Phosphorylation Regulation Of A Histone-like HU Protein From Deinococcus Radiodurans

Deinococcus radiodurans has strong tolerance to various external environmental stresses,such as drought,ionizing radiation,and oxidative stress.Nucleoid-associated proteins(NAPs)are widely distributed in prokaryotes and belong to a class of DNA-binding proteins with small molecular weights,which are involved in various physiological processes including nucleoid morphogenesis,DNA compression,DNA replication and DNA repair.However,the detail mechanisms of post-translational modification regulation of NAPs’ s activity and function remains unclear.In this study,we focused on the DR＿A0065 gene and its encoding nucleoid-associated protein,Dr HU,to study the phosphorylation regulation mechanism of Dr HU and its DNA damage response to environmental stress.The main results are as follows:(1)Deinococcus radiodurans genome encodes a sole nucleoid-associated protein Dr HU(DR＿A0065),which belongs to the Histone-like protein(HU protein)family.Compared with other HU family proteins,in addition to the relatively conserved core domain(approximate 90 amino acids),Dr HU has an additional N-terminal extension region about 32 amino acids,which is only conserved in the Deinococcus HU proteins.(2)According to the previous results,after enrichment of phosphopeptide,LC–MS/MS analysis showed that Dr HU contained a phosphorylation modification at the Thr37 site.This phosphorylation site is located at the very N-terminal region of the core domain,which is present in not only all Deinococcus species but also HU proteins from other bacterias.As noted by previous studies,complete inactivation of Dr HU is lethal.We were unable to generate a homozygous knockout Dr HU mutant using a conventional homologous recombination-based method.To investigate the phenotypical properties of Dr HU phosphorylation with regard to DNA repair,we used a complemented-knockout strategy instead of a knockout-complemented method.Briefly,the complementary plasmid p RADK containing DR＿A0065 was transformed into the D.radiodurans wild-type R1 strain,followed by knockout mutagenesis.In addition to the wild-type protein,we generated two types of mutant proteins: one in which this threonine is replaced by glutamate to mimic a permanent state of phosphorylation(T37E),and one in which this threonine is mutated to alanine to prevent phosphorylation(T37A).Compared with the wild-type strain,complementation of T37 E in a Dr HU-knockout strain caused growth defects and sensitized the cells to UV radiation and oxidative stress,indicating the toxic effects of overphosphorylation of Dr HU in vivo.(3)Transcriptional analysis was used to investigate the phosphorylation regulation of Dr HU.A total of 934 up-regulated genes and 847 down-regulated genes were identified,including proteins involved in DNA damage repair processes such as Ddr genes and the novel DNA damage repair transcriptional factor Ppr A.In addition,TEM results showed that both ΔDr HU/pk-T37 E and ΔDr HU/pk-T37 A exhibited nucleoid dispersion compared to the wild-type strain,and the degree of nucleoid dispersion of ΔDr HU/pk-T37 E was greater,which may explain the reduced DNA damage resistance of ΔDr HU/pk-T37 E.(4)Dr HU protein was expressed and purified to measure the enzymatic kinetics.Compared with the wild type Dr HU protein,DNA binding of T37 A mutant Dr HU was almost eliminated even with a longer DNA.And T37 E Dr HU exhibited much elevated DNA binding(lower Kd value)for short duplex DNA.However,the phosphorylation mimic T37 E exhibited much weaker protection against hydroxyl radical cleavage,which is consistent with the reduced rates of spontaneous mutation and transformation efficiency.Collectively,our findings provide the first evidence that phosphorylation modulates the DNA-binding capabilities of the Histone-like HU protein from D.radiodurans,which may contribute to the robustness of this organism.However,physiological phosphorylation is dynamic and transient in response to various stimuli or cellular events.Given the toxicity of the phosphorylation-mimic protein in the absence of genomic-expressed wild type Dr HU,phosphorylation of Dr HU must be very tightly controlled in response to environmental stresses.