R.KpnI, an HNH superfamily REase, exhibits differential discrimination at non-canonical sequences in the presence of Ca2+ and Mg2+

Nucleic Acids Research
Matheshwaran SaravananValakunja Nagaraja

Abstract

KpnI REase recognizes palindromic sequence, GGTACC, and forms complex in the absence of divalent metal ions, but requires the ions for DNA cleavage. Unlike most other REases, R.KpnI shows promiscuous DNA cleavage in the presence of Mg2+. Surprisingly, Ca2+ suppresses the Mg2+-mediated promiscuous activity and induces high fidelity cleavage. To further analyze these unique features of the enzyme, we have carried out DNA binding and kinetic analysis. The metal ions which exhibit disparate pattern of DNA cleavage have no role in DNA recognition. The enzyme binds to both canonical and non-canonical DNA with comparable affinity irrespective of the metal ions used. Further, Ca2+-imparted exquisite specificity of the enzyme is at the level of DNA cleavage and not at the binding step. With the canonical oligonucleotides, the cleavage rate of the enzyme was comparable for both Mg2+- and Mn2+-mediated reactions and was about three times slower with Ca2+. The enzyme discriminates non-canonical sequences poorly from the canonical sequence in Mg2+-mediated reactions unlike any other Type II REases, accounting for the promiscuous behavior. R.KpnI, thus displays properties akin to that of typical Type II REases and also endonucleases with deg...Continue Reading

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Citations

Mar 9, 2012·Applied Microbiology and Biotechnology·Richa GuptaPrince Sharma
Jul 24, 2008·Proceedings of the National Academy of Sciences of the United States of America·Matheshwaran SaravananValakunja Nagaraja
Apr 18, 2012·Proceedings of the National Academy of Sciences of the United States of America·Kommireddy VasuValakunja Nagaraja
Aug 22, 2013·Nucleic Acids Research·Kommireddy VasuValakunja Nagaraja
Mar 9, 2013·Microbiology and Molecular Biology Reviews : MMBR·Kommireddy Vasu, Valakunja Nagaraja
Feb 9, 2010·PloS One·Siu-Hong ChanShuang-Yong Xu
Jan 1, 2008·Protein Expression and Purification·Yongming BaoShuang-Yong Xu
Mar 22, 2012·Protein Science : a Publication of the Protein Society·Serisha MoodleyVoula Kanelis
Jun 1, 2014·Nucleic Acids Research·Alfred PingoudWolfgang Wende
Sep 29, 2012·Microbiological Research·Abdelkarim Belkebir, Houssine Azeddoug
Aug 18, 2009·Journal of Molecular Biology·Vera PingoudAlfred Pingoud
Oct 19, 2017·Nucleic Acids Research·Céline RalecGhislaine Henneke
Nov 17, 2009·Cellular and Molecular Life Sciences : CMLS·Maria J MarcaidaGuillermo Montoya
Sep 6, 2007·The Journal of Biological Chemistry·Matheshwaran SaravananValakunja Nagaraja
Aug 26, 2010·Biochemistry·Kommireddy VasuValakunja Nagaraja

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Methods Mentioned

BETA
column chromatography
electrophoresis
Fluorescence

Software Mentioned

Phosphorimager Image Gauge
Graphpad Prism

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