Photon Factory Activity Report 2005 #23 Part B (2006)
Crystallography

6A, 18B, NW12/2003G113

Crystallographic analysis of the oxidative DNA repair enzyme from human.
Katsuo KATAYANAGI*, Yasunori KAMIYA, JingJie MAO, Yoshihiko HORI, Kenji YAMAMOTO,
Fuki HAGIWARA, Hiromi KITTAKA
Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University,Higashi-Hiroshima 739-8526, Japan

Introduction
By several aerobic systems in the nature andexogenous agents, reactive oxygen species generate andthey also induce oxidative damage to DNA. In order toavoid the unusual DNA synthesis or unusual DNA
replication derived from the DNA damages, cells haveseveral DNA repair mechanisms. For example, oxidative
pyrimidine lesions such as thymine glycols are primarily
excised from DNA by endonuclease III in E.coli. But
mammalian cells have a homologue of Endo III (NTH1).
Recently, Ide et al. found the broad specificities to theoxidative bases in the mammalian DNA repair enzymes
through the activity studies. In this study, by analyzing
repair enzyme structure of atomic resolution, we would
like to reconstitute the oxide base recognition mechanism.
Results and Discussion
At first, human NTH1 and human SMUG1 wereselected to crystallize for X-ray structure analysis. Bothrecombinant enzymes were expressed in E.coli as GST
fusion protein. After thrombin treatment, those repair
enzymes were isolated, but unstable after the condense
for crystallization. Thus, crystallizations were performedby coupling with purification, and protocols wereoptimized to avoid aggregation.
Crystal of SMUG1 was obtained by the micro batch
method. The crystal size was 0.1 x 0.1 x 0.2mm.
Although the crystal was very hard to obtain (1 crystal of300 trials), the edge shape was quite clear. The celldimensions were, a = 50.19 Å, b = 60.16 Å, c = 91.38 Å,and the space group belonged to orthorhombic P212121.
The data collections were performed using Quantum 4R
CCD detector using synchrotron radiation. The reflection
images were processed using the programs HKL2000 andMosflm. The crystal diffracted x-ray up to 2.0 Åresolution, and 17,125 independent reflections werecollected and the Rsym was 0.111. Initial phase weredetermined by molecular replacement method by using
program MolRep using the coordinate set of Xenopus
SMUG1, which was recently determined by Wibley et al.
Crystallographic refinement were performed using
REFMAC and the R and Rfree were converged to 0.173
and 0.229, respectively. The main chain structure ofhuman and Xenopus SMUG1s are almost identical. In thecase of Xenopus SMUG1, nucleic base such as

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Users' Reporthydroxylmethyl uracil (hmU) and uracil were used assubstrate. But in the case of human enzyme, hydroxyl
uracil were used as substrate. Comparing the binding
thscheme of these three substrates, the recognition of the 5site of base became clearly. And combing with the resultof the site directed mutagenesis, SMUG1 was found tothrequire hydroxyl group at 5 site or, in the case of uracil,
this space is filled up with free water molecule in order toform hydrogen bond. In addition, only in the case ofhuman enzyme, one side chain of glutamic acid werefound to involved for the substare binding.
In the case of NTH1, obtained crystals were quite
small and gave low resolution as 4.5 Å. But recently, wecould obtain larger crystal, of which resolution was 3.2
Å. Now, heavy atom search is in progress.
References[1] Matsubara, M., Tanaka, T., Terato, H., Ohmae, E.,
Izumi, S., Katayanagi, K. & Ide, H. Nucleic Acid
Research., 32, 5291-5302 (2004).

* kkata@sci.hiroshima-u.ac.jp