Mechanistic and Kinetic Study of the ATP-Dependent DNA Ligase of Neisseria meningitidis

Sophie Magnet, John S. Blanchard

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

The gene from Neisseria meningitidis serogroup A, encoding a putative, secreted ATP-dependent DNA ligase was cloned and overexpressed, and the soluble protein was purified. Mass spectrometry indicated that the homogeneous protein was adenylated as isolated, and sedimentation velocity experiments suggested that the enzyme exists as a monomer in solution. The 31.5 kDa protein can catalyze the ATP-dependent ligation of a singly nicked DNA duplex but not blunt-end joining. The first step of the overall reaction, the ATP-dependent formation of an adenylated ligase, was studied by measuring the formation of the covalent intermediate and isotope exchange between [α-32P] ATP and PPi. Mg2+ was absolutely required for this reaction and was the best divalent cation to promote catalysis. Electrophoretic gel mobility shift assays revealed that the enzyme bound both unnicked and singly nicked double stranded DNA with equivalent affinity (Kd ∼ 50 nM) but cannot bind single stranded DNA. Preadenylated DNA was synthesized by transferring the AMP group from the enzyme to the 5′-phosphate of a 3′-dideoxy nicked DNA. The rate of phosphodiester bond formation at the preadenylated nick was also Mg2+-dependent. Kinetic data showed that the overall rate of ligation, which occurred at 0.008 s-1, is the result of three chemical steps with similar rate constants (≈0.025 s -1). The Km values for ATP and DNA substrates, in the overall ligation reaction, were 0.4 μM and 30 nM, respectively.

Original languageEnglish (US)
Pages (from-to)710-717
Number of pages8
JournalBiochemistry
Volume43
Issue number3
DOIs
StatePublished - Jan 27 2004

Fingerprint

DNA Ligases
Neisseria meningitidis
Adenosine Triphosphate
Kinetics
DNA
Ligation
Serogroup A Neisseria meningitidis
Enzymes
Proteins
Single-Stranded DNA
Divalent Cations
Electrophoretic Mobility Shift Assay
Adenosine Monophosphate
Ligases
Catalysis
Sedimentation
Joining
Isotopes
Mass spectrometry
Rate constants

ASJC Scopus subject areas

  • Biochemistry

Cite this

Mechanistic and Kinetic Study of the ATP-Dependent DNA Ligase of Neisseria meningitidis. / Magnet, Sophie; Blanchard, John S.

In: Biochemistry, Vol. 43, No. 3, 27.01.2004, p. 710-717.

Research output: Contribution to journalArticle

@article{1dcb1d4198c44e459edb04694ba32c4f,
title = "Mechanistic and Kinetic Study of the ATP-Dependent DNA Ligase of Neisseria meningitidis",
abstract = "The gene from Neisseria meningitidis serogroup A, encoding a putative, secreted ATP-dependent DNA ligase was cloned and overexpressed, and the soluble protein was purified. Mass spectrometry indicated that the homogeneous protein was adenylated as isolated, and sedimentation velocity experiments suggested that the enzyme exists as a monomer in solution. The 31.5 kDa protein can catalyze the ATP-dependent ligation of a singly nicked DNA duplex but not blunt-end joining. The first step of the overall reaction, the ATP-dependent formation of an adenylated ligase, was studied by measuring the formation of the covalent intermediate and isotope exchange between [α-32P] ATP and PPi. Mg2+ was absolutely required for this reaction and was the best divalent cation to promote catalysis. Electrophoretic gel mobility shift assays revealed that the enzyme bound both unnicked and singly nicked double stranded DNA with equivalent affinity (Kd ∼ 50 nM) but cannot bind single stranded DNA. Preadenylated DNA was synthesized by transferring the AMP group from the enzyme to the 5′-phosphate of a 3′-dideoxy nicked DNA. The rate of phosphodiester bond formation at the preadenylated nick was also Mg2+-dependent. Kinetic data showed that the overall rate of ligation, which occurred at 0.008 s-1, is the result of three chemical steps with similar rate constants (≈0.025 s -1). The Km values for ATP and DNA substrates, in the overall ligation reaction, were 0.4 μM and 30 nM, respectively.",
author = "Sophie Magnet and Blanchard, {John S.}",
year = "2004",
month = "1",
day = "27",
doi = "10.1021/bi0355387",
language = "English (US)",
volume = "43",
pages = "710--717",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "3",

}

TY - JOUR

T1 - Mechanistic and Kinetic Study of the ATP-Dependent DNA Ligase of Neisseria meningitidis

AU - Magnet, Sophie

AU - Blanchard, John S.

PY - 2004/1/27

Y1 - 2004/1/27

N2 - The gene from Neisseria meningitidis serogroup A, encoding a putative, secreted ATP-dependent DNA ligase was cloned and overexpressed, and the soluble protein was purified. Mass spectrometry indicated that the homogeneous protein was adenylated as isolated, and sedimentation velocity experiments suggested that the enzyme exists as a monomer in solution. The 31.5 kDa protein can catalyze the ATP-dependent ligation of a singly nicked DNA duplex but not blunt-end joining. The first step of the overall reaction, the ATP-dependent formation of an adenylated ligase, was studied by measuring the formation of the covalent intermediate and isotope exchange between [α-32P] ATP and PPi. Mg2+ was absolutely required for this reaction and was the best divalent cation to promote catalysis. Electrophoretic gel mobility shift assays revealed that the enzyme bound both unnicked and singly nicked double stranded DNA with equivalent affinity (Kd ∼ 50 nM) but cannot bind single stranded DNA. Preadenylated DNA was synthesized by transferring the AMP group from the enzyme to the 5′-phosphate of a 3′-dideoxy nicked DNA. The rate of phosphodiester bond formation at the preadenylated nick was also Mg2+-dependent. Kinetic data showed that the overall rate of ligation, which occurred at 0.008 s-1, is the result of three chemical steps with similar rate constants (≈0.025 s -1). The Km values for ATP and DNA substrates, in the overall ligation reaction, were 0.4 μM and 30 nM, respectively.

AB - The gene from Neisseria meningitidis serogroup A, encoding a putative, secreted ATP-dependent DNA ligase was cloned and overexpressed, and the soluble protein was purified. Mass spectrometry indicated that the homogeneous protein was adenylated as isolated, and sedimentation velocity experiments suggested that the enzyme exists as a monomer in solution. The 31.5 kDa protein can catalyze the ATP-dependent ligation of a singly nicked DNA duplex but not blunt-end joining. The first step of the overall reaction, the ATP-dependent formation of an adenylated ligase, was studied by measuring the formation of the covalent intermediate and isotope exchange between [α-32P] ATP and PPi. Mg2+ was absolutely required for this reaction and was the best divalent cation to promote catalysis. Electrophoretic gel mobility shift assays revealed that the enzyme bound both unnicked and singly nicked double stranded DNA with equivalent affinity (Kd ∼ 50 nM) but cannot bind single stranded DNA. Preadenylated DNA was synthesized by transferring the AMP group from the enzyme to the 5′-phosphate of a 3′-dideoxy nicked DNA. The rate of phosphodiester bond formation at the preadenylated nick was also Mg2+-dependent. Kinetic data showed that the overall rate of ligation, which occurred at 0.008 s-1, is the result of three chemical steps with similar rate constants (≈0.025 s -1). The Km values for ATP and DNA substrates, in the overall ligation reaction, were 0.4 μM and 30 nM, respectively.

UR - http://www.scopus.com/inward/record.url?scp=1642453835&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=1642453835&partnerID=8YFLogxK

U2 - 10.1021/bi0355387

DO - 10.1021/bi0355387

M3 - Article

VL - 43

SP - 710

EP - 717

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 3

ER -