Identification of two conserved aspartic acid residues required for DNA digestion by a novel thermophilic Exonuclease VII in Thermotoga maritima

Andres A. Larrea, Ilene M. Pedroso, Arun Malhotra, Richard Myers

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

Exonuclease VII was first identified in 1974 as a DNA exonuclease that did not require any divalent cations for activity. Indeed, Escherichia coli ExoVII was identified in partially purified extracts in the presence of EDTA. ExoVII is comprised of two subunits (XseA and XseB) that are highly conserved and present in most sequenced prokaryotic genomes, but are not seen in eukaryotes. To better understand this exonuclease family, we have characterized an ExoVII homolog from Thermotoga maritima. Thermotoga maritima XseA/B homologs TM1768 and TM1769 were co-expressed and purified, and show robust nuclease activity at 80°C. This activity is magnesium dependent and is inhibited by phosphate ions, which distinguish it from E. coli ExoVII. Nevertheless, both E. coli and T. maritima ExoVII share a similar putative active site motif with two conserved aspartate residues in the large (XseA/TM1768) subunit. We show that these residues, Asp235 and Asp240, are essential for the nuclease activity of T. maritima ExoVII. We hypothesize that the ExoVII family of nucleases can be sub-divided into two sub-families based on EDTA resistance and that T. maritima ExoVII is the first member of the branch that is characterized by EDTA sensitivity and inhibition by phosphate.

Original languageEnglish
Pages (from-to)5992-6003
Number of pages12
JournalNucleic Acids Research
Volume36
Issue number18
DOIs
StatePublished - Oct 30 2008

Fingerprint

Thermotoga maritima
Exonucleases
Aspartic Acid
Digestion
Edetic Acid
DNA
Escherichia coli
Exodeoxyribonucleases
Phosphates
Divalent Cations
Eukaryota
Magnesium
Catalytic Domain
Genome
Ions

ASJC Scopus subject areas

  • Genetics

Cite this

Identification of two conserved aspartic acid residues required for DNA digestion by a novel thermophilic Exonuclease VII in Thermotoga maritima. / Larrea, Andres A.; Pedroso, Ilene M.; Malhotra, Arun; Myers, Richard.

In: Nucleic Acids Research, Vol. 36, No. 18, 30.10.2008, p. 5992-6003.

Research output: Contribution to journalArticle

@article{1632b0728e2e4b1a9f7c21880749c01f,
title = "Identification of two conserved aspartic acid residues required for DNA digestion by a novel thermophilic Exonuclease VII in Thermotoga maritima",
abstract = "Exonuclease VII was first identified in 1974 as a DNA exonuclease that did not require any divalent cations for activity. Indeed, Escherichia coli ExoVII was identified in partially purified extracts in the presence of EDTA. ExoVII is comprised of two subunits (XseA and XseB) that are highly conserved and present in most sequenced prokaryotic genomes, but are not seen in eukaryotes. To better understand this exonuclease family, we have characterized an ExoVII homolog from Thermotoga maritima. Thermotoga maritima XseA/B homologs TM1768 and TM1769 were co-expressed and purified, and show robust nuclease activity at 80°C. This activity is magnesium dependent and is inhibited by phosphate ions, which distinguish it from E. coli ExoVII. Nevertheless, both E. coli and T. maritima ExoVII share a similar putative active site motif with two conserved aspartate residues in the large (XseA/TM1768) subunit. We show that these residues, Asp235 and Asp240, are essential for the nuclease activity of T. maritima ExoVII. We hypothesize that the ExoVII family of nucleases can be sub-divided into two sub-families based on EDTA resistance and that T. maritima ExoVII is the first member of the branch that is characterized by EDTA sensitivity and inhibition by phosphate.",
author = "Larrea, {Andres A.} and Pedroso, {Ilene M.} and Arun Malhotra and Richard Myers",
year = "2008",
month = "10",
day = "30",
doi = "10.1093/nar/gkn588",
language = "English",
volume = "36",
pages = "5992--6003",
journal = "Nucleic Acids Research",
issn = "0305-1048",
publisher = "Oxford University Press",
number = "18",

}

TY - JOUR

T1 - Identification of two conserved aspartic acid residues required for DNA digestion by a novel thermophilic Exonuclease VII in Thermotoga maritima

AU - Larrea, Andres A.

AU - Pedroso, Ilene M.

AU - Malhotra, Arun

AU - Myers, Richard

PY - 2008/10/30

Y1 - 2008/10/30

N2 - Exonuclease VII was first identified in 1974 as a DNA exonuclease that did not require any divalent cations for activity. Indeed, Escherichia coli ExoVII was identified in partially purified extracts in the presence of EDTA. ExoVII is comprised of two subunits (XseA and XseB) that are highly conserved and present in most sequenced prokaryotic genomes, but are not seen in eukaryotes. To better understand this exonuclease family, we have characterized an ExoVII homolog from Thermotoga maritima. Thermotoga maritima XseA/B homologs TM1768 and TM1769 were co-expressed and purified, and show robust nuclease activity at 80°C. This activity is magnesium dependent and is inhibited by phosphate ions, which distinguish it from E. coli ExoVII. Nevertheless, both E. coli and T. maritima ExoVII share a similar putative active site motif with two conserved aspartate residues in the large (XseA/TM1768) subunit. We show that these residues, Asp235 and Asp240, are essential for the nuclease activity of T. maritima ExoVII. We hypothesize that the ExoVII family of nucleases can be sub-divided into two sub-families based on EDTA resistance and that T. maritima ExoVII is the first member of the branch that is characterized by EDTA sensitivity and inhibition by phosphate.

AB - Exonuclease VII was first identified in 1974 as a DNA exonuclease that did not require any divalent cations for activity. Indeed, Escherichia coli ExoVII was identified in partially purified extracts in the presence of EDTA. ExoVII is comprised of two subunits (XseA and XseB) that are highly conserved and present in most sequenced prokaryotic genomes, but are not seen in eukaryotes. To better understand this exonuclease family, we have characterized an ExoVII homolog from Thermotoga maritima. Thermotoga maritima XseA/B homologs TM1768 and TM1769 were co-expressed and purified, and show robust nuclease activity at 80°C. This activity is magnesium dependent and is inhibited by phosphate ions, which distinguish it from E. coli ExoVII. Nevertheless, both E. coli and T. maritima ExoVII share a similar putative active site motif with two conserved aspartate residues in the large (XseA/TM1768) subunit. We show that these residues, Asp235 and Asp240, are essential for the nuclease activity of T. maritima ExoVII. We hypothesize that the ExoVII family of nucleases can be sub-divided into two sub-families based on EDTA resistance and that T. maritima ExoVII is the first member of the branch that is characterized by EDTA sensitivity and inhibition by phosphate.

UR - http://www.scopus.com/inward/record.url?scp=54549093895&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=54549093895&partnerID=8YFLogxK

U2 - 10.1093/nar/gkn588

DO - 10.1093/nar/gkn588

M3 - Article

VL - 36

SP - 5992

EP - 6003

JO - Nucleic Acids Research

JF - Nucleic Acids Research

SN - 0305-1048

IS - 18

ER -