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.
ABSTRACT
The DNA damage response (DDR) has a critical role in the maintenance of genomic integrity during chromosome replication. However, responses to replication stress evoked by tight DNA-protein complexes have not been fully elucidated. Here, we used bacterial LacI protein binding to lacO arrays to make site-specific replication fork barriers on the human chromosome. These barriers induced the accumulation of single-stranded DNA (ssDNA) and various DDR proteins at the lacO site. SLX4-XPF functioned as an upstream factor for the accumulation of DDR proteins, and consequently, ATR and FANCD2 were interdependently recruited. Moreover, LacI binding in S phase caused underreplication and abnormal mitotic segregation of the lacO arrays. Finally, we show that the SLX4-ATR axis represses the anaphase abnormality induced by LacI binding. Our results outline a long-term process by which human cells manage nucleoprotein obstacles ahead of the replication fork to prevent chromosomal instability.
