Checkpoint silencing during early C. elegans embryogenesis.

摘要

Cell cycle checkpoints monitor genomic integrity and delay cell cycle progression in response to damaged or incompletely replicated chromosomes. During the early embryonic cell cycle, however, the checkpoint does not respond to DNA damage and instead responds to developmental cues that control the timing of cell division. Thus while early embryonic cell cycles contain active checkpoint pathways these checkpoints are blind to the presence of DNA damage. It was the goal of this work to determine how checkpoint activation by DNA damage is suppressed during early development in C. elegans.; As a starting point in this investigation I have characterized the C. elegans mus-101 gene. The Mus101 family is widely conserved in eukaryotes and is known to play multiple roles in chromosome metabolism. I found that mus-101 is an essential DNA replication factor and that hypomorphic expression of mus-101 causes sensitivity to DNA damaging agents. Further characterization of the damage sensitivity phenotype of mus-101 revealed that loss of mus-101 allows checkpoint activation in response to DNA damage during early embryogenesis. This result suggested that worms have evolved a pathway that actively silences checkpoint activation when DNA damage is present.; To further characterize this checkpoint-silencing pathway I performed a genetic modifier screen for genes that interact with mus-101 and I also examined known components of the DNA damage response for roles in checkpoint silencing. This led to the identification of two genes, gei-17 and polh-1, which suppress checkpoint activation in response to damage during the early cycles. GEI-17 is a SUMO E3 ligase and POLH-1 is a traps-lesion DNA polymerase. Further analysis of the function of these genes revealed the mechanism for checkpoint silencing: the checkpoint is silenced because stalled replication forks do not accumulate on damaged chromosomes as they do in somatic cells. These findings are consistent with the known biochemical function of POLH-1, a traps-lesion DNA polymerase capable of replicating across sites of damage. The results presented here define a novel developmental mechanism that utilizes error-prone, replicative bypass of DNA damage as a strategy to restrict checkpoint activation to developmentally programmed signals during early embryogenesis.