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Krietsch, J.

Publications and source records attributed to Krietsch, J..

5 recordsLinked to original sources
bioRxiv2026

Non-canonical ATR signalling via NBS1 phosphorylation propagates fork slowing from stressed to unperturbed nuclear regions

DNA replication forks frequently encounter obstacles and remodel into four-way junctions to actively slow DNA fork progression. Fork slowing can also spread to undamaged forks via an ATR-dependent mechanism that remained elusive. Here, using mild genotoxic stress, we show that fork slowing and reversal require full ATR activity, but no canonical ATR activators and signalling partners, defining a non-canonical ATR pathway distinct from origin firing control. Phospho-proteomics in S-phase cells under checkpoint-blind replication stress identified a subset of ATR-dependent phospho-sites, such as S343 on NBS1, the regulatory subunit of the MRN complex. This residue is essential to stimulate MRN exonuclease activity in vitro and required in cells for global fork slowing upon mild DNA damage. Ultimately, local UV-C micro-irradiation reveals that ATR-dependent MRN-stimulated resection dampens DNA synthesis at lesions and propagates fork slowing to undamaged chromatin, supporting MRN-mediated ssDNA exposure as a mean to coordinate replication slowdown across the nucleus.

cell biology↗
bioRxiv2023

Replication fork plasticity upon replication stress requires rapid nuclear actin polymerization

Cells rapidly respond to replication stress actively slowing fork progression and inducing fork reversal. How replication fork plasticity is achieved in the context of nuclear organization is currently unknown. Using nuclear actin probes in living and fixed cells, we visualized nuclear actin filaments in unperturbed S phase, rapidly extending in number and thickness upon genotoxic treatments, and taking frequent contact with replication factories. Chemically or genetically impairing nuclear actin polymerization shortly before these treatments prevents active fork slowing and abolishes fork reversal. Defective fork plasticity is linked to reduced recruitment of RAD51 and SMARCAL1 to nascent DNA. Conversely, PRIMPOL gains access to replicating chromatin, promoting unrestrained and discontinuous DNA synthesis, which is associated with increased chromosomal instability and decreased cellular resistance to replication stress. Hence, nuclear F-actin orchestrates replication fork plasticity and is a key molecular determinant in the rapid cellular response to genotoxic treatments.

cancer biology↗
bioRxiv2023

Single molecule MATAC-seq reveals key determinants of DNA replication origin efficiency

SummaryStochastic origin activation gives rise to significant cell-to-cell variability in the pattern of genome replication. The molecular basis for heterogeneity in efficiency and timing of individual origins is a long-standing question. Here, we developed Methylation Accessibility of TArgeted Chromatin domain Sequencing (MATAC-Seq) to determine single-molecule chromatin accessibility of specific genomic loci after targeted purification in their native chromatin context. Applying MATAC-Seq to selected early-efficient (EE) and late-inefficient (LI) budding yeast replication origins revealed large heterogeneity of chromatin states. Disruption of INO80 or ISW2 chromatin remodeling complexes leads to changes at individual nucleosomal positions that correlate with changes in their replication efficiency. We found a chromatin state with an optimal 100-115bp nucleosome-free region in combination with surrounding well-positioned nucleosomes and open +2 linker region is a strong predictor for efficient origin activation. Thus, MATAC-Seq identifies the large spectrum of alternative chromatin states that co-exist on a given locus previously masked in population-based experiments and provides a mechanistic basis for origin activation heterogeneity during DNA replication of eukaryotic cells. Consequently, our single-molecule assay for chromatin accessibility will be ideal to define single-molecule heterogeneity across many fundamental biological processes such as transcription, replication, or DNA repair in vitro and ex vivo.

biochemistry↗
bioRxiv2020

Chromatin Accessibility Maps Provide Evidence of Multilineage Gene Priming in Hematopoietic Stem Cells

Hematopoietic stem cells (HSCs) have the capacity to differentiate into vastly different types of mature blood cells. The epigenetic mechanisms regulating the multilineage ability, or multipotency of HSCs are not well understood. To test the hypothesis that cis regulatory elements that control fate decisions for all lineages are primed in HSCs, we used ATAC-seq to compare chromatin accessibility of HSCs with five unipotent cell types. We observed the highest similarity in accessibility profiles between Megakaryocyte Progenitors and HSCs, whereas B cells had the greatest number of regions with de novo gain in accessibility during differentiation. Despite these differences, we identified cis regulatory elements from all lineages that displayed epigenetic priming in HSCs. These findings provide new insights into the regulation of stem cell multipotency, as well as a resource to identify functional drivers of lineage fate. HIGHLIGHTSO_LIHSCs have higher global chromatin accessibility than any unilineage progeny C_LIO_LIMegakaryocyte Progenitors are the most closely related unipotent cell type to HSCs C_LIO_LIB cell commitment involves de novo chromatin accessibility C_LIO_LIEvidence of cis element priming of lineage-specific genes in HSCs C_LI

cell biology↗