Microscopic Detection of DNA Synthesis in Early Mitosis at Repetitive lacO Sequences in Human Cells

Kazumasa Yoshida; Riko Ishimoto; Masatoshi Fujita

doi:10.21769/BioProtoc.4504

Microscopic Detection of DNA Synthesis in Early Mitosis at Repetitive lacO Sequences in Human Cells

Bio Protoc. 2022 Sep 5;12(17):e4504. doi: 10.21769/BioProtoc.4504.

Authors

Kazumasa Yoshida^{1

2}, Riko Ishimoto³, Masatoshi Fujita³

Affiliations

¹ Department of Cell Biology, Faculty of Medicine, Fukuoka University, Fukuoka, Japan.
² Research Institute for Advanced Molecular Medicine, Fukuoka University, Fukuoka, Japan.
³ Department of Cellular Biochemistry, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.

Abstract

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.

Keywords: BrdU; DNA repair; DNA replication; Immunofluorescence; MiDAS; Mitosis; Repetitive DNA; Replication stress.