Biology subjects

Krasilnikova, M.

Publications and source records attributed to Krasilnikova, M..

4 recordsLinked to original sources

bioRxiv2024

Nuclear DNA replication in Leishmania major relies on a single constitutive origin per chromosome supplemented by thousands of stochastic initiation events

Understanding genome duplication requires characterisation of the locations where DNA replication initiates, termed origins. Genome-wide mapping of DNA replication origins has mainly been derived from population-based techniques, with only a few studies examining origin location and usage at the single-cell or single-molecule level. Leishmania are protozoan parasites where the first attempt to map DNA replication suggested the unprecedented use, for a eukaryote, of just a single origin per chromosome, while a subsequent approach suggested around 200-fold more origins. To reconcile these data and understand DNA replication dynamics in Leishmania major, we have applied DNAscent, a deep learning assay that uses long-read Nanopore sequencing to detect patterns of BrdU incorporation in individual DNA molecules, allowing the description of DNA replication fork movement and prediction of initiation and termination sites across the parasite genome. Our findings confirm the pre-eminence of a single locus of DNA replication initiation in each chromosome and reveal that this locus alone is constitutively activated in S-phase, with bidirectional forks emerging from discrete sites at the ends of multigene transcription units. DNAscent also reveals a much larger number of DNA replication initiation events that have not been detected in any previous mapping and are used stochastically, but whose abundance is greater as chromosome size increases. We show that each of these stochastic initiation sites localise to regions with high AT content, increased G-quadruplex levels and lower chromatin occupancy. In addition, we find markedly increased stochastic DNA replication initiation at sites with lower levels of nascent RNA transcripts. Finally, we show that all DNA replication initiation events result in mutagenesis. This work reveals a novel, bimodal strategy for DNA replication programming in Leishmania that drives genome transmission, replication timing and variation.

microbiology↗

bioRxiv2024

Nanopore sequencing reveals that DNA replication compartmentalisation dictates genome stability and instability in Trypanosoma brucei

The genome of Trypanosoma brucei is structurally complex. Eleven megabase-sized chromosomes each comprise a transcribed core flanked by silent subtelomeres, housing thousands of Variant Surface Glycoprotein (VSG) genes. Additionally, silent VSGs are also found on hundreds of sub-megabase chromosomes that harbour 177 bp repeats of unknown function, and multiple VSG transcription sites localise to the telomeres of both chromosome types. DNA replication dynamics have been described in the megabase chromosome cores but not in the subtelomeres or sub-megabase chromosomes, and targeted early replication of the single active VSG transcription site is unexplained. Here, using Nanopore assembly, we mapped DNA replication across this compartmentalised genome. We show that subtelomeres display a paucity of replication initiation events relative to the core, correlating with increased instability in the silent VSG archive. In addition, early replication of the active VSG transcription site is shown to originate from the telomere, likely causing targeted VSG recombination. Lastly, we demonstrate that the 177 bp repeats act as widespread, conserved DNA replication origins, explaining mitotic stability of the abundant small chromosomes and early DNA replication of megabase chromosome centromeres. Compartmentalized DNA replication dynamics therefore explains how T. brucei balances stable genome transmission with localised instability driving immune evasion.

microbiology↗

bioRxiv2024

R-loops acted on by RNase H1 are a determinant of chromosome length-associated DNA replication timing and genome stability in Leishmania

Genomes in eukaryotes normally undergo DNA replication in a choreographed temporal order, resulting in early and late replicating chromosome compartments. Leishmania, a human protozoan parasite, displays an unconventional DNA replication program in which the timing of DNA replication completion is chromosome size-dependent: larger chromosomes complete replication later then smaller ones. Here we show that both R-loops and RNase H1, a ribonuclease that resolves RNA-DNA hybrids, accumulate in Leishmania major chromosomes in a pattern that reflects their replication timing. Furthermore, we demonstrate that such differential organisation of R-loops, RNase H1 and DNA replication timing across the parasites chromosomes correlates with size-dependent differences in chromatin accessibility, G quadruplex distribution and sequence content. Using conditional gene excision, we show that loss of RNase H1 leads to transient growth perturbation and permanently abrogates the differences in DNA replication timing across chromosomes, as well as altering levels of aneuploidy and increasing chromosome instability in a size-dependent manner. This work provides a link between R-loop homeostasis and DNA replication timing in a eukaryotic parasite and demonstrates that orchestration of DNA replication dictates levels of genome plasticity in Leishmania.

microbiology↗

bioRxiv2023

RAD51-mediated R-loop formation acts to repair transcription-associated DNA breaks driving antigenic variation in Trypanosoma brucei

RNA-DNA hybrids are epigenetic features of all genomes that intersect with many processes, including transcription, telomere homeostasis and centromere function. Increasing evidence suggests RNA-DNA hybrids can provide two conflicting roles in the maintenance and transmission of genomes: they can be the triggers of DNA damage, leading to genome change, or can aid the DNA repair processes needed to respond to DNA lesions. Evasion of host immunity by African trypanosomes, such as Trypanosoma brucei, relies on targeted recombination of silent Variant Surface Glycoprotein (VSG) genes into a specialised telomeric locus that directs transcription of just one VSG from thousands. How such VSG recombination is targeted and initiated is unclear. Here, we show that a key enzyme of T. brucei homologous recombination, RAD51, interacts with RNA-DNA hybrids. In addition, we show that RNA-DNA hybrids display a genome- wide co-localisation with DNA breaks, and that this relationship is impaired by mutation of RAD51. Finally, we show that RAD51 acts to repair highly abundant, localised DNA breaks at the single transcribed VSG, and that mutation of RAD51 alters RNA-DNA hybrid abundance both around the transcribed VSG and across the silent VSG archive. This work reveals a widespread, generalised role for RNA-DNA hybrids in directing RAD51 activity during recombination and uncovers a specialised application of this interplay during targeted DNA break repair needed for the critical T. brucei immune evasion reaction of antigenic variation.

microbiology↗