Abstract
Deinococcus radiodurans is an extraordinarily radioresistant bacterium that is able to repair hundreds of radiation-induced double-stranded DNA breaks. One of the players in this pathway is an X family DNA polymerase (PolX(Dr)). Deletion of PolX(Dr) has been shown to decrease the rate of repair of double-stranded DNA breaks and increase cell sensitivity to gamma-rays. A 3'-->5' exonuclease activity that stops cutting close to DNA loops has also been demonstrated. The present crystal structure of PolX(Dr) solved at 2.46-A resolution reveals that PolX(Dr) has a novel extended conformation in stark contrast to the closed "right hand" conformation commonly observed for DNA polymerases. This extended conformation is stabilized by the C-terminal PHP domain, whose putative nuclease active site is obstructed by its interaction with the polymerase domain. The overall conformation and the presence of non standard residues in the active site of the polymerase X domain makes PolX(Dr) the founding member of a novel class of polymerases involved in DNA repair but whose detailed mode of action still remains enigmatic.
Highlights
The atomic coordinates and structure factors have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ
In the active site of the polymerase catalytic domain, the two universally conserved aspartates are replaced by two glutamates, whereas the active site of the PHP domain is obstructed by its interaction with the polymerase domain
PolXDr is composed of two distinct parts: the polymerase domain, itself composed of subdomains, and the PHP domain
Summary
Expression and Purification—PolXDr was expressed as previously described [3]. The induced cells were harvested and resuspended in buffer A (20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 10 mM imidazole, and a mixture of anti-proteases (Complete, Roche Applied Science)). The bound proteins were eluted by buffer B (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 150 mM imidazole). The eluate was loaded onto a 5-ml HiTrap heparin column (Amersham Biosciences) pre-equilibrated with buffer B without imidazole. PolXDr was eluted by a linear gradient from 150 mM to 1 M NaCl on buffer B using an A KTA Purifier System (Amersham Biosciences). Crystallization, Data Collection, Structure Solution, and Refinement—Native protein (4 mg/ml) crystallized at 293 K by the hanging drop, vapor-diffusion method from 1:1 l drops of protein and precipitant containing 0.1 M NaAc, pH 5, 0.2 M NaCl, 16% 2-methyl-2,4-pentanediol. X-ray diffraction data from a crystal of the native protein was collected on beamline ID23-1 at the European Synchrotron Radiation Facility (Grenoble, France).
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