DNA Repair 11 (2012) 139–145 Contents lists available at SciVerse ScienceDirect DNA Repair jo u rn al hom epa ge: www.elsevier.com/locate/dnarepair DNA binding is essential for PprI function in response to radiation damage in Deinococcus radiodurans Huiming Lu a , Huan Chen b , Guangzhi Xu a , Amir Miraj-Ul-Hussain Shah a , Yuejin Hua a,∗ a Institute of Nuclear-Agricultural Sciences, Key Laboratory of Chinese Ministry of Agriculture for Nuclear-Agricultural Sciences, Zhejiang University, 310029 Hangzhou, China b Zhejiang Institute of Microbiology, 310012 Hangzhou, China a r t i c l e i n f o Article history: Available online 2 November 2011 Keywords: Deinococcus radiodurans PprI Response regulator DNA damage Ionizing radiation a b s t r a c t The extremely radioresistant bacterium Deinococcus radiodurans possesses a rapid and efficient but poorly known DNA damage response mechanism that mobilizes one-third of its genome to survive lethal radia- tion damage. Deinococcal PprI serves as a general switch to regulate the expression of dozens of proteins from different pathways after radiation, including the DNA repair proteins RecA, PprA and SSB. However, the underlying mechanism is poorly understood. In this study, we analyzed the dynamic alteration in global transcriptional profiles in wildtype and pprI mutant strains by combining microarrays and time- course sampling. We found that PprI up-regulated transcription of at least 210 genes after radiation, including 21 DNA repair and replication-related genes. We purified PprI and a helix-turn-helix (HTH) domain mutant and found that PprI specifically bound to the promoters of recA and pprA in vitro but did not bind nonspecific double-strand DNA. Chromatin immunoprecipitation (ChIP) assays confirmed that PprI specifically interacted with the promoter DNA of recA and pprA after radiation. Finally, we showed that a DNA-binding activity-deficient pprI mutant only partially restored resistance of the pprI mutant strain to  radiation, UV radiation, and mitomycin C. Taken together, these results indicate that DNA-binding activity is essential for PprI to program the DNA repair process and cellular survival of D. radiodurans in response to radiation damage. © 2011 Elsevier B.V. All rights reserved. 1. Introduction The extremely radioresistant bacterium Deinococcus radiodu- rans can survive acute  radiation at doses of 18 kGy and grows normally under continuing  radiation at a dose rate of 60 Gy/h [1,2]. This organism also shows high tolerance to other DNA- damaging agents, including UV radiation, mitomycin C, desiccation, and hydroxyl peroxide [3]. The unique functions of this species have been the subject of studies on the underlying mechanisms for 50 years. A 5-kGy radiation introduces 200 double-strand breaks (DSBs) into the D. radiodurans genome, but the bacterium can pre- cisely reassemble the genome from these DNA fragments within hours, indicating efficient DNA repair ability [3]. D. radiodurans possesses almost a full set of DNA repair pathways, including exci- sion repair, mismatch repair, recombinational repair, and potential Abbreviations: PprI, inducer of pleiotropic proteins promoting DNA repair; RecA, recombinase A; DSBs, double-strand DNA breaks; PprA, pleiotropic protein promot- ing DNA repair A; PIR, post-irradiation recovery; dsDNA, double-strand DNA; ssDNA, single-strand DNA; HTH, helix-turn-helix; GAF, cyclic GMP, adenylyl cyclase, FhlA; 2-DE, two-dimensional electrophoresis; COGs, Clusters of Orthologous Groups. ∗ Corresponding author. Tel.: +86 571 8697 1703; fax: +86 571 8697 1703. E-mail address: yjhua@zju.edu.cn (Y. Hua). non-homologous end-joining [4,5]. A RecA-independent phase and RecA-dependent phase are believed to constitute the DNA repair process [6]. Recently, extended synthesis-dependent strand annealing (ESDSA) was suggested to describe the reassembly of the radiation-shattered genome [7,8]. However, much research is still required to understand the detailed functions of D. radiodu- rans. Beyond DNA repair, the ability to deal with oxidative stress facilitates the survival of D. radiodurans under ionizing radia- tion [9]. The high concentration of manganese in D. radiodurans cells protects proteins from reactive oxygen species (ROS), lead- ing to the quick response of this bacterium to radiation damage [10,11]. In addition, abundant cellular carotenoid enhance the effi- ciency of ROS-scavenging [12]. Combined with the efficient DNA repair mechanisms and strong ROS-scavenging ability, the rapid DNA damage response mechanisms of D. radiodurans mobilize involved systems to repair serious damage and are important in the extreme radioresistance [9,13]. The D. radiodurans stress response to DNA-damaging agents involves transcriptional and translational alterations [14–16], and posttranslational modification [13,17,18]. Our laboratory identified several regulators responsible for the transcriptional response to DNA-damaging agents, including PprI (also called IrrE) [13,19,20], DrRRA [21], OxyR [22,23], and DR0171 [24]. Inactivation of pprI causes the most serious loss of radioresis- tance among these regulators (reviewed in [9]). 1568-7864/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.dnarep.2011.10.013