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Bacteriophage T4 endonuclease II, a promiscuous GIY-YIG nuclease, binds as a tetramer to two DNA substrates
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
2009 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 37, no 18, 6174-6183 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2009. Vol. 37, no 18, 6174-6183 p.
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:uu:diva-97871DOI: 10.1093/nar/gkp652ISI: 000271109300027PubMedID: 19666720OAI: oai:DiVA.org:uu-97871DiVA: diva2:172964
Available from: 2008-11-27 Created: 2008-11-27 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Endonuclease II - a GIY-YIG enzyme of bacteriophage T4
Open this publication in new window or tab >>Endonuclease II - a GIY-YIG enzyme of bacteriophage T4
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Endonuclease II (EndoII) of bacteriophage T4 is a GIY-YIG enzyme involved in host DNA breakdown during phage infection of E. coli. EndoII combines features of restriction endonucleases with those of homing endonucleases in that it breaks down DNA foreign to itself but recognizes a 16 bp long asymmetric and ambiguous sequence. This investigation addresses the biological function of EndoII, its mode of interaction with its substrate and roles of individual residues in catalysis, sequence recognition and binding.

It is shown here that EndoII increases the frequency of non-homologous recombination in phage-infected cells, showing that EndoII indeed can induce recombinational events. Although single-stranded nicks are frequent in in vitro reactions with purified protein, the enzyme is found to produce mostly double-stranded breaks in vivo, since nicks are repaired. Mutations of residues positioned on the putative catalytic surface result in severely reduced catalytic activity, while residues in the N-terminal region and a middle region (MR) appear to mainly contribute to substrate binding. Mutation of the putatively magnesium-binding residue E118 renders the enzyme catalytically inactive. Residues K76 (in the MR and positioned on the catalytic surface) and G49 and R57 (on the catalytic surface) also contribute to substrate recognition. All mutants bind as tetramers to two DNA molecules, indicating that the wildtype would also bind as a tetramer. EndoII E118A alone can bind also in monomeric and dimeric form to one DNA molecule, possibly because the glutamate charge normally repels the DNA. The solved crystal structure of tetrameric EndoII E118A shows a striking X-shape with two putative catalytic surfaces to each side positioned so that double-stranded cleavage would require severe DNA distortion. Combination of all data suggests that upon binding in vivo EndoII scans the DNA for a second binding site, binding to both sites but nicking or cleaving only one of them.

Place, publisher, year, edition, pages
Uppsala: Universitetsbiblioteket, 2008. 53 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 582
Keyword
GIY-YIG, EndoII, endonuclease, structure, tetramer, binding, nicking, recombination
National Category
Microbiology
Identifiers
urn:nbn:se:uu:diva-9410 (URN)978-91-554-7361-7 (ISBN)
Public defence
2008-12-19, B41, Biomedicinskt Centrum, Husargatan 3, Uppsala, 10:15
Opponent
Supervisors
Available from: 2008-11-27 Created: 2008-11-27Bibliographically approved

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Lagerbäck, PernillaCarlson, Karin

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