Institut de Biologie StructuraleGrenoble / France

Contact person(s) related to this article / TIMMINS Joanna

Dynamics of DNA Damage Repair Processes

A. Double-Strand Break (DSB) Repair

Homologous recombination plays an essential role in the repair of a variety of DNA lesions, including the lethal DSBs. Both in bacteria and in higher eukaryotes, the mechanisms of recombinational repair are still only poorly understood. Although most of the proteins involved have been identified, the detailed sequence of events leading from DSB recognition to RecA-associated strand exchange remains unclear.

Research Goals

We aim to gain further insight into the role and function of two essential proteins involved in recombinational repair in D. radiodurans: RecN and RecR. RecN has been proposed to be responsible for DSB recognition, while RecR along with its known cellular partners (RecO and RecF) loads RecA onto single-stranded DNA extensions prior to homologous recombination. We wish to study the various macromolecular complexes formed between these different cellular partners (DNA and proteins) using a combination of protein crystallography, small-angle X-Ray/Neutron scattering and single molecule fluorescence.

The first quasi-atomic structure of an intact SMC (Structural Maintenance of Chromosomes) protein, namely RecN, was assembled from three crystal structures of overlapping fragments of RecN and small-angle X-ray scattering data of the full-length protein. Together with functional data, this structural work allowed us to propose a model for the role of RecN in the early steps of repair of DNA double-strand breaks (Pellegrino et al., 2012 Structure).

B. Nucleotide excision repair by the Uvr proteins

Ultra-violet (UV) light is the most pervasive environmental DNA-damaging agent and introduces a diversity of lesions. NER is the primary pathway for repair of such lesions and involves the recognition and subsequent removal of damaged DNA by a dual-incision event. In prokaryotes, this process is carried out by the four Uvr proteins: UvrA, UvrB, UvrC and UvrD.

Research Goals

In collaboration with the PIXEL Team at IBS, we wish to use super-resolution microscopy to localize and follow the individual Uvr proteins within their native cellular environment, i.e. in Deinococcus radiodurans cells, before and after treatment of such cells with genotoxic agents and/or UV radiation. To achieve this goal, we will carry out PALM (photoactivated localization microscopy) imaging on genetically modified D. radiodurans cells expressing Uvr proteins fused to fluorescent proteins.

A-B. Diffraction-limited images (top) and super-resolution images (bottom) of Dendra2-UvrA1 (A) and mEosFP-M159A-UvrA1 (B) in E. coli cells overexpressing D. radiodurans UvrA1 in fusion with fluorescent proteins (Dendra2 and mEOSFP-M159A). C. Example of diffraction-limited (top) and super-resolution (bottom) images of the nucleoid-associated protein HU fused to eYFP in fixed C. crescentus. D. Electron microscopy images of D. radiodurans cells recorded at the IBS. Scale bars represent 0.5µm in A-C and 0.2µm in D.