Biology subjects

Bowerman, S.

Publications and source records attributed to Bowerman, S..

6 recordsLinked to original sources

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

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

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

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

BEES: Bayesian Ensemble Estimation from SAS, a SASSIE-web Module

1Many biomolecular complexes exist in a flexible ensemble of states in solution which are necessary to perform their biological function. Small angle scattering (SAS) measurements are a popular method for characterizing these flexible molecules due to their relative ease of use and ability to simultaneously probe the full ensemble of states. However, SAS data is typically low-dimensional and difficult to interpret without the assistance of additional structural models. In theory, experimental SAS curves can be reconstituted from a linear combination of theoretical models, although this procedure carries significant risk of overfitting the inherently low-dimensional SAS data. Previously, we developed a Bayesian-based method for fitting ensembles of model structures to experimental SAS data that rigorously avoids overfitting. However, we have found that these methods can be difficult to incorporate into typical SAS modeling workflows, especially for users that are not experts in computational modeling. To this end, we present the "Bayesian Ensemble Estimation from SAS" (BEES) program. Two forks of BEES are available, the primary one existing as module for the SASSIE webserver and a developmental version that is a standalone python program. BEES allows users to exhaustively sample ensemble models constructed from a library of theoretical states and to interactively analyze and compare each models performance. The fitting routine also allows for secondary data sets to be supplied, thereby simultaneously fitting models to both SAS data as well as orthogonal information. The flexible ensemble of K63-linked ubiquitin trimers is presented as an example of BEES capabilities.

biophysics↗