Activation of DNA damage and spindle checkpoints during Metaphase.

A few years ago we obtained the surprising result that Grp/Chk1 (a DNA damage checkpoint thought to function only during S-phase and G2) controls delay exit from metaphase in response to DNA damage.  While it is well known that this DNA-damage S-phase checkpoint controls entry into metaphase, it was surprising that it also controls exit from metaphase.  Prior to this result, activation of the spindle checkpoint was thought to be the only checkpoint controlling metaphase exit.  We followed up on our result by demonstrating that general DNA damaging agents, such as X-irradiation, not only produce an expected delay in interphase (due to an activation of DNA damage checkpoints), but also a pronounced metaphase delay.  These observations landed us squarely in the middle of a controversy of whether DNA damage checkpoints function during metaphase, as well as during interphase.  A similar DNA-damage-induced metaphase delay had been observed in mammalian cells.  Unresolved was whether this delay was due to activation of a DNA damage or spindle assembly checkpoint.  The controversy arose because it was unclear if the damaging agents were disrupting the centromere, thus activating the spindle assembly checkpoint.  We then undertook experiments using the recently developed I-Cre system in Drosophila.  With the I-Cre system, one can produce a double stranded break at a defined locus in the chromosome. In this way, one is able to produce a break without possibility of collateral kinetochore damage.  Anne Royou compared the effects of random DNA breaks induced by X-irradiation to site-specific I-Cre endonuclease-induced chromosome breaks in wild-type, grp, and bubR1 mutant Drosophila neuroblasts.  She found that both the BubR1 spindle checkpoint and the Grp/Chk1 DNA damage checkpoint pathway are involved in delaying the metaphase/anaphase transition after extensive X-irradiation induced DNA damage.  However Grp/Chk1, but not BubR1, is required to delay anaphase onset in the presence of I-Cre-induced double strand breaks.  Based on these results, we propose that DNA damage in non-kinetochore regions produces a Grp/Chk1 DNA damage checkpoint-mediated delay in the metaphase/anaphase transition.  This work is published in Current Biology (Royou et al. 2005).  We are currently pursuing the molecular mechanism by which Grp/Chk1 delays anaphase entry.