TY - JOUR
T1 - Microscopic Detection of DNA Synthesis in Early Mitosis at Repetitive lacO Sequences in Human Cells
AU - Yoshida, Kazumasa
AU - Ishimoto, Riko
AU - Fujita, Masatoshi
N1 - Funding Information:
This work was supported by grants from the Japan Society for the Promotion of Science (KAKENHI JP15K18478 and JP22H02603), the Fukuoka Foundation for Sound Health Cancer Research Fund, and the Mochida Memorial Foundation for Medical and Pharmaceutical Research. This protocol was adapted from our previous work (Ishimoto et al., 2021). Basic methods for detection of MiDAS were originally developed by the laboratory of Dr. Ian Hickson (Minocherhomji et al., 2015; Bhowmick et al., 2016)
Publisher Copyright:
Copyright: © 2022 The Authors; exclusive licensee Bio-protocol LLC.
PY - 2022/9/5
Y1 - 2022/9/5
N2 - In the human cell cycle, complete replication of DNA is a fundamental process for the maintenance of genome integrity. Replication stress interfering with the progression of replication forks causes difficult-to-replicate regions to remain under-replicated until the onset of mitosis. In early mitosis, a homology-directed repair DNA synthesis, called mitotic DNA synthesis (MiDAS), is triggered to complete DNA replication. Here, we present a method to detect MiDAS in human U2OS 40-2-6 cells, in which repetitive lacO sequences integrated into the human chromosome evoke replication stress and concomitant incomplete replication of the lacO array. Immunostaining of BrdU and LacI proteins is applied for visualization of DNA synthesis in early mitosis and the lacO array, respectively. This protocol has been established to easily detect MiDAS at specific loci using only common immunostaining methods and may be optimized for the investigation of other difficult-to-replicate regions marked with site-specific binding proteins.
AB - In the human cell cycle, complete replication of DNA is a fundamental process for the maintenance of genome integrity. Replication stress interfering with the progression of replication forks causes difficult-to-replicate regions to remain under-replicated until the onset of mitosis. In early mitosis, a homology-directed repair DNA synthesis, called mitotic DNA synthesis (MiDAS), is triggered to complete DNA replication. Here, we present a method to detect MiDAS in human U2OS 40-2-6 cells, in which repetitive lacO sequences integrated into the human chromosome evoke replication stress and concomitant incomplete replication of the lacO array. Immunostaining of BrdU and LacI proteins is applied for visualization of DNA synthesis in early mitosis and the lacO array, respectively. This protocol has been established to easily detect MiDAS at specific loci using only common immunostaining methods and may be optimized for the investigation of other difficult-to-replicate regions marked with site-specific binding proteins.
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U2 - 10.21769/BioProtoc.4504
DO - 10.21769/BioProtoc.4504
M3 - Article
AN - SCOPUS:85139342388
VL - 12
JO - Bio-protocol
JF - Bio-protocol
SN - 2331-8325
IS - 17
M1 - e4504
ER -