Detecting Mutations the Easy Way

Recent Brandeis Ph.D graduate, Tracey Seier (Molecular and Cell Biology Program), Professor Sue Lovett, Research Assistant Vincent Sutera, together with former Brandeis undergraduates Noor Toha, Dana Padgett and Gal Zilberberg have developed a set of bacterial strains that can be used as “mutational reporters”.  Students in the Fall 2009 BIOL155a, Project Laboratory in Genetics and Genomics, course also assisted in the development of this resource. This work has recently been published in the journal Genetics.

These Escherichia coli strains carry mutations in the lacZ (β-galactosidase) gene that regain the ability to metabolize lactose by one, and only one, specific type of mutation. This set allows environmental compounds to be screened for effects on a broad set of potential mutations, establishing mutagen status and the mutational specificity in one easy step.

This strain set is improved over previous ones in the inclusion of reporters that are specific for certain types of mutations associated with mutational hotspots in gene. Mutations at these sites occur much more frequently than average and involve DNA strand misalignments at repeated DNA sequences rather than DNA polymerase errors. Such mutations are associated with human diseases, including cancer progression, and have been under-investigated because of the lack of specific assays. Using this strain set, Seier et al. also identified a mutagen, hydroxyurea, used in the treatment of leukemia and sickle cell disease, which affects only the “hotspot” class of mutations. This strain set, which will be deposited in the E. coli Genetic Stock Center,  will facilitate the screening of potential mutagens, environmental conditions or genetic loci for effects on a wide spectrum of mutational events.



Left: E. coli colonies showing lacZ mutant revertants (blue pimples) arising on a white colony on growth medium containing the beta-galactosidase indicator dye,  X-gal


Alex’s life as a fly barista

Alex Dainis ’11 writes about her experiences in the Garrity lab studying the genetics of nociception in fruit flies in her story “My life as a fly barista” on the Life@Deis blog.

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.

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