Biology subjects

Staples, C. J.

Publications and source records attributed to Staples, C. J..

3 recordsLinked to original sources

bioRxiv2025

A nuclease-driven mechanism of post-replicative ssDNA gap suppression.

During replication stress, failure to resolve post-replicative ssDNA gaps generated by PRIMPOL-mediated replication repriming is linked with chemosensitivity, and in all models reported to date the nuclease MRE11 has been implicated as a gap-promoting factor. We have dissected ssDNA gap dynamics following nucleoside analogue-mediated nascent strand termination and report a novel mechanism via which loss of the MRE11 negative regulator MRN-Interacting Protein (MRNIP) leads to MRE11 exonuclease-dependent suppression of post-replicative ssDNA gaps. This process is driven by UBC13 and the B-family TLS polymerase REV3L, suggesting that in the absence of MRNIP, dysregulated MRE11 activity at chain termination sites licenses gap filling via template switching. In the absence of PRIMPOL-dependent repriming, MRNIP loss leads to paradoxical SMARCAL1, MRE11 and primosome-dependent ssDNA gaps, suggesting that nucleolytic digestion of reversed forks generates DNA intermediates that platform primase redundancy in the context of chain termination. We also highlight novel site-specific roles for the anti-resection factor 53BP1 in enabling primase redundancy via suppression of the nuclease EXO1, and in limiting the EXO1-dependent processing of post-replicative ssDNA gaps. Finally, we demonstrate that CDK-dependent MRNIP phosphorylation is required for MRNIP functionality in the regulation of ssDNA gaps and sensitivity to chain terminators. This work represents the first report of nuclease-driven post-replicative gap filling, illuminates an additional level of versatility in the replication stress response, and expands our understanding of the context-dependent links between nuclease regulation and chemoresistance.

cancer biology↗

bioRxiv2025

IFI16 senses and protects stalled replication forks

Replication stress is a key driver of DNA damage and genome instability. Replication stress-induced fork remodelling generates a new DNA end that is vulnerable to the action of nucleases, and which is protected by a range of factors including the canonical tumour suppressors BRCA1 and BRCA2. Here we report that replication stress drives elevated production of cytokines and chemokines in the absence of DNA damage. The DNA sensor IFI16 binds nascent DNA at stalled replication forks and signals via the DNA sensing adaptor STING, to induce the activation of NF-{kappa}B and the production of pro-inflammatory cytokines in response to replication stress. IFI16 also acts directly at stalled replication forks to protect nascent DNA from degradation by the nucleases MRE11 and DNA2. Furthermore, IFI16 is required for the interferon-mediated rescue of fork protection in BRCA-deficient cells, highlighting the critical role of IFI16 in the cross-talk between innate immunity and fork protection during replication stress. HighlightsO_LIReplication stress induces an early innate immune response, which is dependent on the DNA sensing factors IFI16 and STING, but not cGAS C_LIO_LIIFI16 binds directly to nascent DNA at stalled replication forks C_LIO_LIIFI16 prevents nucleolytic degradation of reversed forks C_LIO_LIIFI16 is required for interferon-mediated fork protection in BRCA-deficient cells C_LI

cancer biology↗

bioRxiv2024

CIZ1 regulates G1 length and the CDK threshold for initiation of DNA replication to prevent DNA replication stress

Eukaryotic cell division is regulated by oscillating CDK activity, which must reach critical CDK threshold activity levels to progress through cell cycle stages. In low mitogen, low CDK environments cells exit the cell cycle into a non-proliferative quiescent state, G0, that plays essential roles in stem cell maintenance and cellular homeostasis. CIZ1 regulates cell cycle and epigenetic programmes, and CIZ1 ablation enhances genomic instability after release from quiescence. Here, we determined the mechanisms that promote genome instability in CIZ1 ablated cells using a combination of Fucci(CA) live cell imaging, cell-free DNA replication assays and DNA combing. Cell cycle dynamics are unaffected in CIZ1-/- (CIZ1 KO) fibroblasts; however, a specific post-quiescent phenotype is observed resulting in a reduced G1 phase and cell cycle length. The reduction in G1 length in CIZ1 KO cells is associated with increased cyclin E1/E2 and A2 expression, and enhanced phosphorylation of Rb leading to early restriction point bypass. CIZ1-/- cells are deficient in cyclin A chromatin binding and required increased cyclin-CDK activity for the initiation of DNA replication, which is associated with DNA replication stress in vitro and in vivo. Significantly, the CDK threshold for initiation of DNA replication was 2-fold higher in CIZ1 KO nuclei than parental controls. Importantly, addition of recombinant CIZ1 in vitro and in vivo promotes recruitment of cyclin A to chromatin and reinstates the CDK threshold for initiation of DNA replication, reversing DNA replication stress and increasing replication fork rates. Loss of CIZ1 is associated with dysregulated cyclin-CDK signalling, resulting in reduced G1 length, an increased CDK activity threshold required to promote initiation of DNA replication that results in DNA replication stress. These data suggest that CIZ1 facilitates recruitment of cyclin-CDK complexes to chromatin and contributes to the mechanisms that determine the threshold CDK activity required for the G1/S transition in post-quiescent cells. Taken together the data support a role for CIZ1 in the prevention of DNA replication stress and maintenance of genome stability.

cell biology↗