Unmasking anticooperative DNA-binding interactions of vaccinia DNA topoisomerase I

Rajesh Nagarajan, James Stivers

Research output: Contribution to journalArticle

Abstract

Vaccinia DNA topoisomerase (vTopo) catalyzes highly specific nucleophilic substitution at a single phosphodiester linkage in the pentapyrimidine recognition sequence 5′-(CyT)+5C4+C 3+T+2T+1p↓N-1 using an active-site tyrosine nucleophile, thereby expelling a 5′ hydroxyl leaving group of the DNA. Here, we report the energetic effects of subtle modifications to the major-groove hydrogen-bond donor and acceptor groups of the 3′-GGGAA-5′ consensus sequence of the nonscissile strand in the context of duplexes in which the scissile strand length was progressively shortened. We find that the major-groove substitutions become energetically more damaging as the scissile strand is shortened from 32 to 24 and 18 nucleotides, indicating that enzyme interactions with the duplex region present in the 32-mer but not the 24- or 18-mer weaken specific interactions with the DNA major groove. Regardless of strand length, the destabilizing effects of the major-groove substitutions increase as the reaction proceeds from the Michaelis complex to the transition state for DNA cleavage and, finally, to the phosphotyrosine-DNA covalent complex. These length-dependent anticooperative interactions involving the DNA major groove and duplex regions 3′ to the cleavage site indicate that the major-groove binding energy is fully realized late during the reaction for full-length substrates but that smaller more flexible duplex substrates feel these interactions earlier along the reaction coordinate. Such anticooperative binding interactions may play a role in strand exchange and supercoil unwinding activities of the enzyme.

Original languageEnglish (US)
Pages (from-to)192-199
Number of pages8
JournalBiochemistry®
Volume46
Issue number1
DOIs
StatePublished - Jan 9 2007

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Vaccinia
Type I DNA Topoisomerase
DNA
Substitution reactions
DNA Cleavage
Phosphotyrosine
Consensus Sequence
Enzymes
Nucleophiles
Hydroxyl Radical
Tyrosine
Hydrogen
Substrates
Catalytic Domain
Binding energy
Nucleotides
Hydrogen bonds

ASJC Scopus subject areas

  • Biochemistry

Cite this

Unmasking anticooperative DNA-binding interactions of vaccinia DNA topoisomerase I. / Nagarajan, Rajesh; Stivers, James.

In: Biochemistry®, Vol. 46, No. 1, 09.01.2007, p. 192-199.

Research output: Contribution to journalArticle

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