Figure 1 from A novel processive mechanism for DNA synthesis revealed Biology Diagrams
Figure 1 from A novel processive mechanism for DNA synthesis revealed Biology Diagrams 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 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
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).
resolution mapping of mitotic DNA synthesis regions and common ... Biology Diagrams
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. 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
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 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). 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).
