Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Experiment Videos

Structure-based mutational and functional analysis identify human NM23-H2 as a multifunctional enzyme.

Edith H Postel1, Bozena A Abramczyk, Susan K Gursky

  • 1Department of Molecular Biology, Lewis Thomas Laboratory, Princeton University, Washington Road, Princeton, New Jersey 08546-5414, USA. epostel@molbio.princeton.edu

Biochemistry
|May 16, 2002
PubMed
Summary

The human NM23-H2 protein, a transcriptional regulator (PuF), has distinct DNA-binding and NDP kinase sites. Mutagenesis identified key residues for DNA cleavage and NDP kinase activity, revealing conserved functional importance.

Related Concept Videos

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

Experimental Study on the Effect of Ammonia on Combustion and Emission Characteristics of a Spark Ignition Engine Fueled with Hydrogen.

ACS omega·2024
Same author

Functional analysis of PI-like gene in relation to flower development from bamboo (Bambusa oldhamii).

Journal of genetics·2016
Same author

Role of Interaction and Nucleoside Diphosphate Kinase B in Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator Function by cAMP-Dependent Protein Kinase A.

PloS one·2016
Same author

A long downstream probe-based platform for multiplex target capture.

Analytical biochemistry·2015
Same author

Purification and characterization of the plastid-localized NAD-dependent malate dehydrogenase from Arabidopsis thaliana.

Biotechnology and applied biochemistry·2015
Same author

Purification and characterization of Cyclin-H1 from Arabidopsis thaliana.

Protein expression and purification·2015

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Protein Function

Background:

  • Human NM23-H2 (PuF) is a dual-function protein, acting as a transcriptional regulator and catalyzing phosphoryl transfer (NDP kinase).
  • Previous studies identified separate DNA-binding regions: a sequence-dependent surface and a covalent DNA-binding site within the nucleotide-binding pocket.

Purpose of the Study:

  • To elucidate the role of the nucleotide-binding site in NM23-H2's DNA cleavage activity.
  • To establish a mechanistic link between the protein's nuclease and NDP kinase functions.

Main Methods:

  • Site-directed mutagenesis based on the crystal structure of NM23-H2 complexed with GDP.
  • Biochemical assays to assess DNA binding, DNA cleavage, and NDP kinase activity.

Related Experiment Videos

Main Results:

  • Identified Arg88 and Arg105 as critical for covalent DNA binding, DNA cleavage, and NDP kinase activity, in addition to Lys12.
  • Gln17 is essential solely for DNA cleavage, while Tyr52, Asn115, and His118 are crucial only for NDP kinase activity.
  • Nucleoside triphosphates, but not diphosphates, inhibited DNA binding and cleavage independently of kinase activity, suggesting distinct binding modes.

Conclusions:

  • NM23-H2 possesses multiple biochemical activities mediated by distinct, yet interconnected, functional sites.
  • Seven functionally important residues within the nucleotide-binding site are evolutionarily conserved, highlighting their biological significance.
  • The findings support a model where nucleotide and DNA binding sites within NM23-H2 differ, influencing its diverse functions.