Events that compromise genomic integrity, such as in DNA damage, result in arrest of the cell cycle to prevent replication of damaged DNA. However, the mechanisms of this checkpoint are not yet fully understood. Recent findings from a team from Nara Institute of Science and Technology report a new molecular pathway for DNA damage response using Arabidopsis as a model.
In Arabidopsis a family of transcriptional repressors and activators, called MYB3R, are known to regulate mitotic genes specific to the transition to the cell cycle’s M phase, when cell division takes place. A subset of MYB3R that are repressor-types, called Rep-MYB, are particularly striking. In the event of DNA damage, plant growth stops and Rep-MYB accumulates. However, when Rep-MYB is absent plant growth resumes.
To understand how Rep-MYB accumulation occurs in response to DNA damage, the research team considered the role of cyclin-dependent kinases (CDKs) which also regulate the cell cycle. Recent findings have shown that DNA damage results in repression of key CDKs and then an arrest at M phase. The team proposed that it is this reduction of CDK activity that causes an accumulation of Rep-MYB, which in turn suppresses the mitotic genes that cause cell cycle arrest.
When CDKs are inhibited in the absence of DNA damage, Rep-MYB3R accumulates. This is demonstrated by increased fluorescent expression of Rep-MYB proteins fused to GFP. In an in vitro assay that combines E. coli expressed Rep-MYB and Arabidopsis total protein, immunoblotting shows depletion of the Rep-MYB over time. However, when a CDK inhibitor is added, Rep-MYB remains undepleted, suggesting CDK has an active function in degrading Rep-MYB. CDK appears to cause this by direct phosphorylation. An in vitro kinase assay, followed by LC-MS/MS analysis reveals phosphorylation of Rep-MYB in its C-terminal region. When Rep-MYB is mutated so that it cannot be phosphorylated, it is shown to also resist degradation.
The study authors further find that Rep-MYB proteins target genes responsible for entering the M phase. In a chromatin immunoprecipitation (ChIP) experiment, promoter fragments of M-specific genes are shown to be highly enriched with Rep-MYB. When the plant is subjected to DNA damage, this promoter-binding is even more pronounced. This is complemented by quantitative RT-PCR that shows a resulting reduction of transcripts of these M-specific genes.
Altogether, the experimental results demonstrate that CDK normally phosphorylates Rep-MYB resulting in its degradation, and that Rep-MYB represses key genes responsible for transitioning to the M phase of the cell cycle. Thus, in the event that the cell is hit with DNA damage and CDK is reduced, Rep-MYB jumps into action. It accumulates and represses, effectively stopping the cell cycle.
These findings were published recently in Nature Communications.
Image: Green spots indicate a transcription factor that accumulates and inhibits cell division upon DNA damage. Researchers found an indispensable role of the transcription factor in arresting plant growth under stressful conditions. Image courtesy of Masaaki Umeda.