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Shunsuke Kurosawa^1,, Reiko Murakami^2,, Kiyoshi Onai^2,, Megumi Morishita², Daisuke Hasegawa¹, Ryo Iwase^2,3, Tatsuya Uzumaki^1,2, Fumio Hayashi^2,, Tomomi Kitajima-Ihara², Shuhei Sakata¹, Midori Murakami¹, Tsutomu Kouyama^1,4,* and Masahiro Ishiura^2,3,*

¹ Department of Physics, Graduate School of Science, Nagoya University, Furo, Chikusa, Nagoya 464-8602, Japan
² Center for Gene Research, Nagoya University, Furo, Chikusa, Nagoya 464-8602, Japan
³ Division of Biological Science, Graduate School of Science, Nagoya University, Furo, Chikusa, Nagoya 464-8602, Japan
⁴ RIKEN Harima Institute/SPring-8, 1-1-1, Kouto, Mikazuki, Sayo, Hyogo 679-5148, Japan

Pex, a clock-related protein involved in the input pathway of the cyanobacterial circadian clock system, suppresses the expression of clock gene kaiA and lengthens the circadian period. Here, we determined the crystal structure of Anabaena Pex (AnaPex; Anabaena sp. strain PCC 7120) and Synechococcus Pex (SynPex; Synechococcus sp. strain PCC 7942). Pex is a homodimer that forms a winged-helix structure. Using the DNase I protection and electrophoresis mobility shift assays on a Synechococcus kaiA upstream region, we identified a minimal 25-bp sequence that contained an imperfectly inverted repeat sequence as the Pex-binding sequence. Based on crystal structure, we predicted the amino acid residues essential for Pex's DNA-binding activity and examined the effects of various Ala-substitutions in the 3 helix and wing region of Pex on in vitro DNA-binding activity and in vivo rhythm functions. Mutant AnaPex proteins carrying a substitution in the wing region displayed no specific DNA-binding activity, whereas those carrying a substitution in the 3 helix did display specific binding activity. But the latter were less thermostable than wild-type AnaPex and their in vitro functions were defective. We concluded that Pex binds a kaiA upstream DNA sequence via its wing region and that its 3 helix is probably important to its stability.

These authors contributed equally to this work.

Present address: Graduate School of Science, Kyoto University, Kitashirakawa, Sakyo, Kyoto 606-8502, Japan.

Present address: Graduate school of Engineering, Gunma University, 1-5-1 Tenjin, Kiryu, Gunma 376-8515, Japan.

^* Correspondence: kouyama{at}bio.phys.nagoya-u.ac.jp or ishiura{at}gene.nagoya-u.ac.jp