Protein Flexing: A New Look at Transcription-Coupled DNA Repair

Alexandra M. Deaconescu, a research associate in the Rosenstiel Basic Medical Sciences Research Center and 2008-2010 Fellow of the Damon Runyon Cancer Research Foundation, together with Professor of Biochemistry and HHMI Investigator Nikolaus Grigorieff and collaborators in the Laboratory of Dr. Irina Artsimovitch at Ohio State University have just published a new study in PNAS, which delineates novel mechanistic details of transcription-coupled DNA repair.

In any cell, there is intense interplay between various DNA-based transactions, such as replication, transcription and DNA repair. More than twenty years ago, it was discovered that DNA lesions that cause stalling of RNA polymerase molecules elicit a form of preferential nucleotide excision repair (NER) that exists in both eubacteria and eukaryotes, and specifically targets the transcribed DNA strand. Termed transcription-coupled DNA repair (TCR), the process is found to be carried out in bacteria by an ATPase called Mfd or TRCF (see Figure, right). In TCR, TRCF performs two functions: 1) it recognizes a damage-stalled RNA polymerase (RNAP), then dissociates it off the DNA using energy derived from ATP hydrolysis and 2) it recruits DNA repair enzymes via binding to the UvrA subunit of the Uvr(A)BC NER machinery [1].   The Uvr(A)BC machinery is one of the main players in bacterial DNA repair, and distinguishes itself from other DNA repair proteins by its ability to repair a remarkably diverse repertoire of lesions by utilizing a “cut and patch” mechanism, whereby an oligonucleotide containing the damage is excised and the gap later filled.

The cellular role of TRCFs extends beyond TCR. Because of their ability to forward translocate and dissociate stalled RNAPs (or  “backtracked” RNAPs that have slid backwards on the template) [2], TRCFs are also involved in transcription elongation regulation [3, 4], resolution of head-on collisions of the transcription apparatus with the DNA replication machinery [5], and antibiotic resistance [6, 7]. In humans, the effects of impaired TCR are systemic and complex. Mutations in the transcription-repair coupling factor CSB lead to Cockayne Syndrome [8], a progeroid (accelerated-aging) disease characterized by severe developmental abnormalities and neurodegeneration, and whose etiology is currently poorly understood.

To elucidate the mechanism underpinning UvrA recruitment by TRCF, Deaconescu crystallized and solved the X-ray structure of a core UvrA-TRCF complex (Figure, left) demonstrating that UvrA binding involves unmasking of a conserved intramolecular surface within TRCF via a gating motion of the C-terminal domain (red in Figure above). Despite significant effort so far, Deaconescu is still trying to coax nucleotide-bound TRCF to form crystals suitable for X-ray diffraction. These would be highly informative because ATP is required for DNA binding, and its hydrolysis leads to TRCF translocation on dsDNA and ultimately release of RNAP off the damaged template.  Because diffracting crystals eluded her, and to further find out how ADP/ATP modulate the structure of TRCF, Deaconescu learned small-angle X-ray scattering techniques suitable for probing TRCF in solution in the absence and presence of nucleotides, thus circumventing the need for highly-ordered crystals. Then, the Brandeis team and their collaborators at Ohio State employed domain-locking disulfide engineering in conjunction with functional assays to gain a deeper understanding of what TRCF looks like during its catalytic cycle and upon binding to UvrA.  They find that the two main functions of TRCF (RNAP release and UvrA binding) can be uncoupled, suggesting that UvrA recruitment may only occur during/post RNAP release, and not upon RNAP binding as had been proposed earlier in the literature [9]. Furthermore, they show that the ternary elongation complex (consisting of RNAP, template and nascent RNA), but not naked DNA, significantly stimulates ATP hydrolysis by TRCF. Thus, bacterial TRCF operates in a manner reminiscent of that utilized by eukaryotic chromatin remodeling factors, and are preferentially stimulated by nucleosomes over naked DNA substrates.

Deaconescu previously “looked” at TCR using X-rays – as a graduate student she solved the first structure of an intact transcription-repair coupling factor from any organism using X-ray crystallography [10]. She now hopes to reconstitute the larger intermediates that form during TCR and bridge low- with high-resolution information using hybrid structural methods, particularly electron cryo-microscopy, and ultimately formulate a cogent model of how TRCFs operate in cells.

Bacteria have RNAs that sense fluoride, and channels that tranport it

Fluoride: unless you’re a synthetic chemist or a dentist, you probably don’t worry about this ion very often.  But, according to a new paper published in Science, bacteria do, and have done for a very long time.

The work, spearheaded by Ron Breaker’s group at Yale University, identified a novel RNA motif that selectively binds fluoride ion.  In response to Fbinding, this motif, called a riboswitch, undergoes a structural change that leads to increased transcription of downstream genes.  These genes encode crucial metabolic enzymes that are strongly inhibited by fluoride ion, like enolase and pyrophosphatase, as well as members of a family of chloride transport proteins, the CLC’s.  The CLC’s that are associated with F riboswitches are clustered together in a phylogenetic clade distant from well-characterized CLC’s.  Could these “chloride” channel proteins actually assist with fluoride export?  Randy Stockbridge, a Brandeis postdoc working in Chris Miller’s lab, contributed to the findings by showing that this subset of riboswitch-associated CLC’s do, in fact, transport F, whereas “conventional” CLC’s strictly exclude F.   The F riboswitches, and the F CLC’s, are found among a huge variety of bacteria and archaea, from plant and human pathogens to benign soil and seawater-dwelling bugs, leading to the inference that F toxicity has been a consistent evolutionary pressure.

You’re probably wondering just how much fluoride there is in the environment.  Fluoridated municipal drinking water contains about 80 micromolar F, and natural F- concentrations in the environment can be  higher and lower than that number.   In acidic environments especially, F might accumulate to much higher levels in bacteria.  With a pKa of 3.4, a small amount of F is present as HF at low pH, and the uncharged HF can diffuse cross the cell membrane into the cell.  Once in the cytoplasm, where the pH is around 7, HF dissociates, and F can’t diffuse across the membrane back into the environment.  Unless, of course, evolution has provided that bacterium a system to transport F out of the cell…

see also

Otten named Damon Runyon Fellow

Renee Otten, a postdoctoral fellow in the Kern lab at Brandeis, has been awarded a November 2011 Damon Runyon Fellowship to support his postdoctoral research. Otten received his Ph.D. in 2011 from the University of Groningen, working on applying NMR spectroscopic methods to studying the relationship between protein structure and dynamics. The fellowship will support his continued efforts to use NMR to study dynamics and enzyme catalysis in protein kinases.

The ancient insect nose

In a recent short article in The Journal of Experimental Biology titled JUMPING BRISTLETAILS – A GLIMPSE INTO THE ANCIENT INSECT NOSE“, postdoc Katherine Parisky discusses the evolution of the olfactory system in insects.

In order for aquatic organisms to have made the transition from living in water to surviving on land, mutations in several physiological processes needed to occur. For one sensory system, that of smell, olfactory brain structures that detect odors based on sensing air-borne, volatile and hydrophobic molecules evolved from structures that had the ability to detect aqueous hydrophilic solutions […]


More postdocs than ever

and still not paid very well. The annual nationwide Survey of Earned Doctorates from a group of US government agencies shows that an increasing majority of Ph.D. recipients in the sciences go on to postdoctoral positions, as do the majority of Brandeis Ph.D. recipients in the life sciences, the only disciplines for which I  have statistics handy. The average salaries for postdocs are, as you might expect, less than luxurious when compared to other career paths taken by Ph.D. recipients.

Summer course on building a microscope from simple components

This past June the MRSEC Center offered a condensed summer course based on the popular graduate course QB120: Quantitative Biology Instrumentation Laboratory.

Professor Dogic

The course was taught by Zvonimir Dogic of the Physics Department (pictured).   Prof. Dogic has extensive experience with several forms of microscopy and his Lab features several home-built or heavily modified optical setups.

The course is designed to offer students hands on experience with building their own optical setups from basic components as well as learning how to optimally acquire imaging data from commercial microscopes.  The focus was on understanding the physics behind microscope function and leveraging that knowledge towards improving data acquisition in the lab.

Initially, students used basic lenses, apertures, an objective, a camera and a light source to build the simplest possible light microscope.  This initial setup was quickly extended to include Köhler illumination, a core principle in microscopy which allows even illumination of the sample as well as access to the conjugate image plane for image filtering.

The next project required students to build a fluorescence microscope, a highly relevant and ubiquitous technique in biological imaging.  To image a slide with fluorescently labeled beads students used a dichroic mirror to separate excitation light at one wavelength from emission light at another wavelength.  A schematic diagram, a photo of this setup with the light path superimposed and actual data acquired with one of these microscopes can be seen in the video below.

Next, a more advanced technique in microscopy, total internal reflection microscopy (TIRF), was introduced and an imaging setup using this technique was built.  TIRF microscopes excel at imaging small molecules that are immobilized in a small area.  A laser beam was pointed to shine through a prism at an angle sufficient to cause total internal reflection and the resulting evanescent wave caused fluorescent excitation of the sample.  The video below shows a schematic and imaging data of a TIRF microscope built by students.

Finally, students used commercial microscopes to understand the principles behind phase contrast and difference interference contrast microscopy, both techniques well suited for imaging samples that are nearly transparent.

Overall the Course provided an excellent introduction to the physical principles behind microscope function.  I highly recommend it to anyone interested in using microscopes in their research!

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