Biology subjects

Krejci, L.

Publications and source records attributed to Krejci, L..

5 recordsLinked to original sources

bioRxiv2026

An integrated genome-wide resource reveals distinct replication environments of DNA breakage in human cancer cell lines

Replication stress is a major source of genome instability in cancer, yet the genomic features that determine where DNA double-strand breaks (DSBs) arise remain incompletely defined. Here, we establish an integrated genome-wide resource of DSBs, DNA replication, replication timing, transcription, and R-loops in two widely used cancer cell lines, U2-OS and HeLa, under steady-state conditions and following prolonged low-dose DNA polymerase inhibition. We combine these datasets with systematic statistical testing and comparative analytical approaches to define the replication environments associated with genome fragility. Endogenous DSBs preferentially accumulated at origin-rich initiation zones, where R-loops were enriched, whereas prolonged DNA polymerase inhibition redirected DSB formation toward origin-poor, late-replicating regions. Among the replication features examined, replication initiation zones and late-replicating areas were most sensitive to prolonged replication stress. R-loops were specifically enriched at initiation zones but depleted from late-replicating regions and recurrent DNA break clusters (RDCs), demonstrating that their association with genome fragility is context dependent. Replication stress further induced RDCs within long, actively transcribed genes, while their locations only partially overlapped with common fragile sites. Functional analyses identified MUS81 as a major regulator of RDC formation. MUS81 loss increased RDCs, whereas restoration of its catalytic activity suppressed them, indicating that MUS81 resolves replication intermediates before they persist into late-replicating fragile regions. Together, this resource and analytical framework provide a systematic basis for dissecting how replication architecture, transcription, and DNA processing shape genome fragility in cancer cells.

genomics↗

bioRxiv2026

BRC-2/BRCA2-RIPR-1 mediated constraints on homologous recombination execution are spatiotemporally regulated during meiosis

Homologous recombination-dependent processing of meiotic double-strand breaks is crucial to generate functional gametes. The breast and ovarian cancer susceptibility gene BRCA2 is essential for the loading of RAD51-DMC1 recombinases at DNA breaks to allow accurate repair. Here, we identify the novel protein RIPR-1 as a binding partner of Caenorhabditis elegans BRC-2/BRCA2. RIPR-1 and BRC-2 form an obligate complex both in vitro and in vivo and display interdependent loading in developing oocytes. Loss of ripr-1 results in the complete destabilization of BRC-2, while brc-2 is only partially required to preserve RIPR-1 stability. Strikingly, we found a dramatic accumulation of RAD-51 in late pachytene cells of both ripr-1 and brc-2 nulls, unveiling a BRCA2-independent pathway for recruitment of RAD-51 at resected meiotic DNA breaks. RIPR-1 enhances BRC-2 binding to single-stranded DNA and unlike the individual components, the assembled RIPR-1-BRC-2 complex can also bind double-stranded DNA in vitro. Surprisingly, we observe that RAD-51-dependent crossover designation still occurs in absence of RIPR-1-BRC-2, indicating partial, and stage-dependent constraints on homologous recombination execution. Altogether, our work identifies a novel BRC-2 interactor and sheds new light on HR-mediated regulation of DNA repair during meiosis.

Cell Biology↗

bioRxiv2025

Discovery of two structurally distinct classes of inhibitors targeting the nuclease MUS81 and enhancing efficacy of chemotherapy in cancer cells

Nucleases are emerging as promising pharmacological targets due to their essential role in maintaining genomic stability, which is crucial for cellular viability and can be exploited in the prevention and treatment of various diseases, including cancer. The conserved structure-specific endonuclease MUS81 is required for resolving branched DNA intermediates during replication, repair, and recombination. Aberrant activity of MUS81 leads to DNA damage, chromosomal abnormalities and genome instability, and contributes to oncogenesis. Pharmacological targeting of MUS81 thus represents an attractive underexplored therapeutic approach. Here we describe the discovery of two chemically distinct classes of small-molecule inhibitors of MUS81, exemplified by the compounds MU262 and MU876. Both compounds can effectively inhibit MUS81 in vitro and in the cell-based context and sensitize cancer cells to DNA-damaging agents through impairing their ability to repair DNA lesions. These compounds can be also used as chemical biology tools for further exploration of MUS81 function, and as leads in the process of drug discovery focused on development of new therapies that exploit DNA repair vulnerabilities in the treatment of cancer.

molecular biology↗

bioRxiv2023

Concurrent D-loop cleavage by Mus81 and Yen1 yields half-crossover precursors

Homologous recombination involves the formation of branched DNA molecules that may interfere with chromosome segregation. To resolve these persistent joint molecules, cells rely on the activation of structure-selective endonucleases (SSEs) during the late stages of the cell cycle. However, the premature activation of SSEs compromises genome integrity, due to untimely processing of replication/recombination intermediates. Here, we employed a biochemical approach to demonstrate that the budding yeast SSEs Mus81 and Yen1 possess the ability to cleave the central recombination intermediate known as the displacement loop or D-loop. Moreover, we confirm that, consistently with previous genetic data, the simultaneous action of Mus81 and Yen1, followed by ligation, is sufficient to recreate the formation of a half-crossover precursor in vitro. Our results provide not only mechanistic explanation for the formation of a half-crossover, but also highlight the critical importance for precise regulation of these SSEs to prevent chromosomal rearrangements.

molecular biology↗

bioRxiv2020

Antibiotic-induced DNA damage results in a controlled loss of pH homeostasis and genome instability

Extracellular pH has been assumed to play little if any role in how bacteria respond to antibiotics and antibiotic resistance development. Here, we show that the intracellular pH of Escherichia coli equilibrates to the environmental pH following antibiotic treatment. We demonstrate that this allows the environmental pH to influence the transcription of various DNA damage response genes and physiological processes such as filamentation. Using purified RecA and a known pH-sensitive mutant variant RecA K250R we show how pH can affect the biochemical activity of a protein central to control of the bacterial DNA damage response system. Finally, two different mutagenesis assays indicate that environmental pH affects antibiotic resistance development. Specifically, at environmental pHs greater than six we find that mutagenesis plays a significant role in producing antibiotic resistant mutants. At pHs less than or equal to 6 the genome appears more stable but extensive filamentation is observed, a phenomenon that has previously been linked to increased survival in the presence of macrophages.

microbiology↗