Simultaneous binding of three recognition sites is necessary for a concerted plasmid DNA cleavage by EcoRII restriction endonuclease

Journal of Molecular Biology
Gintautas TamulaitisVirginijus Siksnys

Abstract

According to the current paradigm type IIE restriction endonucleases are homodimeric proteins that simultaneously bind to two recognition sites but cleave DNA at only one site per turnover: the other site acts as an allosteric locus, activating the enzyme to cleave DNA at the first. Structural and biochemical analysis of the archetypal type IIE restriction enzyme EcoRII suggests that it has three possible DNA binding interfaces enabling simultaneous binding of three recognition sites. To test if putative synapsis of three binding sites has any functional significance, we have studied EcoRII cleavage of plasmids containing a single, two and three recognition sites under both single turnover and steady state conditions. EcoRII displays distinct reaction patterns on different substrates: (i) it shows virtually no activity on a single site plasmid; (ii) it yields open-circular DNA form nicked at one strand as an obligatory intermediate acting on a two-site plasmid; (iii) it cleaves concertedly both DNA strands at a single site during a single turnover on a three site plasmid to yield linear DNA. Cognate oligonucleotide added in trans increases the reaction velocity and changes the reaction pattern for the EcoRII cleavage of one and...Continue Reading

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Citations

Sep 30, 2008·Nucleic Acids Research·Gintautas TamulaitisVirginijus Siksnys
Jun 24, 2010·Nucleic Acids Research·Mindaugas ZarembaVirginijus Siksnys
Mar 9, 2013·Microbiology and Molecular Biology Reviews : MMBR·Kommireddy Vasu, Valakunja Nagaraja
Jul 27, 2006·Proceedings of the National Academy of Sciences of the United States of America·Gregory J GemmenDouglas E Smith
Oct 22, 2008·Biochimica Et Biophysica Acta·Chun-Yan XuJian-Hua He
Sep 22, 2006·Journal of Molecular Biology·Mindaugas ZarembaVirginijus Siksnys
Jun 1, 2014·Nucleic Acids Research·Alfred PingoudWolfgang Wende
Jun 5, 2014·Nucleic Acids Research·John R HortonXiaodong Cheng
Sep 18, 2007·Journal of Molecular Biology·Stuart R W BellamyStephen E Halford
Feb 3, 2007·Journal of Molecular Biology·Jacqueline J T MarshallStephen E Halford
Sep 5, 2009·Nucleic Acids Research·Dmitrij GolovenkoVirginijus Siksnys
Jan 1, 2013·ISRN Biochemistry·Peechapack SomyoonsapSomchai Pornbanlualap
Sep 22, 2017·Nucleic Acids Research·Giedrius SasnauskasVirginijus Siksnys
Sep 12, 2007·Biochemistry·Luda S ShlyakhtenkoYuri L Lyubchenko
Jul 23, 2008·Biochemistry·Arturas JakubauskasArvydas Janulaitis
Jun 28, 2014·The Journal of Physical Chemistry. B·Danielis RutkauskasVirginijus Siksnys

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