The fate of the mitochondrial DNA replication or destruction can be Biology Diagrams Bergoglio, V. et al. DNA synthesis by Pol eta promotes fragile site stability by preventing under-replicated DNA in mitosis. J. Cell Biol. 201 , 395-408 (2013). UDRs also have the potential to generate DNA bridges in anaphase cells or micronuclei in the daughter cells, which could promote genomic instability. To suppress such damaging changes to the genome, human cells have developed a strategy to conduct 'unscheduled' DNA synthesis in mitosis (termed MiDAS) that serves to rescue under-replicated loci. To examine the DNA polymerase(s) responsible for mitotic DNA synthesis, we released G2-arrested cells pre-treated with/without low-dose APH into mitosis in the presence of a high dose of APH (2 ฮผM).
Mitotic DNA synthesis (or abbreviated as MiDAS) may be one such mechanism, as it is strongly activated under replication stress ().This unusual timing of DNA synthesis is universally observed in a variety of mammalian cells after treatment with a low dose of aphidicolin (Aph), a replication inhibitor ().After pulse labeling with EdU (5-ethynyl-2โฒ-deoxyuridine), punctuated sites of mitotic Cells experiencing DNA replication stress enter mitosis with under-replicated DNA, which activates a repair mechanism known as mitotic DNA synthesis (MiDAS). Here we describe a protocol to identify at genome wide and at high resolution the genomic sites where MiDAS occurs in cells exposed to aphidicolin. Interestingly, treatment of cells with aphidicolin leads not only to breaks and gaps on mitotic chromosomes, but also to foci of nascent DNA synthesis that decorate these chromosomes. 37 These
Replication stress activates DNA repair synthesis in mitosis Biology Diagrams
Mitotic DNA synthesis is a recently discovered mechanism that resolves late replication intermediates, thereby supporting cell proliferation under replication stress. This unusual form of DNA synthesis occurs in the absence of RAD51 or BRCA2, which led to the identification of RAD52 as a key player in this process. Notably, mitotic DNA Aberrant replication causes cells lacking BRCA2 to enter mitosis with under-replicated DNA, which activates a repair mechanism known as mitotic DNA synthesis (MiDAS). Here, we identify genome-wide the sites where MiDAS reactions occur when BRCA2 is abrogated. High-resolution profiling revealed that โฆ To exclusively capture DNA synthesis in mitosis, we inhibited S/G2 replication with hydroxyurea, as previously described (Macheret et al., 2020). EdU-labeled DNA was isolated from mitotic "shake-off" cells and analyzed by high-throughput sequencing for high-resolution mapping of MiDAS sites genome-wide (Macheret et al., 2020).
