The Deinococcus radiodurans-Encoded HU Protein Has Two DNA-Binding Domains

Abstract

Deinococcus radiodurans can reconstitute its genome from double-strand breaks, most likely due to unusually efficient DNA repair and recombination. Factors that may contribute to such processes include the histone-like protein HU. The D. radiodurans-encoded HU (DrHU), which binds preferentially to DNA recombination intermediates, contains a 47-amino acid extension preceding the fold characteristic of HU proteins. Here we use electrophoretic mobility shift assays and DNA footprinting to show that the DrHU N-terminal domain significantly modulates DNA binding. The truncated DrHU (deltaDrHU), comprising only the conserved DNA-binding fold, has a site size of approximately 11 bp in contrast to full-length DrHU which does not stably engage DNA shorter than approximately 50 bp. Unlike wild-type DrHU, deltaDrHU distinguishes between linear DNA and DNA with nicks or gaps. DeltaDrHU also binds preferentially to four-way junction (4WJ) DNA, with half-maximal saturation of 1.4 +/- 0.4 nM compared to 20 +/- 2 nM for 37 bp duplex DNA. However, in contrast to full-length protein which binds the junction arms, deltaDrHU primarily protects the junction crossover. Evidently, the DrHU N-terminus changes the mode of binding to both 4WJ DNA, duplex DNA, and DNA with nicks or gaps, thereby resulting in DrHU binding preferentially only to 4WJ structures. Combined with Western blots that confirm the presence of the extended form of DrHU in vivo, our data provide mechanistic insight into discrimination between 4WJ DNA and other distorted DNA constructs and suggest that an in vivo role of DrHU may be to stabilize DNA junctions.

One of the Two Genes Encoding Nucleoid-Associated HU Proteins in Streptomyces coelicolor Is Developmentally Regulated and Specifically Involved in Spore Maturation

“…Desiccation leads to increased frequencies of double-stranded DNA breaks, and desiccation resistance is related to resistance to radioactivity, as indicated by studies of Deinococcus radiodurans (35). Interestingly, although HupSlike proteins were not found outside of the phylum Actinobacteria, the famously radiation-resistant D. radiodurans has an HU protein with lysine-rich repeats in an N-terminal extension, instead of at the C terminus, and this N-terminal part contributes to DNA-binding (20). It remains to be tested whether the HupS-like proteins contribute to radioresistance and desiccation resistance in K. radiotolerans and if they might also do so in S. coelicolor spores or dormant mycobacteria.…”

DNA organization by the apicoplast-targeted bacterial histone-like protein of Plasmodium falciparum

“…The gel was electrophoresed in 0.25× TBE. K d determination was carried out by incubating 100 femtomoles of 5′-end-labeled 30 bp double-stranded oligo probe with increasing concentrations of protein as described in Ghosh and Grove (31). The region of a lane from the complex upto the free probe was taken as complex.…”

Oxidative Stress Resistance inDeinococcus radiodurans

“…However, the nucleoid condensation role of HU is not supported by data from in vitro studies, which showed that HU cannot bend DNA or bind to DNA with nicks or gaps (209). HU binds to four-way DNA junctions and may be additionally optimized for DNA recombination events by stabilizing recombination intermediates (208,209). Contrary to HU, one of the D. radiodurans Dps ("DNA protection during starvation") proteins, Dps1 (DR2263), was shown to promote DNA compaction in vitro (54) but not in vivo, as its depletion has no effect on genome condensation and cell viability (459).…”