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.

Time for Worms in Circadian Biology

Almost every organism on earth, from archae to humans, exhibits circadian rhythms – periodic cycles of behavior or gene expression that repeat approximately every 24 hours. These rhythms are generated by a circadian clock – an internal time-keeping mechanism – which can be entrained and synchronized by environmental cues such as temperature or light/dark cycles. This clock may provide organisms with an adaptive advantage throughout their life, and disruption of the function of this clock can lead to severe behavioral and metabolic disorders in humans.

For more than two decades researchers have wondered whether the tiny soil-dwelling nematode worm Caenorhabditis elegans, one of the foremost model organisms, contains a circadian clock. Circadian rhythmic behaviors described previously in C. elegans are variable and hard to quantify, and no genes were known to exhibit gene expression oscillations with 24 hr cycles as shown in many other animals.

Now, in a recent study published in the open-access journal PLoS Biology, several students and postdoctoral fellows in the labs of Piali Sengupta and Michael Rosbash joined forces and took on the challenge to identify C. elegans genes under clock control.

Light and temperature cycles both drive and entrain 24 hr oscillations in gene expression in C. elegans.

They showed that indeed C. elegans contains genes whose expression cycles in a circadian manner. They found that light and temperature cycles appear to regulate different sets of genes (see above), indicating that these stimuli may entrain two distinct clocks. Moreover, the underlying clock mechanisms may not be dependent on oscillations of known clock genes. “These findings were surprising to us since Drosophila only has a single conserved clock running in multiple cells and tissues” says Alexander van der Linden – lead author and former postdoctoral fellow in the Sengupta Lab.

C. elegans has a wealth of genetic and behavioral tools. The next critical step will be to identify the mechanisms underlying the C. elegans circadian clock(s). These investigations may also provide information of how the clock evolved since nematodes and humans split about 600-1200 million years ago.

Alexander M. van der Linden is now an Assistant Professor at the University of Nevada, Reno. The work was conducted in the labs of Profs. Michael Rosbash, a member of the Howard Hughes Medical Institute and Piali Sengupta in the Department of Biology. Other authors who contributed to this work include Molecular and Cell Biology graduate students Matthew Beverly, Joseph Rodriquez and Sara Wasserman (now a postdoctoral fellow at UCLA), and Sebastian Kadener, a former postdoctoral fellow who is now an Assistant Professor at the Silberman Institute of Life Science, The Hebrew University of Jerusalem, Israel.

Chirality leads to self-limited self-assembly

Simple building blocks that self-assemble into ordered structures with controlled sizes are essential for nanomaterials applications, but what are the general design principles for molecules that undergo self-terminating self-assembly? The question is addressed in a recent paper in Physical Review Letters by Yasheng Yang, graduate student in Physics, working together with Profs. Meyer and Hagan,  The paper considers molecules that self assemble to form filamentous bundles, and shows that chirality, or asymmetry with respect to a molecule’s mirror image, can result in stable self-limited structures. Using modern computational techniques, the authors demonstrate that chirality frustrates long range order and thereby terminates assembly upon formation of regular self-limited bundles.  With strong interactions, however, the frustration is relieved by defects, which give rise to branched networks or irregular bundles.

Figure: (a) Snapshots of regular chiral bundles. Free energy calculations and dynamics demonstrate that the optimal diameter decreases with increasing chirality. (b) Branched bundles form with strong interactions

How crabs deal with the chill

Chemical (and biochemical) reaction rates can increase dramatically with even small changes in temperature, but many biological systems require rhythms with a precise ordering of events. How are these rhythms maintained in organisms that can prosper in a wide range of temperatures? In a recent study published in PLoS Biology (comment). Lamont Tang, a Neuroscience grad student, and other members of the Marder lab studied this question by looking at neurons in the pyloric network of the crab Cancer borealis. They argue from a combination of experiments and computational models that even though firing frequencies change with temperature, the phase between elements of the network is maintained by balancing opposing currents with similar temperature dependencies.

crab pyloric rhythms in hot and cold

graphic courtesy of Gabrielle Gutierrez

PhD Defense Season

It’s the season for PhD defenses…

  • Apr 20: Megan Zahniser (Biochemistry), On the structure of Benzaldehyde Dehydrogenase, a Class 3 Aldehyde Dehydrogenase from Pseudomonas putida – 2pm, Rosenstiel Penthouse
  • Apr 21: Chris Hoefler (Biochemistry/Bioorganic Chemistry). Inhibitors of IMPDH: Tools for Probing Mechanism and Function – 3:40 pm, Gerstenzang 122
  • Apr 22: Tepring Piquado (Neuroscience), Language and the aging brain – Thu 4/22/2010, 2 pm, Volen 201
  • Apr 23: Suvi Jain (Molecular and Cell Biology), Regulation of DNA Double-Strand Break Repair by the Recombination Execution Checkpoint in Saccharomyces cerevisiae – 3:30 pm, Rosenstiel 118
  • Apr 29: Ben Cuiffo (Molecular and Cell Biology), Targeting RAS palmitoylation in hematological malignancies – 2 pm, Abelson 131

Dilute-’N’-Go sequencing

Prof. Larry Wangh and his lab are interested in detecting changes in mitochondrial genomic sequences that result from aging, disease, or drugs.  To do this, they use LATE-PCR, an advanced form of asymmetric PCR, to detect mutations in the mitochondria by using multiplexes to study many mitochondrial genes at the same time.  LATE-PCR generates single DNA strands that are easily diluted for sequencing.  In the past. they have only been able to sequence one DNA strand from these multiplex reactions.

In a recent publication in Nucleic Acid Research, staff members Yanwei Jia and John Rice, along with Molecular and Cell Biology grad student Adam Osborne, describe the development of a blocking reagent that allows them to sequence both strands of the product DNA, thus allowing for the easy verification of mutations.

The figure at right shows that without a blocker (BLK), one is not able to obtain the excess (XP) strand sequence from a multiplex reaction.  Using a blocker one is able to get not only the limiting (LP) strand, but also the excess strand from the same multiplex

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