Tài liệu Báo cáo khoa học: Kinetics and thermodynamics of nick sealing by T4 DNA ligase pptx

Kinetics and thermodynamics of nick sealing by T4 DNA ligase

Alexey V. Cherepanov* and Simon de Vries

Kluyver Department of Biotechnology, Delft University of Technology, the Netherlands

T4 DNA ligase is an Mg2+-dependent and ATP-dependent

enzyme that seals DNA nicks in three steps: it covalently

binds AMP,transadenylates the nick phosphate,and cata￾lyses formation of the phosphodiester bond releasing AMP.

In this kinetic study,we further detail the reaction mechan￾ism,showing that the overall ligation reaction is a super￾imposition of two parallel processes: a
processive ligation,in

which the enzyme transadenylates and seals the nick without

dissociating from dsDNA,and a
nonprocessive ligation,in

which the enzyme takes part in the abortive adenylation

cycle (covalent binding of AMP,transadenylation of the

nick,and dissociation). At low concentrations of ATP

(< 10 lM) and when the DNA nick is sealed with

mismatching base pairs (e.g. five adjacent),this super￾imposition resolves into two kinetic phases,a burst

ligation (
0.2 min)1

) and a subsequent slow ligation

(
2 · 10)3 min)1

). The relative rate and extent of each

phase depend on the concentrations of ATP and Mg2+. The

activation energies of self-adenylation (16.2 kcalÆmol)1

),

transadenylation of the nick (0.9 kcalÆmol)1

),and nick￾sealing (16.3–18.8 kcalÆmol)1

) were determined for several

DNA substrates. The low activation energy of transadeny￾lation implies that the transfer of AMP to the terminal DNA

phosphate is a spontaneous reaction,and that the T4 DNA

ligase–AMP complex is a high-energy intermediate. To

summarize current findings in the DNA ligation field,we

delineate a kinetic mechanism of T4 DNA ligase catalysis.

Keywords: DNA ligase; end-joining; kinetics; mechanism of

action; mismatching nick.

T4 DNA ligase is an enzyme that catalyses formation of the

phosphodiester bond between the adjacent 5¢-PO4 and

3¢-OH groups of two dsDNA fragments [1]. It is able to join

two dsDNAs (blunt-end or sticky-end ligation),or seal a

break between two ssDNA fragments annealed on the

complementary DNA strand (nick-ligation). It can join

phosphodiester linkages on triple-stranded nucleic acids [2],

seal single-stranded 1–5-nucleotide gaps [3],and act as a

lyase,removing apurininc/apyrimidinic (AP) sites in DNA

[4]. The enzyme requires a bivalent metal cation such as

Mg2+ or Mn2+,and joins DNA using ATP as a coenzyme.

One phosphodiester bond in DNA is formed per ATP

molecule hydrolysed to AMP and pyrophosphate.

The mechanism of catalysis of T4 DNA ligase comprises

three steps and involves two covalent reaction intermedi￾ates:

E þ ATP $ EAMP þ PPi ð1Þ

E--AMP þ ndsDNA $ EAMPndsDNA

! E þ AMPndsDNA ð2Þ

E þ AMP--ndsDNA $ E  AMPndsDNA

! E þ dsDNA þ AMP ð3Þ

where ndsDNA is nicked dsDNA,AMP–ndsDNA is

ndsDNA adenylated at the 5¢-phosphate of the nick,and

a one-sided arrow indicates that the
reverse reaction is

at least three orders of magnitude slower than the

forward reaction.

On the basis of the electrophoretic mobility shift assay

experiments,it has been suggested that adenylated ligase

forms transient Tcomplexes,E–AMPndsDNA,in search

of a phosphorylated 5¢-end of (n)dsDNA. When the nick

phosphate is found,it is adenylated,and a stable
Scomplex

is formed,EAMP–ndsDNA [5]. The enzyme in this

complex has been suggested to
stall on DNA until the

nick is sealed and dsDNA is released. Formation of the first

phosphodiester bond during joining of the blunt ends has

been proposed to happen accordingly; the main difference is

the 2 : 1 dsDNA to enzyme stoichiometry in step 3. It has

been suggested [5] that during blunt end joining,the ternary

complex ligaseDNA is formed via two second-order

associative reactions:

E þ AMPdsDNA ! EAMPsDNA (Scomplex)

Scomplex þ dsDNA ! EAMPdDNAdsDNA

Despite a good understanding of the overall reaction

mechanism,relatively few articles have been dedicated

to the kinetic studies of catalysis performed by this

enzyme. The optimal concentration of Mg2+ in the nick￾joining reaction (8–10 mM),apparent Km for the nicked

Correspondence to S. de Vries,Kluyver Department of Biotechno￾logy,Delft University of Technology,Julianalaan 67,

2628 BC Delft,the Netherlands.

Fax: + 31 15 2782355,Tel.: + 31 15 2785139,

E-mail: [email protected]

Abbreviations: ndsDNA,nicked dsDNA.

Enzymes: DNA ligase (EC 6.5.1.1).

*Present address: Metalloprotein & Protein Engineering Group,

Leiden Institute of Chemistry,Gorlaeus Laboratories,Leiden

University,Einsteinweg 55,PO Box 9502,2300 RA Leiden,

the Netherlands.

(Received 28 May 2003,revised 8 September 2003,

accepted 9 September 2003)

Eur. J. Biochem. 270,4315–4325 (2003) FEBS 2003 doi:10.1046/j.1432-1033.2003.03824.x