A combined structural and biochemical approach reveals translocation and stalling of UvrB on the DNA lesion as a mechanism of damage verification in bacterial nucleotide excision repair

Nucleotide excision repair (NER) is a DNA repair pathway present in all domains of life. In bacteria, UvrA protein localizes the DNA lesion, followed by verification by UvrB helicase and excision by UvrC double nuclease. UvrA senses deformations and flexibility of the DNA duplex without precisely localizing the lesion in the damaged strand, an element essential for proper NER. Using a combination of techniques, we elucidate the mechanism of the damage verification step in bacterial NER. UvrA dimer recruits two UvrB molecules to its two sides. Each of the two UvrB molecules clamps a different DNA strand using its \u3b2-hairpin element. Both UvrB molecules then translocate to the lesion, and UvrA dissociates. The UvrB molecule that clamps the damaged strand gets stalled at the lesion to recruit UvrC. This mechanism allows UvrB to verify the DNA damage and identify its precise location triggering subsequent steps in the NER pathway

How much of protein sequence space has been explored by life on Earth?

We suggest that the vastness of protein sequence space is actually completely explorable during the populating of the Earth by life by considering upper and lower limits for the number of organisms, genome size, mutation rate and the number of functionally distinct classes of amino acids. We conclude that rather than life having explored only an infinitesimally small part of sequence space in the last 4 Gyr, it is instead quite plausible for all of functional protein sequence space to have been explored and that furthermore, at the molecular level, there is no role for contingency

"4D Biology for health and disease" workshop report

The "4D Biology Workshop for Health and Disease", held on 16-17th ofMarch 2010 in Brussels, aimed at finding the best organising principlesfor large-scale proteomics, interactomics and structural genomics/biology initiatives, and setting the vision for future high-throughputresearch and large-scale data gathering in biological and medical science.Major conclusions of the workshop include the following. (i)Development of new technologies and approaches to data analysis iscrucial. Biophysical methods should be developed that span a broadrange of time/spatial resolution and characterise structures andkinetics of interactions. Mathematics, physics, computational andengineering tools need to be used more in biology and new tools needto be developed. (ii) Database efforts need to focus on improveddefinitions of ontologies and standards so that system-scale data andassociated metadata can be understood and shared efficiently. (iii)Research infrastructures should play a key role in fosteringmultidisciplinary research, maximising knowledge exchange betweendisciplines and facilitating access to diverse technologies. (iv)Understanding disease on a molecular level is crucial. Systemapproaches may represent a new paradigm in the search for biomarkersand new targets in human disease. (v) Appropriate education andtraining should be provided to help efficient exchange of knowledgebetween theoreticians, experimental biologists and clinicians. Theseconclusions provide a strong basis for creating major possibilities inadvancing research and clinical applications towards personalisedmedicine.Biophysical Structural Chemistr

Crystal Structure of the Escherichia coli 23S rRNA:m5C Methyltransferase RlmI (YccW) Reveals Evolutionary Links between RNA Modification Enzymes

10.1016/j.jmb.2008.08.062Journal of Molecular Biology3833652-666JMOB

Crystal structure of human Acinus RNA recognition motif domain

Acinus is an abundant nuclear protein involved in apoptosis and splicing. It has been implicated in inducing apoptotic chromatin condensation and DNA fragmentation during programmed cell death. Acinus undergoes activation by proteolytic cleavage that produces a truncated p17 form that comprises only the RNA recognition motif RRM domain. We have determined the crystal structure of the human Acinus RRM domain AcRRM at 1.65 resolution. It shows a classical four stranded antiparallel amp; 946; sheet fold with two flanking amp; 945; helices and an additional, non classical amp; 945; helix at the C terminus, which harbors the caspase 3 target sequence that is cleaved during Acinus activation. In the structure, the C terminal amp; 945; helix partially occludes the potential ligand binding surface of the amp; 946; sheet and hypothetically shields it from non sequence specific interactions with RNA. Based on the comparison with other RRM RNA complex structures, it is likely that the C terminal amp; 945; helix changes its conformation with respect to the RRM core in order to enable RNA binding by Acinu

The crystal structure of Escherichia coli spermidine synthase SpeE reveals a unique substrate-binding pocket

10.1016/j.jsb.2009.12.024Journal of Structural Biology1693277-285JSBI

Crystal structure of a fructokinase homolog from Halothermothrix orenii

10.1016/j.jsb.2010.05.007Journal of Structural Biology1713397-401JSBI

Specific interaction of zinc finger protein Com with RNA and the crystal structure of a self complementary RNA duplex recognized by Com

The bacteriophage Mu Com is a small zinc finger protein that binds to its cognate mom mRNA and activates its translation. The Mom protein, in turn, elicits a chemical modification momification of the bacteriophage genome, rendering the DNA resistant to cleavage by bacterial restriction endonucleases, and thereby protecting it from defense mechanisms of the host. We examined the basis of specificity in Com RNA interactions by in vitro selection and probing of RNA structure. We demonstrated that Com recognizes a sequence motif within a hairpin loop structure of its target RNA. Our data support the model of Com interaction with mom mRNA, in which Com binds to the short hairpin structure proximal to the so called translation inhibition structure. We also observed that Com binds its target motif weakly if it is within an RNA duplex. These results suggest that the RNA structure, in addition to its sequence, is crucial for Com to recognize its target and that RNA conformational changes may constitute another level of Mom regulation. We determined a crystal structure of a Com binding site variant designed to form an RNA duplex preferentially. Our crystal model forms a 19 mer self complementary double helix composed of the canonical and non canonical base pairs. The helical parameters of crystalized RNA indicate why Com may bind it more weakly than a monomeric hairpin for

The structure of sucrose phosphate synthase from Halothermothrix orenii reveals its mechanism of action and binding mode

Plant Cell2041059-1072PLCE