Characterization of DNA Binding by the Isolated N‑Terminal
Domain of Vaccinia Virus DNA Topoisomerase IB
Benjamin Reed
Lyudmila Yakovleva
Stewart Shuman
Ranajeet Ghose
10.1021/acs.biochem.7b00042.s001
https://acs.figshare.com/articles/journal_contribution/Characterization_of_DNA_Binding_by_the_Isolated_N_Terminal_Domain_of_Vaccinia_Virus_DNA_Topoisomerase_IB/5117650
Vaccinia
TopIB (vTopIB), a 314-amino acid eukaryal-type IB topoisomerase,
recognizes and transesterifies at the DNA sequence 5′-(T/C)CCTT↓,
leading to the formation of a covalent DNA–(3′-phosphotyrosyl<sup>274</sup>)–enzyme intermediate in the supercoil relaxation
reaction. The C-terminal segment of vTopIB (amino acids 81–314),
which engages the DNA minor groove at the scissile phosphodiester,
comprises an autonomous catalytic domain that retains cleavage specificity,
albeit with a cleavage site affinity lower than that of the full-length
enzyme. The N-terminal domain (amino acids 1–80) engages the
major groove on the DNA face opposite the scissile phosphodiester.
Whereas DNA contacts of the N-terminal domain have been implicated
in the DNA site affinity of vTopIB, it was not known whether the N-terminal
domain per se could bind DNA. Here, using isothermal titration calorimetry,
we demonstrate the ability of the isolated N-terminal domain to bind
a CCCTT-containing 24-mer duplex with an apparent affinity that is
∼2.2-fold higher than that for an otherwise identical duplex
in which the pentapyrimidine sequence is changed to ACGTG. Analyses
of the interactions of the isolated N-terminal domain with duplex
DNA via solution nuclear magnetic resonance methods are consistent
with its DNA contacts observed in DNA-bound crystal structures of
full-length vTopIB. The chemical shift perturbations and changes in
hydrodynamic properties triggered by CCCTT DNA versus non-CCCTT DNA
suggest differences in DNA binding dynamics. The importance of key
N-terminal domain contacts in the context of full-length vTopIB is
underscored by assessing the effects of double-alanine mutations on
DNA transesterification and its sensitivity to ionic strength.
2017-06-01 00:00:00
supercoil relaxation reaction
CCCTT-containing 24- mer duplex
DNA site affinity
N-terminal domain
N-terminal domain contacts
DNA binding dynamics
DNA contacts
acid eukaryal-type IB topoisomerase
DNA-bound crystal structures
scissile phosphodiester
Vaccinia Virus DNA Topoisomerase IB Vaccinia TopIB
chemical shift perturbations
ACGTG
cleavage site affinity