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Amino acid residues in the GIY-YIG endonuclease II of phage T4 affecting sequence recognition and binding as well as catalysis
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.
2008 (English)In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 190, no 16, 5533-5544 p.Article in journal (Refereed) Published
Abstract [en]

Phage T4 endonuclease II (EndoII), a GIY-YIG endonuclease lackinga carboxy-terminal DNA-binding domain, was subjected to site-directedmutagenesis to investigate roles of individual amino acids insubstrate recognition, binding, and catalysis. The structureof EndoII was modeled on that of UvrC. We found catalytic rolesfor residues in the putative catalytic surface (G49, R57, E118,and N130) similar to those described for I-TevI and UvrC; inaddition, these residues were found to be important for substraterecognition and binding. The conserved glycine (G49) and arginine(R57) were essential for normal sequence recognition. Our resultsare in agreement with a role for these residues in forming theDNA-binding surface and exposing the substrate scissile bondat the active site. The conserved asparagine (N130) and an adjacentproline (P127) likely contribute to positioning the catalyticdomain correctly. Enzymes in the EndoII subfamily of GIY-YIGendonucleases share a strongly conserved middle region (MR,residues 72 to 93, likely helical and possibly substitutingfor heterologous helices in I-TevI and UvrC) and a less stronglyconserved N-terminal region (residues 12 to 24). Most of theconserved residues in these two regions appeared to contributeto binding strength without affecting the mode of substratebinding at the catalytic surface. EndoII K76, part of a conservedNUMOD3 DNA-binding motif of homing endonucleases found to overlapthe MR, affected both sequence recognition and catalysis, suggestinga more direct involvement in positioning the substrate. Ourdata thus suggest roles for the MR and residues conserved inGIY-YIG enzymes in recognizing and binding the substrate.

Place, publisher, year, edition, pages
2008. Vol. 190, no 16, 5533-5544 p.
Keyword [en]
phage T4, GIY-YIG endonuclease, nicking, structure modeling, sequence recognition, information content
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-97870DOI: 10.1128/JB.00094-08ISI: 000258274400001OAI: oai:DiVA.org:uu-97870DiVA: diva2:172963
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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