD1 accumulate significantly more R-loops than wild-type cells, particularly in regions distinct from known fork blockage-prone sites. These R-loop-enriched regions in SMARCAD1 mutants also exhibit increased mutagenesis in germline tumors, suggesting they are mutation hotspots in cancer. Therefore, SMARCAD1 acts as an R-loop sensor and resolvase at actively progressing forks, maintaining genome stability and preventing tumorigenesis.

genetics↗

bioRxiv2024

Characterization of Medusavirus encoded histones reveals nucleosome-like structures and a unique linker histone

The organization of DNA into nucleosomes is a ubiquitous and ancestral feature that was once thought to be exclusive to the eukaryotic domain of life. Intriguingly, several representatives of the Nucleocytoplasmic Large DNA Viruses (NCLDV) encode histone-like proteins that in Melbournevirus were shown to form nucleosome-like particles. Medusavirus medusae (MM), a distantly related giant virus, encodes all four core histone proteins and, unique amongst most giant viruses, a putative acidic protein with two domains resembling linker histone H1. Here we report the structure of nucleosomes assembled with Medusavirus histones and highlight similarities and differences with eukaryotic and Melbournevirus nucleosomes. Our structure provides insight into how variations in histone tail and loop lengths are accommodated within the context of the nucleosome. We show that Medusavirus histones assemble into tri-nucleosome arrays, and that the putative linker histone H1 does not function in chromatin compaction. These findings expand our understanding of viral histones and suggest that Medusavirus histones represent a snapshot in the evolutionary timeline of nucleosome architecture. ONE SENTENCE SUMMARYThe four Medusavirus medusae core histones form nucleosome-like structures that combine features of eukaryotic and other viral nucleosomes.

molecular biology↗

bioRxiv2023

A FACT-ETS-1 Antiviral Response Pathway Restricts Viral Replication and is Countered by Poxvirus A51R Proteins

The FACT complex is an ancient chromatin remodeling factor comprised of Spt16 and SSRP1 subunits that regulates specific eukaryotic gene expression programs. However, whether FACT regulates host immune responses to infection was unclear. Here, we identify an antiviral pathway mediated by FACT, distinct from the interferon response, that restricts poxvirus replication. We show that early viral gene expression triggers nuclear accumulation of specialized, SUMOylated Spt16 subunits of FACT required for expression of ETS-1, a downstream transcription factor that activates a virus restriction program. However, poxvirus-encoded A51R proteins block ETS-1 expression by outcompeting SSRP1 for binding to SUMOylated Spt16 in the cytosol and by tethering SUMOylated Spt16 to microtubules. Moreover, we show that A51R antagonism of FACT enhances both poxvirus replication in human cells and viral virulence in mice. Finally, we demonstrate that FACT also restricts unrelated RNA viruses, suggesting a broad role for FACT in antiviral immunity. Our study reveals the FACT-ETS-1 Antiviral Response (FEAR) pathway to be critical for eukaryotic antiviral immunity and describes a unique mechanism of viral immune evasion.

microbiology↗

bioRxiv2023

Histone-organized chromatin in bacteria

Histones are the principal constituents of chromatin in eukaryotes and most archaea, while bacteria generally rely on an orthogonal set of proteins to organize their chromosomes. However, several bacterial genomes encode proteins with putative histone fold domains. Whether these proteins are structurally and functionally equivalent to archaeal and eukaryotic histones is unknown. Here, we demonstrate that histones are essential and are major components of chromatin in the bacteria Bdellovibrio bacteriovorus and Leptospira interrogans. Patterns of sequence evolution suggest important roles in several additional bacterial clades. Structural analysis of the B. bacteriovorus histone (Bd0055) dimer shows that histone fold topology is conserved between bacteria, archaea, and eukaryotes. Yet, unexpectedly, Bd0055 binds DNA end-on and forms a sheath of tightly packed histone dimers to encase straight DNA. This binding mode is in stark contrast to archaeal, eukaryotic, and viral histones, which invariably bend and wrap DNA around their outer surface. Our results demonstrate that histones are integral chromatin components across the tree of life and highlight organizational innovation in the domain Bacteria.

molecular biology↗

bioRxiv2022

Dynamics of endogenous PARP1 and PARP2 during DNA damage revealed by live-cell single-molecule imaging

Summary/AbstractPARP1 contributes to genome architecture and DNA damage repair through its dynamic association with chromatin. PARP1 and PARP2 (PARP1/2) recognize damaged DNA and recruit the DNA repair machinery. Using single molecule microscopy in live cells, we monitored the movement of PARP1/2 on undamaged and damaged chromatin. We identify two classes of freely diffusing PARP1/2 and two classes of bound PARP1/2. The majority (> 60%) of PARP1/2 diffuse freely in both undamaged and damaged nuclei and in the presence of inhibitors of PARP1/2 used for cancer therapy (PARPi). Laser induced DNA damage results in a small fraction of slowly diffusing PARP1 and PARP2 to become transiently bound. Treatment of cells with PARPi in the presence of DNA damage causes subtle changes in the dynamics of bound PARP1/2, in contrast to bulk studies that suggest PARP trapping. Our results imply that next-generation PARPi could specifically target the small fraction of DNA-bound PARP1/2.

biophysics↗

bioRxiv2021

CENP-N promotes the compaction of centromeric chromatin

The histone variant CENP-A is the epigenetic determinant for the centromere, where it is interspersed with canonical H3 to form a specialized chromatin structure that nucleates the kinetochore. The arrangement of nucleosomes at the centromere into higher order structure is unknown. Here we demonstrate that the CENP-A interacting protein CENP-N promotes the stacking of CENP-A containing mono-nucleosomes and nucleosomal arrays through a previously undefined interaction between the 6 helix of CENP-N with the DNA of a neighboring nucleosome. We describe the cryoEM structures and biophysical characterization of such CENP-N mediated nucleosome stacks and nucleosomal arrays and demonstrate that this interaction is responsible for the formation of densely packed chromatin at the centromere in the cell. Our results provide first evidence that CENP-A, together with CENP-N, promotes specific chromatin higher order structure at the centromere. One-Sentence SummaryThe centromere-associated protein CENP-N promotes centromere-specific nucleosome stacking and higher order structures in vitro and in the cell.

molecular biology↗

bioRxiv2021

Melbournevirus-encoded histone doublets are recruited to virus particles and form destabilized nucleosome-like structures

The organization of genomic DNA into defined nucleosomes has long been viewed as a hallmark of eukaryotes. This paradigm has been challenged by the identification of minimalist histones in archaea, and more recently by the discovery of genes that encode fused remote homologs of the four eukaryotic histones in Marseilleviridae, a subfamily of giant viruses that infect amoebae. We demonstrate that viral doublet histones localize to the cytoplasmic viral factories after virus infection, and ultimately to mature virions. CryoEM structures of viral nucleosome-like particles show strong similarities to eukaryotic nucleosomes, despite the limited sequence identify. The unique connectors that link the histone chains contribute to the observed instability of viral nucleosomes, and some histone tails assume structural roles. Our results further expand the range of organisms that have nucleosomes and suggest a specialized function of histones in the biology of these unusual viruses. One Sentence SummarySome large DNA viruses encode fused histone doublets that are targeted to viral factories and assemble into open nucleosome-like structures.

molecular biology↗

bioRxiv2021

Structure and function of the chromatin remodeler SMARCAD1 with its nucleosome substrate

The ATP-dependent chromatin remodeler SMARCAD1 acts on nucleosomes during DNA repair and transcription, but despite its implication in disease, information on its structure and function is scarce. Chromatin remodelers use a variety of ways to engage nucleosomes, and outcomes of the ATP-dependent reactions vary widely. Here we show that SMARCAD1 transfers the entire histone octamer from one DNA segment to another in an ATP-dependent manner but is also capable of de novo nucleosome assembly from histone octamer, due to its ability to bind all histones simultaneously. We describe the cryoEM structure of SMARCAD1 in complex with a nucleosome and show that it engages its substrate unlike any other chromatin remodeler. Our combined data allow us to put forward a testable model for SMARCAD1 mechanism. One-Sentence SummaryThe single subunit chromatin remodeler SMARCAD1 engages nucleosomes in a unique manner and transfers the entire histone octamer.

biochemistry↗

bioRxiv2021

Automated Modeling of Protein Accumulation at DNA Damage Sites using qFADD.py

Cells are exposed to a plethora of influences that can cause damage to DNA and alter the genome, often with detrimental consequences for health. Cells mitigate this damage through a variety of repair protein pathways, and accurate measurement of the accumulation, action, and dissipation timescales of these repair proteins is required to fully understand the DNA damage response. Recently, we described the Q-FADD (Quantitation of Fluorescence Accumulation after DNA Damage) method, which enhances the analytical power of the widely used laser microirradiation technique. In that study, Q-FADD and its preprocessing operations required licensed software and a significant amount of user overhead to find the model of best fit. Here, we present "qFADD.py", an open-source implementation of the Q-FADD algorithm that is available as both a stand-alone software package and on a publicly accessible webserver (https://qfadd.colorado.edu/). Furthermore, we describe significant improvements to the fitting and preprocessing methods that include corrections for nuclear drift and an automated grid-search for the model of best fit. To improve statistical rigor, the grid-search algorithm also includes automated simulation of replicates. As an example, we discuss the recruitment dynamics of the signaling protein PARP1 to DNA damage sites, and we show how to compare different populations of qFADD.py models. Statement of SignificanceCells are constantly bombarded by factors that can alter or damage their genome, and they have evolved a variety of proteins that can identify and fix this damage. To fully understand how these proteins interact in repair pathways, we need robust methods to quantify the timescales between the initial identification of the DNA damage event and the subsequent protein-protein interactions that lead to repair. Laser microirradiation is a popular method for studying these repair protein cascades in vivo, and methods for quantifying the timescales of recruitment in these experiments have historically been simple to implement but lacking in physical interpretation. Here, we present qFADD.py, the next iteration of the Q-FADD method, which uses Monte Carlo diffusion models to interpret repair protein recruitment timescales to sites of DNA damage. By moving towards automated fitting procedures with minimal bias from the user, qFADD.py provides a statistically robust but low-effort means to analyze laser microirradiation experiments through a biophysical framework.

biophysics↗

bioRxiv2020

HPF1 and nucleosomes mediate a dramatic switch in activity of PARP1 from polymerase to hydrolase

Poly(ADP-ribose) polymerase 1 (PARP1) is an important player in the response to DNA damage. Recently, histone PARylation factor (HPF1) was shown to be a critical modulator of the activity of PARP1 by facilitating PARylation of histones and redirecting the target amino acid specificity from acidic to serine residues. Here we investigate the mechanism and specific consequences of HPF1-mediated PARylation using nucleosomes as both activators and substrates for PARP1. HPF1 provides that catalytic base Glu284 to substantially redirect PARylation by PARP1 such that the histones in nucleosomes become the primary recipients of PAR chains. Surprisingly, HPF1 partitions most of the reaction product to free ADPR, resulting in much shorter PAR chains compared to reactions in the absence of HPF1. This HPF1-mediated switch from polymerase to hydrolase has important implications for the PARP1-mediated response to DNA damage and raises interesting new questions about the role of intracellular ADPR and depletion of NAD+.

biochemistry↗

bioRxiv2020

Archaeal chromatin 'slinkies' are inherently dynamic complexes with deflected DNA wrapping pathways

Eukaryotes and many archaea package their DNA with histones. While the four eukaryotic histones wrap [~]147 DNA base pairs into nucleosomes, archaeal histones form "nucleosome-like" complexes that continuously wind between 60 - 500 base pairs of DNA ("archaeasomes"), suggested by crystal contacts and analysis of cellular chromatin. Solution structures of large archaeasomes (>90 DNA base pairs) have never been directly observed. Here, we utilize molecular dynamics simulations, analytical ultracentrifugation, and cryoEM to structurally characterize the solution state of archaeasomes on longer DNA. Simulations reveal dynamics of increased accessibility without disruption of DNA-binding or tetramerization interfaces. Mg2+ concentration influences compaction, and cryoEM densities illustrate that DNA is wrapped in consecutive substates arranged 90{degrees} out-of-plane with one another. Without ATP-dependent remodelers, archaea may leverage these inherent dynamics to balance chromatin packing and accessibility.

biophysics↗

bioRxiv2019

Bridging of nucleosome-proximal DNA double-strand breaks by PARP2 enhances its interaction with HPF1

Poly(ADP-ribose) Polymerase 2 (PARP2) is one of three DNA-dependent PARPs involved in the detection of DNA damage. Upon binding to DNA double-strand breaks, PARP2 uses nicotinamide adenine dinucleotide to synthesize poly(ADP-ribose) (PAR) onto itself and other proteins, including histones. PAR chains in turn promote the DNA damage response by recruiting downstream repair factors. These early steps of DNA damage signaling are relevant for understanding how genome integrity is maintained and how their failure leads to genome instability or cancer. There is no structural information on DNA double-strand break detection in the context of chromatin. Here we present a cryo-EM structure of two nucleosomes bridged by human PARP2 and confirm that PARP2 bridges DNA ends in the context of nucleosomes bearing short linker DNA. We demonstrate that the conformation of PARP2 bound to damaged chromatin provides a binding platform for the regulatory protein Histone PARylation Factor 1 (HPF1), and that the resulting HPF1*PARP2*nucleosome complex is enzymatically active. Our results contribute to a structural view of the early steps of the DNA damage response in chromatin.

biochemistry↗

bioRxiv2018

Q-FADD: A mechanistic approach for modeling the accumulation of proteins at sites of DNA damage by free diffusion

The repair of DNA damage requires the ordered recruitment of many different proteins that are responsible for signaling and subsequent repair. A powerful tool for studying the orchestrated accumulation of these proteins at damage sites is laser microirradiation in live cells, followed by monitoring of the accumulation of the fluorescently labeled protein in question. Despite the widespread use of this approach, there exists no rigorous method for characterizing this process quantitatively. Here we introduce a free diffusion model that explicitly accounts for the unique topology of individual nuclei and quantitatively describes the accumulation of two test proteins, poly-ADP-ribose polymerases 1 and 2. Application of our model to other proteins will yield novel insights into the timing and mechanism of DNA repair.

biochemistry↗