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Nucleic Acids Research Advance Access originally published online on August 7, 2009
Nucleic Acids Research 2009 37(18):6174-6183; doi:10.1093/nar/gkp652
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Nucleic Acids Research, 2009, Vol. 37, No. 18 6174-6183
© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Nucleic Acid Enzymes

Bacteriophage T4 endonuclease II, a promiscuous GIY-YIG nuclease, binds as a tetramer to two DNA substrates

Pernilla Lagerbäck1, Evalena Andersson2, Christer Malmberg1 and Karin Carlson1,*

1Department of Cell and Molecular Biology, University of Uppsala and 2Department of Molecular Biology, Swedish University of Agricultural Sciences, Uppsala, Sweden

*To whom correspondence should be addressed. Tel: +46 18 471 40 18; Fax: +46 18 53 03 96; Email: karin.carlson{at}icm.uu.se

Received June 21, 2009. Revised July 21, 2009. Accepted July 21, 2009.

The oligomerization state and mode of binding to DNA of the GIY-YIG endonuclease II (EndoII) from bacteriophage T4 was studied using gel filtration and electrophoretic mobility shift assays with a set of mutants previously found to have altered enzyme activity. At low enzyme/DNA ratios all mutants except one bound to DNA only as tetramers to two DNA substrates. The putatively catalytic E118 residue actually interfered with DNA binding (possibly due to steric hindrance or repulsion between the glutamate side chain and DNA), as shown by the ability of E118A to bind stably also as monomer or dimer to a single substrate. The tetrameric structure of EndoII in the DNA–protein complex is surprising considering the asymmetry of the recognized sequence and the predominantly single-stranded nicking. Combining the results obtained here with those from our previous in vivo studies and the recently obtained crystal structure of EndoII E118A, we suggest a model where EndoII translocates DNA between two adjacent binding sites and either nicks one strand of one or both substrates bound by the tetramer, or nicks both strands of one substrate. Thus, only one or two of the four active sites in the tetramer is catalytically active at any time.


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