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Structure of bacteriophage T4 endonuclease II mutant E118A, a tetrameric GIY-YIG enzyme
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
2010 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 397, no 4, 1003-1016 p.Article in journal (Refereed) Published
Abstract [en]

Coliphage T4 endonuclease II (EndoII), encoded by gene denA, is a small (16Da, 136aa) enzyme belonging to the GIY-YIG family of endonucleases, which lacks a C-terminal domain corresponding to that providing most of the binding energy in the structurally characterized GIY-YIG endonucleases, I-TevI and UvrC. In vivo, it is involved in degradation of host DNA, permitting scavenging of host-derived nucleotides for phage DNA synthesis. EndoII primarily catalyzes single-stranded nicking of DNA; 5- to 10-fold less frequently double-stranded breaks are produced. The Glu118Ala mutant of EndoII was crystallized in space group P21 with four monomers in the asymmetric unit. The fold of the EndoII monomer is similar to that of the catalytic domains of UvrC and I-TevI. In contrast to these enzymes, EndoII forms a striking X-shaped tetrameric structure composed as a dimer of dimers, with a protruding hairpin domain not present in UvrC or I-TevI providing most of the dimerization and tetramerization interfaces. A bound phosphate ion in one of the four active sites of EndoII likely mimics the scissile phosphate in a true substrate complex. In silico docking experiments showed that a protruding loop containing a nuclease-associated modular domain 3 element is likely to be involved in substrate binding, as well as residues forming a separate nucleic acid binding surface adjacent to the active site. The positioning of these sites within the EndoII primary dimer suggests that the substrate would bind to a primary EndoII dimer diagonally over the active sites, requiring significant distortion of the enzyme or the substrate DNA, or both, for simultaneous nicking of both DNA strands. The scarcity of potential nucleic acid binding residues between the active sites indicates that EndoII may bind its substrate inefficiently across the two sites in the dimer, offering a plausible explanation for the catalytic preponderance of single-strand nicks. Mutations analyzed in earlier functional studies are discussed in their structural context.

Place, publisher, year, edition, pages
2010. Vol. 397, no 4, 1003-1016 p.
Keyword [en]
Endonuclease, GIY-YIG, Phage, Tetramer, X-ray crystallography
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:uu:diva-97873DOI: 10.1016/j.jmb.2010.01.076ISI: 000276566900013PubMedID: 20156453OAI: oai:DiVA.org:uu-97873DiVA: diva2:172966
Available from: 2008-11-27 Created: 2008-11-27 Last updated: 2013-11-15Bibliographically 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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