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ISSN
1672-9145
Acta Biochim Biophys Sin
2004, 36(9): 609–617
CN 31-1940/Q
Cleavage of Supercoiled Circular Double-stranded DNA Induced by a
Eukaryotic Cambialistic Superoxide Dismutase from Cinnamomum camphora
Bao-Zhong WANG, Xu-Bin WEI&, and Wang-Yi LIU*
State Key Laboratory of Molecular Biology, Institute of Biochemistry
and Cell Biology, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences, Shanghai 200031, China
Abstract A eukaryotic cambialistic superoxide dismutase (SOD) has been
purified to homogeneity from mature seeds of the disease- and insect-resistant
camphor tree (Cinnamomum camphora). Besides the known role of this SOD
in protecting cells against oxidative stress, it can induce the cleavage of
supercoiled double-stranded DNA into nicked and linear DNA. It can not cleave
linear DNA or RNA, demonstrating there is no DNase or RNase in the purified
cambialistic SOD. Furthermore, the SOD can linearize circular pGEM-4Z DNA that
is relaxed by topoisomerase I. This result indicates that the DNA-cleaving
activity requires substrates being topologically constrained. The supercoiled
DNA-cleaving activity of the cambialistic SOD can be inhibited by either SOD
inhibitor (azide) or catalase and hydroxyl radical scavengers (ethanol and
mannitol). The chelator of iron, diethylenetriaminepentaacetic acid (DTPA),
also inhibits the supercoiled DNA-cleaving activity. These results show that
the dismutation activity is crucial for the supercoiled DNA cleavage. The
modification of tryptophan residue of the cambialistic SOD with
N-bromosuccinimide (NBS) shows that these two activities are structurally
correlative. The reaction mechanism is proposed that the hydroxyl radical
formed in a transition-metal-catalyzing Fenton-type reaction contributes to the
DNA-cleaving activity. In addition, the cleavage sites in supercoiled pGEM-4Z
DNA are random.
Key words cambialistic SOD; Fenton-type reaction; hydroxyl radical; relaxed circular
duplex DNA; supercoiled DNA cleavage
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Received: Mar 17, 2004 Accepted: July 9,
2004
This work was supported by grants from Academia Sinica (39570173) and
the National Natural Science Foundation of China (39870186)
&A research student from the
Institute of life science, Lanzhou University
*Corresponding author: Tel, 86-21-54921238; Fax, 86-21-54921011;
E-mail, [email protected]