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Meroni, A.

Publications and source records attributed to Meroni, A..

3 recordsLinked to original sources

CIP2A Mediates the Recruitment of the SLX4-MUS81-XPF Tri-Nuclease Complex in Mitosis and Protects Against Replication Stress

DNA replication stress frequently elicits problems in mitosis because incompletely replicated chromosomes or replication intermediates physically link sister chromatids together and prevent their proper segregation during anaphase. We and others recently discovered a mitotic role for the CIP2A oncoprotein, which is critically implicated in chromosome stability maintenance and chromosome fragment clustering during mitosis. In addition, in homologous recombination deficient (HRD) cells, CIP2A is essential and may thus constitute a new drug target in HRD cancers. Yet, the precise mechanisms by which CIP2A suppresses chromosomal instability during mitosis and thus allows for the survival of HRD cancer cells remain largely elusive. Here we characterize CIP2As role in DNA replication stress responses. We show that upon replication stress, wild-type cells show an elevated accumulation of CIP2A foci during mitosis, indicating its involvement in the mitotic response to replication stress. Defective DNA replication leads to the accumulation of under-replicated DNA, which can be carried into mitosis. We demonstrate that in the absence of CIP2A, cells fail to recruit the SLX4-MUS81-XPF (SMX) tri-nuclease complex to sites of under-replicated DNA in mitosis, resulting in a high incidence of lagging chromosomes during anaphase and subsequent micronuclei formation. In a subset of cell lines, we also observed CIP2A-dependent mitotic DNA synthesis (MiDAS) upon replication stress. However, our data suggest that MiDAS and SMX recruitment are not functionally linked. This novel role of CIP2A in managing under-replicated DNA may provide insights into the molecular mechanisms underlying therapeutic vulnerabilities in cancer cells.

cell biology↗

The SMC5/6 complex is required for maintenance of genome integrity upon APOBEC3A-mediated replication stress

Mutational patterns caused by APOBEC3 cytidine deaminase activity are evident throughout human cancer genomes. In particular, the APOBEC3A family member is a potent genotoxin that causes substantial DNA damage in experimental systems and human tumors. However, the mechanisms that ensure genome stability in cells with active APOBEC3A are unknown. Through an unbiased genome-wide screen, we define the Structural Maintenance of Chromosomes 5/6 (SMC5/6) complex as essential for cell viability when APOBEC3A is active. We observe an absence of APOBEC3A mutagenesis in human tumors with SMC5/6 dysfunction, consistent with synthetic lethality. Cancer cells depleted of SMC5/6 incur substantial genome damage from APOBEC3A activity during DNA replication. Further, APOBEC3A activity results in replication tract lengthening which is dependent on PrimPol, consistent with re-initiation of DNA synthesis downstream of APOBEC3A-induced lesions. Loss of SMC5/6 abrogates elongated replication tracts and increases DNA breaks upon APOBEC3A activity. Our findings indicate that replication fork lengthening reflects a DNA damage response to APOBEC3A activity that promotes genome stability in an SMC5/6-dependent manner. Therefore, SMC5/6 presents a potential therapeutic vulnerability in tumors with active APOBEC3A.

molecular biology↗

DNA Combing versus DNA Spreading and the Separation of Sister Chromatids

DNA combing and DNA spreading are two central approaches for studying DNA replication fork dynamics genome-wide at single-molecule resolution by distributing labeled genomic DNA on coverslips or slides for immunodetection. Perturbations in DNA replication fork dynamics can differentially affect either leading or lagging strand synthesis, for example in instances where replication is blocked by a lesion or obstacle on only one of the two strands. Thus, we sought to investigate whether the DNA combing and/or spreading approaches are suitable for resolving adjacent sister chromatids during DNA replication, thereby enabling the detection of DNA replication dynamics within individual nascent strands. To this end, we developed a thymidine labeling scheme that discriminates between these two possibilities. Our data suggests that DNA combing resolves single chromatids, allowing the detection of strand-specific alterations, whereas DNA spreading does not. These findings have important implications when interpreting DNA replication dynamics from data obtained by these two commonly used techniques.

molecular biology↗