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Title: Characterization of a periplasmic S1-like nuclease coded by the Mesorhizobium loti symbiosis island

Abstract

DNA sequences encoding hypothetical proteins homologous to S1 nuclease from Aspergillus oryzae are found in many organisms including fungi, plants, pathogenic bacteria, and eukaryotic parasites. One of these is the M1 nuclease of Mesorhizobium loti which we demonstrate herein to be an enzymatically active, soluble, and stable S1 homolog that lacks the extensive mannosyl-glycosylation found in eukaryotic S1 nuclease homologs. We have expressed the cloned M1 protein in M. loti and purified recombinant native M1 to near homogeneity and have also isolated a homogeneous M1 carboxy-terminal hexahistidine tag fusion protein. Mass spectrometry and N-terminal Edman degradation sequencing confirmed the protein identity. The enzymatic properties of the purified M1 nuclease are similar to those of S1. At acidic pH M1 is 25 times more active on single-stranded DNA than on double-stranded DNA and 3 times more active on single-stranded DNA than on single-stranded RNA. At neutral pH the RNase activity of M1 exceeds the DNase activity. M1 nicks supercoiled RF-I plasmid DNA and rapidly cuts the phosphodiester bond across from the nick in the resultant relaxed RF-II plasmid DNA. Therefore, M1 represents an active bacterial S1 homolog in spite of great sequence divergence. The biochemical characterization of M1 nuclease supports ourmore » sequence alignment that reveals the minimal 21 amino acid residues that are necessarily conserved for the structure and functions of this enzyme family. The ability of M1 to degrade RNA at neutral pH implies previously unappreciated roles of these nucleases in biological systems.« less

Authors:
 [1];  [1];  [1];  [1];  [1];  [2]
  1. Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111 (United States)
  2. Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111 (United States). E-mail: AT_Yeung@fccc.edu
Publication Date:
OSTI Identifier:
20798919
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 343; Journal Issue: 1; Other Information: DOI: 10.1016/j.bbrc.2006.02.117; PII: S0006-291X(06)00408-6; Copyright (c) 2006 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; AMINO ACIDS; ASPERGILLUS; BACTERIA; DNA; DNA SEQUENCING; MASS SPECTROSCOPY; NUCLEASES; PARASITES; PH VALUE; RNA; SYMBIOSIS

Citation Formats

Pimkin, Maxim, Miller, C. Glenn, Blakesley, Lauryn, Oleykowski, Catherine A., Kodali, Nagendra S., and Yeung, Anthony T. Characterization of a periplasmic S1-like nuclease coded by the Mesorhizobium loti symbiosis island. United States: N. p., 2006. Web. doi:10.1016/j.bbrc.2006.02.117.
Pimkin, Maxim, Miller, C. Glenn, Blakesley, Lauryn, Oleykowski, Catherine A., Kodali, Nagendra S., & Yeung, Anthony T. Characterization of a periplasmic S1-like nuclease coded by the Mesorhizobium loti symbiosis island. United States. doi:10.1016/j.bbrc.2006.02.117.
Pimkin, Maxim, Miller, C. Glenn, Blakesley, Lauryn, Oleykowski, Catherine A., Kodali, Nagendra S., and Yeung, Anthony T. Fri . "Characterization of a periplasmic S1-like nuclease coded by the Mesorhizobium loti symbiosis island". United States. doi:10.1016/j.bbrc.2006.02.117.
@article{osti_20798919,
title = {Characterization of a periplasmic S1-like nuclease coded by the Mesorhizobium loti symbiosis island},
author = {Pimkin, Maxim and Miller, C. Glenn and Blakesley, Lauryn and Oleykowski, Catherine A. and Kodali, Nagendra S. and Yeung, Anthony T.},
abstractNote = {DNA sequences encoding hypothetical proteins homologous to S1 nuclease from Aspergillus oryzae are found in many organisms including fungi, plants, pathogenic bacteria, and eukaryotic parasites. One of these is the M1 nuclease of Mesorhizobium loti which we demonstrate herein to be an enzymatically active, soluble, and stable S1 homolog that lacks the extensive mannosyl-glycosylation found in eukaryotic S1 nuclease homologs. We have expressed the cloned M1 protein in M. loti and purified recombinant native M1 to near homogeneity and have also isolated a homogeneous M1 carboxy-terminal hexahistidine tag fusion protein. Mass spectrometry and N-terminal Edman degradation sequencing confirmed the protein identity. The enzymatic properties of the purified M1 nuclease are similar to those of S1. At acidic pH M1 is 25 times more active on single-stranded DNA than on double-stranded DNA and 3 times more active on single-stranded DNA than on single-stranded RNA. At neutral pH the RNase activity of M1 exceeds the DNase activity. M1 nicks supercoiled RF-I plasmid DNA and rapidly cuts the phosphodiester bond across from the nick in the resultant relaxed RF-II plasmid DNA. Therefore, M1 represents an active bacterial S1 homolog in spite of great sequence divergence. The biochemical characterization of M1 nuclease supports our sequence alignment that reveals the minimal 21 amino acid residues that are necessarily conserved for the structure and functions of this enzyme family. The ability of M1 to degrade RNA at neutral pH implies previously unappreciated roles of these nucleases in biological systems.},
doi = {10.1016/j.bbrc.2006.02.117},
journal = {Biochemical and Biophysical Research Communications},
number = 1,
volume = 343,
place = {United States},
year = {Fri Apr 28 00:00:00 EDT 2006},
month = {Fri Apr 28 00:00:00 EDT 2006}
}