Effect of a Double Strand-
Break on the Cell Cycle

Adaptation and Recovery

Regulation of the eukaryotic cell cycle by checkpoints ensure that preceding processes get completed before initiation of mechanistically unconnected ensuing processes. For example, in yeast, DNA damage induces a DNA damage checkpoint-dependent G2/M arrest that prevents the segregation of damaged DNA capable of causing genomic instability. This kind of a machinery implies that this arrest is reversible upon repair of the DNA damage. This channel of escape from arrest is defined as 'recovery'. We anticipate activation and downregulation of DNA damage checkpoint response during recovery.

Interestingly, unrepairable DNA damage does not cause a permanent arrest in yeast. Cells remain arrested in G2/M phase for an extended duration but eventually they exit the arrest and enter the subsequent cell cycle, despite continual presence of the DNA damage that induced the arrest. This manner of exit from checkpoint arrest is referred to as adaptation. During this phenomenon, a broken chromosome has been shown to be inherited for as many as ten generations prior to its loss. These cells do not show any indication of arrest in the subsequent generations.

Several adaptation defective mutants have been identified. They include cdc5-ad, ckb1, ckb2, yku70, rad51, tid1, dad1, dad2, and dad3. Cdc5-ad, yku70, and tid1 mutants show a stronger phenotype compared to the others. CDC5 encodes an essential polo-like protein tyrosine kinase. It has phylogenetically conserved and essential functions in a number of stages throughout mitosis. It has been inferred that the cdc5-ad mutation (L251W) alters phosphorylation of a subset of its targets. Yku70 mutation inactivates the Ku protein and is believed to block adaptation by causing increased extent (or rate) of accumulation of signal. No cause of inability of other mutants to adapt has been put forth.

Two recovery defective mutations have been identified so far: Two mutations that confer a recovery-defect have been identified in S. pombe. They are slp1 (Matsumoto, MCB 17, 742 1997) and crb2-T215A (Esashi and Yanagida, Mol. Cell, 4, 167, 1999). slp1+ is S. cerevisiae CDC20 homologue. CDC20 genetically interacts with CDC5. This suggests a possible mechanistic link between recovery and adaptation. Crb2+ is homologous to S. cerevisiae RAD9.

An interesting issue is whether these two modes of exit from arrest define two separate pathways. The reason to pose this question is that we do not really understand the nature of the cellular signal for completion of DNA repair. To study adaptation and recovery in comparison with each other, it was necessary to develop a set of strains that either adapt or recover after the introduction of a defined damage. For easy comparison, the following features were necessary in the strains that recover:

1. The strain should have a defined DNA damage.
2. Repair of the damage should take a very long time. This will ensure a robust G2/M arrest.
3. Efficiency of repair should be very high and measurable.

Towards these ends, we have developed an single strand annealing (SSA) based system. The rate limiting step in SSA is the resection of DSB ends. It occurs at 4 kb/h. We have separated the homologous segments from DSB site by 25 kb (see figure below). As predicted, DNA repair gets completed in 6 h. During this time, all the cells arrest at the G2/M point of cell cycle and recover after repair is completed. Deletion of RAD52 prevents completion of repair but the cells are still able to adapt. We think this system will be useful for us to study recovery and adaptation in comparison with each other.