Tài liệu Báo cáo Y học: Dynamic mechanism of nick recognition by DNA ligase ppt

REVIEW ARTICLE

Dynamic mechanism of nick recognition by DNA ligase

Alexei V. Cherepanov* and Simon de Vries

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

DNA ligases are the enzymes responsible for the repair of

single-stranded and double-stranded nicks in dsDNA. DNA

ligases are structurally similar, possibly sharing a common

molecular mechanism of nick recognition and ligation

catalysis. This mechanism remains unclear, in part because

the structure of ligase in complex with dsDNA has yet to be

solved. DNA ligases share common structural elements with

DNA polymerases, which have been cocrystallized with

dsDNA. Based on the observed DNA polymerase–dsDNA

interactions, we propose a mechanism for recognition of a

single-stranded nick by DNA ligase. According to this

mechanism, ligase induces a B-to-A DNA helix transition of

the enzyme-bound dsDNA motif, which results in DNA

contraction, bending and unwinding. For non-nicked

dsDNA, this transition is reversible, leading to dissociation

of the enzyme. For a nicked dsDNA substrate, the con￾traction of the enzyme-bound DNA motif (a) triggers an

opened–closed conformational change of the enzyme, and

(b) forces the motif to accommodate the strained A/B-form

hybrid conformation, in which the nicked strand tends to

retain a B-type helix, while the non-nicked strand tends to

form a shortened A-type helix. We propose that this con￾formation is the catalytically competent transition state,

which leads to the formation of the DNA–AMP interme￾diate and to the subsequent sealing of the nick.

Keywords: DNA ligase; nick recognition; A-form DNA;

A/ B-form DNA hybrid; protein–DNA interactions; B-A

DNA helix transition.

DNA ligases are the enzymes that catalyze the joining of

single- and double-stranded nicks in dsDNA [1]. These

enzymes play a pivotal role in replication, sealing the nicks

in the lagging DNA strand [2–5]. They also participate in

DNA excision [6–8], double-strand break repair [9–12] and

take part in DNA recombination [10,13–15]. The mechan￾ism of enzyme catalysis (Scheme 1) includes three main

steps: (1) covalent binding of the nucleoside monophos￾phate, AMP or GMP, via the e-amino lysyl phosphorami￾date bond, (2) transfer of the nucleotidyl moiety onto the

5¢-phosphate end of the nick, forming an inverted pyro￾phosphate bridging structure, A(G)ppN and (3) formation

of the phosphodiester bond between the 3¢-OH and the

5¢-phosphate ends of the nick, releasing the nucleotide.

Scheme 1. Mechanism of the ATP-dependent

end-joining activity of T4 DNA ligase. nds￾DNA, dsDNA containing a 5¢-phosphorylated

nick. n-MgAMP-dsDNA, nicked dsDNA

adenylylated at the 5¢-phosphate of the nick.

Correspondence to S. de Vries, Kluyver Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft,

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

E-mail: [email protected]

Abbreviations: EMSA, electrophoretic mobility shift assay.

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 8 July 2002, accepted 11 October 2002)

Eur. J. Biochem. 269, 5993–5999 (2002) FEBS 2002 doi:10.1046/j.1432-1033.2002.03309.x