Phosphatases and DNA double strand break repair

When cells suffer DNA damage – as little as a single break in one chromosome – they respond by activating the DNA damage checkpoint, which prevents cells from entering mitosis until there is enough time to to repair the damage.  The principal biochemical events in the checkpoint pathway are the phosphorylations of protein kinases by other protein kinases and eventually the phosphorylation of other proteins that regulate mitosis.    When repair is complete, the checkpoint must be turned off.  Not surprisingly, the enzymes that turn off the checkpoint are phosphatases that can remove the phosphates added by the protein kinases.

The Haber lab has previously shown that, in budding yeast, a pair of PP2C phosphatases known as Ptc2 and Ptc3 were important in turning off a key protein kinase, Rad53.  A member of another phosphatase subgroup, the PP4 phosphatase Pph3, dephosphorylates a target of the checkpoint kinases, histone protein H2A.  There is one aspect that they didn’t understand at all: It seems that the intensity of the checkpoint signals must grow the longer it takes to repair DNA damage, because deletions of ptc2 and ptc3 or a deletion of pph3 prevented cells from turning off the damage signal when it took a long time – 6 hours – to repair the damage, but they had much less effect on different repair events that could complete in 3-4 hours or in less than 2 hours.  So they decided to see what would happen if they created a yeast strain lacking all three phosphatases (ptc2 ptc3 pph3), leading to a paper appearing this month in the journal Molecular and Cell Biology.

To their surprise, these cells had a new defect: they couldn’t complete the repair event itself, rather than simply being defective in resuming mitosis after repair was completed.  The mutants could not properly initiate the small amounts of DNA copying that are required for repair.  Again, the severity of the defect depends on the length of the delay it takes to initiate the repair event itself.  The figure (right) shows that the triple mutant is also much more sensitive to DNA damaging agents such as the anti-cancer drug camptothecin (CPT) and to methylmethansulfonate (MMS). These data show a complex connection between DNA damage signaling and the repair process itself, and reveal new roles for the phosphatases in DNA repair.  The work was carried out primarily by graduate student Jung-Ae Kim, now a postdoc at Rockefeller University, with help by another grad student, Wade Hicks, and by an undergraduate Sue Yen Tay, and postdoc Jin Li. The work was supported by a research and a graduate student training grant from the NIH.

Is my DNA fixed yet?

A broken chromosome (a double-strand DNA break) activates the DNA damage checkpoint to prevent cells from carrying out mitosis until the break has been repaired.  Repair of the break involves the modification and the removal of histone protein octamers from DNA around the break and these must be reestablished when repair is complete.  In a new paper in PNAS, Brandeis alumnus Jung-Ae Kim (Ph.D., Molecular and Cell Biology, 2008) and Professor James Haber show that when two of the major histone chaperone protein complexes (Asf1 and CAF-1) are deleted in yeast cells, their absence prevents cells from turning off the DNA damage checkpoint and hence cells stay permanently arrested.   These results suggest that cells specifically monitor the re-establishment of normal chromatin status after DNA repair.

Sigma factors

In a new study appearing in PNAS this week, Brandeis Molecular and Cell Biology graduate student Houra Merrikh and co-workers from the Lovett lab identified the E.coli gene iraD as a regulator of the response to oxidative DNA damage in exponentially growing bacteria. Interestingly, the mechanism seems to involve the alternative RNA polymerase sigma factor RpoS, previously characterized as a regulator of expression during the “stationary phase”. Merrikh et al. argue that this response works in parallel with the previously characterized SOS response in protecting growing bacteria from DNA damage.

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