Biology subjects

Whale, A.

Publications and source records attributed to Whale, A..

4 recordsLinked to original sources

bioRxiv2025

TrAEL-seq captures DNA replication dynamics in mammalian cells

Precise DNA replication is critical to the maintenance of genome stability, and the DNA replication machinery is a focal point of many current and upcoming chemotherapeutics. TrAEL-seq is a robust method for profiling DNA replication genome-wide that works in unsynchronised cells and does not require treatment with drugs or nucleotide analogues. Here, we provide an updated method for TrAEL-seq including multiplexing of up to 6 samples that dramatically improves sample quality and throughput, and we validate TrAEL-seq in multiple mammalian cell lines. The updated protocol is straightforward and robust yet provides excellent resolution comparable to OK-seq in mammalian cell samples. High resolution replication profiles can be obtained across large panels of samples and in dynamic systems, for example during the progressive onset of oncogene induced senescence. In addition to mapping zones where replication initiates and terminates, TrAEL-seq is sensitive to replication fork speed, revealing effects of both transcription and proximity to replication Initiation Zones on fork progression. Although forks move more slowly through transcribed regions, this does not have a significant impact on the broader dynamics of replication fork progression, which is dominated by rapid fork movement in long replication regions (>1Mb). Short and long replication regions are not intrinsically different, and instead replication forks accelerate across the first [~]1 Mb of travel such that forks progress faster in the middle of regions lying between widely spaced Initiation Zones. We propose that this is a natural consequence of fewer replication forks being active later in S phase when these distal regions are replicated and there being less competition for replication factors.

molecular biology↗

bioRxiv2025

RNA helicase DDX1 regulates germinal centre selection and affinity maturation by promoting tRNA ligase activity

Clonal expansion of antigen-specific B-cells defines effective germinal centre responses and is key for the generation of high-affinity antibodies. While positive selection in germinal centres has been associated with anabolic metabolism and cell growth, the downstream drivers of B-cell proliferation are not well understood. Here we report that the RNA helicase DDX1 is required for germinal centre maturation and accrual of dark-zone cellularity. Upon interaction with T-follicular helper cells, DDX1-deficient B-cells upregulate c-MYC but do not clonally expand. We show that positive selection is coupled with an increase in mRNA translation, that is dependent on DDX1. DDX1 endows B-cells with the protein biosynthetic capability that is required for rapid cell proliferation. It does so by modulating the activity of the tRNA ligase complex and tRNA splicing. Our data reveal that mRNA translation efficiency is a key determinant of B-cell fitness during germinal centre responses.

immunology↗

bioRxiv2024

RNA-binding proteins control the G2-M checkpoint of the germinal centre B cell

How germinal centre (GC) B cells undergo rapid cell division while maintaining genome stability is poorly understood. Here, we show that the RNA-binding proteins ZFP36L1 and ZFP36L2 act downstream of antigen-sensing and protect GC B cells from replication stress by controlling a cell cycle-related RNA post-transcriptional regulon. ZFP36L1 and ZFP36L2 safeguard faithful completion of mitosis by restraining the expression of CDK1 and cyclin B1, whilst controlling their activity through regulation of a p21-mediated negative feedback loop. In the absence of ZFP36L1 and ZFP36L2, GC B cells arrest in G2-M and die by apoptosis, resulting in curtailed GC responses. This is associated with stalling of the DNA replication fork at active replication initiation zones, which causes replication stress and increased activity of the ATR/CHK1 DNA damage response. Our findings reveal that gene regulation by RNA-binding proteins is essential for a functional G2-M checkpoint to operate in GC B cells.

immunology↗

bioRxiv2023

The Fork Protection Complex protects long replicons from DNA damage at the cost of genome instability induced by DNA topological stress.

Tof1/Timeless protects eukaryotic cells from DNA replication stress as part of the Fork Protection Complex (FPC). Tof1 supports rapid DNA replication, fork pausing, and resolution of DNA topological stress. Here, we show that disruption of FPC function through loss of either Tof1 or Mrc1 results in DNA damage in long replicons. Despite increasing DNA damage in long replicons, loss of either Tof1 or Mrc1 concurrently reduces DNA damage in regions prone to damage caused by DNA topological stress, indicating that the rapid replication promoted by the FPC fosters completing DNA replication at the cost of increased vulnerability to DNA topological stress. Supporting this we find that a tof1 mutation that selectively inhibits DNA topological stress resolution increases DNA damage in contexts prone to DNA topological stress. Our data indicates that the FPC balances rapid replication with recruitment of topoisomerase I to resolve the topological stress generated by increased DNA unwinding.

molecular biology↗