Accurate replication of DNA requires stringent regulation to ensure genome integrity. In human cells, thousands of origins of replication are coordinately activated during S phase, and the velocity of replication forks is adjusted to fully replicate DNA in pace with the cell cycle1. Replication stress induces fork stalling and fuels genome instability2. The mechanistic basis of replication stress remains poorly understood despite its emerging role in promoting cancer2. Here we show that inhibition of poly(ADP-ribose) polymerase (PARP) increases the speed of fork elongation and does not cause fork stalling, which is in contrast to the accepted model in which inhibitors of PARP induce fork stalling and collapse3. Aberrant acceleration of fork progression by 40% above the normal velocity leads to DNA damage. Depletion of the treslin or MTBP proteins, which are involved in origin firing, also increases fork speed above the tolerated threshold, and induces the DNA damage response pathway. Mechanistically, we show that poly(ADP-ribosyl)ation (PARylation) and the PCNA interactor p21Cip1 (p21) are crucial modulators of fork progression. PARylation and p21 act as suppressors of fork speed in a coordinated regulatory network that is orchestrated by the PARP1 and p53 proteins. Moreover, at the fork level, PARylation acts as a sensor of replication stress. During PARP inhibition, DNA lesions that induce fork arrest and are normally resolved or repaired remain unrecognized by the replication machinery. Conceptually, our results show that accelerated replication fork progression represents a general mechanism that triggers replication stress and the DNA damage response. Our findings contribute to a better understanding of the mechanism of fork speed control, with implications for genomic (in)stability and rational cancer treatment.

High speed of fork progression induces DNA replication stress and genomic instability.

Selective Loss of PARG Restores PARylation and Counteracts PARP Inhibitor-Mediated Synthetic Lethality

Due to the DNA repair defect, BRCA1/2 deficient tumor cells are more sensitive to PARP inhibitors (PARPi) through the mechanism of synthetic lethality At present, several PAPRi targeting poly (ADP-ribose) polymerase (PARP) have been approved for ovarian cancer and breast cancer indications However, PARPi resistance is ubiquitous in clinic More than 40% BRCA1/2-deficient patients fail to respond to PARPi In addition, lots of patients acquire PARPi resistance with prolonged oral administration of PARPi Homologous recombination repair deficient (HRD), as an essential prerequisite of synthetic lethality, plays a vital role in killing tumor cells Therefore, Homologous recombination repair restoration (HRR) becomes the predominant reason of PARPi resistance Recently, it was reported that DNA replication fork protection also contributed to PARPi resistance in BRCA1/2-deficient cells and patients Moreover, various factors, such as reversion mutations, epigenetic modification, restoration of ADP-ribosylation (PARylation) and pharmacological alteration lead to PARPi resistance as well In this review, we reviewed the underlying mechanisms of PARP inhibitor resistance in detail and summarized the potential strategies to overcome PARPi resistance and increase PARPi sensitivity

/pdf/parp-inhibitor-resistance-the-underlying-mechanisms-and-181intqjud.pdf

PARP inhibitor resistance: the underlying mechanisms and clinical implications

BET Bromodomain Inhibition Synergizes with PARP Inhibitor in Epithelial Ovarian Cancer

Senescence and senotherapeutics: a new field in cancer therapy

Topoisomerase IIβ-binding protein 1 (TOPBP1) participates in DNA replication and DNA damage response; however, its role in DNA repair and relevance for human cancer remain unclear. Here, through an unbiased small interfering RNA screen, we identified and validated TOPBP1 as a novel determinant whose loss sensitized human cells to olaparib, an inhibitor of poly(ADP-ribose) polymerase. We show that TOPBP1 acts in homologous recombination (HR) repair, impacts olaparib response, and exhibits aberrant patterns in subsets of human ovarian carcinomas. TOPBP1 depletion abrogated RAD51 loading to chromatin and formation of RAD51 foci, but without affecting the upstream HR steps of DNA end resection and RPA loading. Furthermore, TOPBP1 BRCT domains 7/8 are essential for RAD51 foci formation. Mechanistically, TOPBP1 physically binds PLK1 and promotes PLK1 kinase-mediated phosphorylation of RAD51 at serine 14, a modification required for RAD51 recruitment to chromatin. Overall, our results provide mechanistic insights into TOPBP1's role in HR, with potential clinical implications for cancer treatment.

/pdf/topbp1-regulates-rad51-phosphorylation-and-chromatin-loading-2nrbby1ru7.pdf

TOPBP1 regulates RAD51 phosphorylation and chromatin loading and determines PARP inhibitor sensitivity.

RAD51: Beyond the break

The RAD51 recombinase plays critical roles in safeguarding genome integrity, which is fundamentally important for all living cells. While interphase functions of RAD51 in repairing broken DNA and protecting stalled replication forks are well characterised, its role in mitosis remains contentious. In this study, we show that RAD51 protects under-replicated DNA in mitotic human cells and, in this way, promotes mitotic DNA synthesis (MiDAS) and successful chromosome segregation. MiDAS was globally detectable irrespective of DNA damage and was promoted by de novo RAD51 recruitment, RAD51-mediated fork protection, and RAD51 phosphorylation by the key mitotic regulator Polo-like kinase 1. Importantly, acute inhibition of RAD51-promoted MiDAS led to mitotic DNA damage, delayed anaphase onset and induced centromere fragility, revealing a mechanism that prevents the satisfaction of the spindle assembly checkpoint when chromosomal replication remains incomplete. This study hence identifies an unexpected function of RAD51 in promoting the stability of mitotic chromatin.

/pdf/the-rad51-recombinase-protects-mitotic-chromatin-in-human-2frwef74a2.pdf

The RAD51 recombinase protects mitotic chromatin in human cells

The RAD51 recombinase plays critical roles in safeguarding genome integrity, which is fundamentally important for all living cells. While interphase functions of RAD51 in maintaining genome stability are well-characterised, its role in mitosis remains contentious. In this study, we show that RAD51 protects under-replicated DNA in mitotic human cells and, in this way, promotes mitotic DNA synthesis (MiDAS) and successful chromosome segregation. In cells experiencing mild replication stress, MiDAS was detected irrespective of mitotically generated DNA damage. MiDAS broadly required de novo RAD51 recruitment to single-stranded DNA, which was supported by the phosphorylation of RAD51 by the key mitotic regulator Polo-like kinase 1. Importantly, acute inhibition of MiDAS delayed anaphase onset and induced centromere fragility, suggesting a mechanism that prevents the satisfaction of the spindle assembly checkpoint while chromosomal replication remains incomplete. This study hence identifies an unexpected function of RAD51 in promoting genomic stability in mitosis.

The RAD51 recombinase protects mitotic chromatin in human cells.

5-Methylcytosine (5mC) and DNA methyltransferases (DNMTs) are broadly conserved in eukaryotes but are also frequently lost during evolution. The mammalian SNF2 family ATPase HELLS and its plant ortholog DDM1 are critical for the maintenance of 5mC. Mutations in HELLS, its activator subunit CDCA7, and the de novo DNA methyltransferase DNMT3B, cause immunodeficiency-centromeric instability-facial anomalies (ICF) syndrome, a genetic disorder associated with the loss of DNA methylation. We here examine the coevolution of CDCA7, HELLS and DNMTs. While DNMT3, the maintenance DNA methyltransferase DNMT1, HELLS, and CDCA7 are all highly conserved in vertebrates and green plants, they are frequently co-lost in other evolutionary clades. The presence-absence patterns of these genes are not random; almost all CDCA7 harboring eukaryote species also have HELLS and DNMT1 (or another maintenance methyltransferase, DNMT5), whereas species that maintain DNMT1 or HELLS without CDCA7 are identified in several clades, such as Fungi and Ecdysozoa. Coevolution of presence-absence patterns (CoPAP) analysis in Ecdysozoa further indicates coevolutionary linkages among CDCA7, HELLS, DNMT1 and its activator UHRF1. We hypothesize that CDCA7 becomes dispensable in species that lost HELLS or DNA methylation, and/or the loss of CDCA7 triggers the replacement of DNA methylation by other chromatin regulation mechanisms. Our study suggests that a unique specialized role of CDCA7 in HELLS-dependent DNA methylation maintenance is broadly inherited from the last eukaryotic common ancestor.

/pdf/coevolution-of-the-cdca7-hells-icf-related-nucleosome-17ivdbt2.pdf

Isabel E. Wassing

Papers

TOPBP1 regulates RAD51 phosphorylation and chromatin loading and determines PARP inhibitor sensitivity.

RAD51: Beyond the break

The RAD51 recombinase protects mitotic chromatin in human cells

The RAD51 recombinase protects mitotic chromatin in human cells.

Coevolution of the CDCA7-HELLS ICF-related nucleosome remodeling complex and DNA methyltransferases