Elimination of inter-domain interactions increases the cleavage fidelity of the restriction endonuclease DraIII

Protein & Cell
Wei ZhuoDapeng Sun

Abstract

DraIII is a type IIP restriction endonucleases (REases) that recognizes and creates a double strand break within the gapped palindromic sequence CAC↑NNN↓GTG of double-stranded DNA (↑ indicates nicking on the bottom strand; ↓ indicates nicking on the top strand). However, wild type DraIII shows significant star activity. In this study, it was found that the prominent star site is CAT↑GTT↓GTG, consisting of a star 5' half (CAT) and a canonical 3' half (GTG). DraIII nicks the 3' canonical half site at a faster rate than the 5' star half site, in contrast to the similar rate with the canonical full site. The crystal structure of the DraIII protein was solved. It indicated, as supported by mutagenesis, that DraIII possesses a ββα-metal HNH active site. The structure revealed extensive intra-molecular interactions between the N-terminal domain and the C-terminal domain containing the HNH active site. Disruptions of these interactions through site-directed mutagenesis drastically increased cleavage fidelity. The understanding of fidelity mechanisms will enable generation of high fidelity REases.

References

Oct 12, 1987·Nucleic Acids Research·M Nasri, D Thomas
Mar 11, 1985·Nucleic Acids Research·R GrosskopfC Kessler
Jan 1, 1986·Gene·S E HalfordS A McCallum
Apr 1, 1982·Proceedings of the National Academy of Sciences of the United States of America·J George, J G Chirikjian
Jul 1, 1982·Proceedings of the National Academy of Sciences of the United States of America·W E JackP Modrich
Apr 11, 1995·Proceedings of the National Academy of Sciences of the United States of America·C R Robinson, S G Sligar
Nov 5, 1999·The Journal of Biological Chemistry·D J WrightP Modrich
Jul 6, 2000·Molecular Cell·H Viadiu, A K Aggarwal
Dec 6, 2001·Journal of Molecular Biology·A J PommerC Kleanthous
Oct 24, 2002·Acta Crystallographica. Section D, Biological Crystallography·Paul D AdamsThomas C Terwilliger
Mar 26, 2003·Nucleic Acids Research·Richard J RobertsShuang-yong Xu
Jun 26, 2003·Nucleic Acids Research·Tamas VinczeRichard J Roberts
Dec 24, 2003·Protein Science : a Publication of the Protein Society·Preeti MehtaSankaran Krishnaswamy
Sep 1, 1994·Acta Crystallographica. Section D, Biological Crystallography·UNKNOWN Collaborative Computational Project, Number 4
Nov 25, 2004·Nucleic Acids Research·Matheshwaran SaravananValakunja Nagaraja
Dec 2, 2004·Acta Crystallographica. Section D, Biological Crystallography·Paul Emsley, Kevin Cowtan
Apr 21, 2005·Proceedings of the National Academy of Sciences of the United States of America·Richard J Roberts
Aug 25, 2006·Acta Crystallographica. Section D, Biological Crystallography·Kevin Cowtan
Mar 3, 2007·Nucleic Acids Research·Ken-ichi MiyazonoIchizo Kobayashi
May 15, 2007·Journal of Molecular Biology·Arturas JakubauskasArvydas Janulaitis
Jul 14, 2007·BMC Structural Biology·Elena M IbryashkinaJanusz M Bujnicki
Sep 6, 2007·The Journal of Biological Chemistry·Matheshwaran SaravananValakunja Nagaraja
Apr 22, 2009·Nucleic Acids Research·Monika SokolowskaMatthias Bochtler
Oct 31, 2009·Journal of Molecular Biology·Donald C Rau, Nina Y Sidorova
Feb 9, 2010·PloS One·Siu-Hong ChanShuang-Yong Xu
Jan 14, 2012·Nucleic Acids Research·Jasmina DikićPierre Desbiolles
Jun 30, 2012·Science·Martin JinekEmmanuelle Charpentier
Sep 6, 2012·Proceedings of the National Academy of Sciences of the United States of America·Giedrius GasiunasVirginijus Siksnys
Jan 1, 1997·Methods in Enzymology·Zbyszek Otwinowski, Wladek Minor

❮ Previous
Next ❯

Citations

Oct 19, 2017·Nucleic Acids Research·Jagoda JablonskaKrzysztof Ginalski
Dec 3, 2016·Protein & Cell·Hao Cheng

❮ Previous
Next ❯

Methods Mentioned

BETA
restriction-modification
electrophoresis
differential scanning calorimetry
PCR
size exclusion chromatography

Software Mentioned

COOT
Origin
PHENIX
CCP4
PyMOL
BUCCANEER
ImageQuant TL
DM
HKL2000

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.