Biology subjects

Khatri, B. S.

Publications and source records attributed to Khatri, B. S..

10 recordsLinked to original sources

bioRxiv2025

A new material constant of DNA is the major determinant of looping dynamics

DNA bending and looping is crucial for gene expression, packaging, and chromatin organisation, as well as the design of artificial nanomaterials and devices. But what determines how quickly DNA bends? While DNAs static flexibility is well-characterised by its persistence length, we lack an understanding of how quickly DNA responds to mechanical forces: remarkably current semiflexible polymer theory based on solvent dissipation underestimates spontaneous looping times by ~1000-fold. By analysing fluctuations of DNA several kilobases long and developing new theory for bending dissipation in semiflexible polymers, we show DNA bending dynamics cannot be explained by solvent friction alone and requires significant contributions from intramolecular friction. The theory defines a new material constant of DNA -- the bending friction, which we determine to be{zeta} B = 241 {+/-} 17 g nm3/ms. Strikingly, our measurement does not depend on the buffer ionic conditions. We predict bending friction will dominate DNA dynamics between {approx} 50 nm and 420 nm and significantly longer under external force. We show that mean first passage time calculations are greatly simplified when bending friction dominates and so using this constant, with no fitting parameters, we accurately predict the slow experimental spontaneous looping times. Our discovery of significant bending dissipation is unexpected as DNA has no obvious large (> kBT) internal energy barriers. The salt-independence of this dissipation also rules out long range electrostatic interactions as its origins. Instead our findings point to a complex local energy landscape for bending and a potential previously unappreciated role of water binding DNA constraining its local mobility. Our findings radically change our understanding of DNA dynamics and reveal DNA as a viscoelastic semiflexible polymer with dramatically slower dynamics compared to an ideal elastic rod. This work establishes bending friction as a fundamental material property that must underpin any model of DNA dynamics in biology, physics, and nanotechnology.

biophysics↗

bioRxiv2024

Progressive chromosome shape changes during cell divisions, recapitulated by loop capture simulations

Mitotic chromosomes give genome portions the required compaction and mechanical stability for faithful inheritance during cell divisions. Here, we record human chromosome dimensions from their appearance in prophase over successive times in a mitotic arrest. Chromosomes first appear long and uniformly thin. Then, individual chromosome arms become discernible, which continuously shorten and thicken - the longer a chromosome arm, the thicker it becomes. The observed chromosome arm length to width relationship can be described by a power law with progressively increasing exponent. In the search for a molecular explanation of this behavior, the popular loop extrusion model provides no obvious means by which longer arms become thicker. Instead, we find that simulations of an alternative loop capture model recapitulate key features of our observations, including the gradually developing arm length to width relationship. Our analyses portray chromosomes as out-of-equilibrium structures in the process of transitioning towards, but on biologically relevant time scales not typically reaching, steady state.

biophysics↗

bioRxiv2024

Engineering Resilient Gene Drives Towards Sustainable Malaria Control: Predicting, Testing and Overcoming Target Site Resistance

CRISPR-based gene drives are selfish genetic elements with the potential to spread through entire insect populations for sustainable vector control. Gene drives designed to disrupt the reproductive capacity of females can suppress laboratory populations of the malaria mosquito. However, any suppressive intervention will inevitably exert an evolutionary pressure for resistance. Here, we present a pipeline for the accelerated discovery, engineering, and testing of both natural and drive-induced variants that could reverse gene drive spread. We applied our method to stress-test a highly effective gene drive that has evaded resistance in all laboratory-contained releases to date, known as Ag(QFS)1. We showed that previously undetected resistant alleles can arise at low frequency, and discovered novel, partially resistant alleles that can perturb drive-invasion dynamics. We then engineered next-generation gene drives that can actively remove resistant alleles by targeting several highly conserved and non-overlapping sites in the female-specific exon of the doublesex gene. Our models predict that such gene drive designs could suppress large, natural populations of the malaria mosquito in the field.

synthetic biology↗

bioRxiv2021

Weakly deleterious natural genetic variation amplifies probability of resistance in multiplexed gene drive systems

Evolution of resistance is a major barrier to successful deployment of gene drive systems to suppress natural populations, which could greatly reduce the burden of many vector borne diseases. Multiplexed guide RNAs that require resistance mutations in all target cut sites is a promising anti-resistance strategy, since in principle resistance would only arise in unrealistically large populations. Using novel stochastic simulations that accurately model evolution at very large population sizes, we explore the probability of resistance due to three important mechanisms: 1) non-homologous end-joining mutations, 2) single nucleotide mutants arising de novo or, 3) single nucleotide polymorphisms pre-existing as standing variation. Our results explore the relative importance of these mechanisms and highlight a complexity of the mutation-selection-drift balance between haplotypes with complete resistance and those with an incomplete number of resistant alleles. We find this leads to a qualitatively new phenomenon where weakly deleterious naturally occurring variants greatly amplify the probability of multi-site resistance. This challenges the intuition that many target sites would guarantee prevention of resistance, where in the face of standing genetic variation, it can be probable even in not very large populations. This result has broad application to resistance arising in many multi-site evolutionary scenarios including multi-drug resistance to antibiotics, antivirals and cancer treatments, as well as the evolution of vaccine escape mutations in large populations.

evolutionary biology↗

bioRxiv2021

A theory of the dynamics of DNA loop initiation in condensin/cohesin complexes

The structural maintenance of chromosome complexes exhibit the remarkable ability to actively extrude DNA, which has led to the appealing and popular "loop extrusion" model to explain one of the most important processes in biology: the compaction of chromatin during the cell cycle. A potential mechanism for the action of extrusion is the classic Brownian ratchet, which requires short DNA loops to overcome an initial enthalpic barrier to bending, before favoured entropic growth of longer loops. We present a simple model of the constrained dynamics of DNA loop formation based on a frictional worm like chain, where for circular loops of order, or smaller than the persistence length, internal friction to bending dominates solvent dynamics. Using Rayleighs dissipation function, we show how bending friction can be translated to simple one dimensional diffusion of the angle of the loop resulting in a Smoluchowski equation with a coordinate dependent diffusion constant. This interplay between Brownian motion, bending dissipation and geometry of loops leads to a qualitatively new phenomenon, where the friction vanishes for bends with an angle of exactly 180 degrees, due to a decoupling between changes in loop curvature and angle. Using this theory and given current parameter uncertainties, we tentatively predict mean first passage times of between 1 and 10 seconds, which is of order the cycle time of ATP, suggesting spontaneous looping could be sufficient to achieve efficient initiation of looping.

biophysics↗

bioRxiv2020

Computational and experimental analyses of mitotic chromosome formation pathways in fission yeast

Underlying higher order chromatin organization are Structural Maintenance of Chromosomes (SMC) complexes, large protein rings that entrap DNA. The molecular mechanism by which SMC complexes organize chromatin is as yet incompletely understood. Two prominent models posit that SMC complexes actively extrude DNA loops (loop extrusion), or that they sequentially entrap two DNAs that come into proximity by Brownian motion (diffusion capture). To explore the implications of these two mechanisms, we perform biophysical simulations of a 3.76 Mb-long chromatin chain, the size of the long S. pombe chromosome I left arm. On it, the SMC complex condensin is modeled to perform loop extrusion or diffusion capture. We then compare computational to experimental observations of mitotic chromosome formation. Both loop extrusion and diffusion capture can result in native-like contact probability distributions. In addition, the diffusion capture model more readily recapitulates mitotic chromosome axis shortening and chromatin density enrichment. Diffusion capture can also explain why mitotic chromatin shows reduced, as well as more anisotropic, movements, features that lack support from loop extrusion. The condensin distribution within mitotic chromosomes, visualized by stochastic optical reconstruction microscopy (STORM), shows clustering predicted from diffusion capture. Our results inform the evaluation of current models of mitotic chromosome formation.

biophysics↗

bioRxiv2018

Robust estimation of recent effective population size from number of independent origins in soft sweeps

Estimating recent effective population size is of great importance in characterising and predicting the evolution of natural populations. Methods based on nucleotide diversity may underestimate current day effective population sizes due to historical bottlenecks, whilst methods that reconstruct demographic history typically only detect long-term variations. However, soft selective sweeps, which leave a fingerprint of mutational history by recurrent mutations on independent haplotype backgrounds, holds promise of an estimate more representative of recent population history. Here we present a simple and robust method of estimation based only on knowledge of the number of independent recurrent origins and the current frequency of the beneficial allele in a population sample, independent of the strength of selection and age of the mutation. Using a forward time theoretical framework, we show the mean number of origins is a function of{theta} = 2N and current allele frequency, through a simple equation, and the distribution is approximately Poisson. This estimate is robust to whether mutants pre-existed before selection arose, and is equally accurate for diploid populations with incomplete dominance. For fast (e.g., seasonal) demographic changes compared to time scale for fixation of the mutant allele, and for moderate peak-to-trough ratios, we show our constant population size estimate can be used to bound the maximum and minimum population size. Applied to the Vgsc gene of Anopheles gambiae, we estimate an effective population size of roughly 6 x 107, and including seasonal demographic oscillations, a minimum effective population size greater than 6 x 106 and a maximum less than 3 x 109.

evolutionary biology↗

bioRxiv2018

Survival of the frequent at finite population size and mutation rate: filing the gap between quasispecies and monomorphic regimes

In recent years, there has been increased attention on the non-trivial role that genotype-phenotype maps play in the course of evolution, where natural selection acts on phenotypes, but variation arises at the level of mutations. Understanding such mappings is arguably the next missing piece in a fully predictive theory of evolution. Although there are theoretical descriptions of such mappings for the monomorphic (N << 1) and deterministic or very strong mutation (N [>>>] 1) limit, given by developments of Iwasas free fitness and quasispecies theories, respectively, there is no general description for the intermediate regime where N ~ 1. In this paper, we address this by transforming Wrights well-known stationary distribution of genotypes under selection and mutation to give the probability distribution of phenotypes, assuming a general genotype-phenotype map. The resultant distribution shows that the degeneracies of each phenotype appear by weighting the mutation term; this gives rise to a bias towards phenotypes of larger degeneracy analogous to quasispecies theory, but at finite population size. On the other hand we show that as population size is decreased, again phenotypes of higher degeneracy are favoured, which is a finite mutation description of the effect of sequence entropy in the monomorphic limit. We also for the first time (to the authors knowledge) provide an explicit derivation of Wrights stationary distribution of the frequencies of multiple alleles.

evolutionary biology↗

bioRxiv2017

Rate Of Fixation Of Rare Variants In A Population

The process of molecular evolution has been dominated by the Kimura paradigm for nearly 60 years; mutations arise at a certain rate in the population and they go to fixation with a probability given by Kimuras classic formula, which assumes there are no further mutations that interfere with the fixation process. An alternative view is that rare variants exist in the population in a mutation-drift-selection balance and rise to fixation through a combination of chance (genetic drift), selection and mutation. When mutations increase in strength, but still in the weak regime, we would expect the Kimura rate approximation to be an overestimate, as a rare variant which grows in frequency will suffer a greater backward flux of mutations, slowing progress to fixation. However, to date calculating important quantities for a general model of selection and mutation, like the rate of fixation of these rare variants has not been tractable in the conventional diffusion approximation of population genetics. Here, we use Fishers angular transformation to convert the frequency-dependent diffusion inherent in population genetics to simple diffusion in an effective potential, which describes the forces of selection, drift and mutation. Once this potential is defined it is simple to show that the mean first passage time is given by a double integral which relate to populations at the barrier. Exact numerical integration shows excellent agreement with discrete Wright-Fisher simulations, which do show a slowing down of the fixation of mutants at higher mutation rates and for strong positive selection, compared to the Kimura prediction. We then seek a closed-form analytical expression for the rate of fixation of mutants, by adapting Kramers approximation for the mean first passage time. This overall gives an accurate approximation, but however, does not improve on the Kimura rate.

evolutionary biology↗

bioRxiv2017

Pairwise Hybrid Incompatibilities Dominate During Allopatric Speciation For A Simple Genotype-Phenotype Map Of Embryonic Spatial Patterning

Developmental system drift is a likely mechanism for the origin of hybrid incompatibilities between closely related species. We examine here the detailed mechanistic basis of hybrid incompatibilities for a genotype-phenotype map for developmental system drift under stabilising selection, where the organismal phenotype is conserved, but the underlying molecular phenotypes and genotype can drift. This leads to number of emergent phenomenon not obtainable by modelling genotype or phenotype alone. Our results show that: 1) speciation is more rapid at smaller population sizes with a characteristic, Orr-like, power law, but at large population sizes slow, characterised by a sub-diffusive growth law; 2) the molecular phenotypes under weakest selection contribute to the earliest incompatibilities; and 3) pairwise incompatibilities dominate over higher order, contrary to previous predictions that the latter should dominate. Our results indicate that biophysics and population size provide a much stronger constraint to speciation than suggested by previous models.

evolutionary biology↗