Abstract
Topoisomerases play a fundamental role in genome stability, DNA replication and repair. As a result, topoisomerases have served as therapeutic targets of interest in Eukarya and Bacteria, two of the three domains of life. Since members of Archaea, the third domain of life, have not been implicated in any diseased state to-date, there is a paucity of data on archaeal topoisomerases. Here we report Methanosarcina acetivorans TopoIIIα (MacTopoIIIα) as the first biochemically characterized mesophilic archaeal topoisomerase. Maximal activity for MacTopoIIIα was elicited at 30–35°C and 100 mM NaCl. As little as 10 fmol of the enzyme initiated DNA relaxation, and NaCl concentrations above 250 mM inhibited this activity. The present study also provides the first evidence that a type IA Topoisomerase has activity in the presence of all divalent cations tested (Mg2+, Ca2+, Sr2+, Ba2+, Mn2+, Fe2+, Co2+, Ni2+, Cu2+, Zn2+ and Cd2+). Activity profiles were, however, specific to each metal. Known type I (ssDNA and camptothecin) and type II (etoposide, novobiocin and nalidixic acid) inhibitors with different mechanisms of action were used to demonstrate that MacTopoIIIα is a type IA topoisomerase. Alignment of MacTopoIIIα with characterized topoisomerases identified Y317 as the putative catalytic residue, and a Y317F mutation ablated DNA relaxation activity, demonstrating that Y317 is essential for catalysis. As the role of Domain V (C-terminal domain) is unclear, MacTopoIIIα was aligned with the canonical E. coli TopoI 67 kDa fragment in order to construct an N-terminal (1–586) and a C-terminal (587–752) fragment for analysis. Activity could neither be elicited from the fragments individually nor reconstituted from a mixture of the fragments, suggesting that native folding is impaired when the two fragments are expressed separately. Evidence that each of the split domains plays a role in Zn2+ binding of the enzyme is also provided.
Highlights
Topoisomerases manage DNA supercoiling and are classified according to the mechanism of action employed [1,2,3,4]
MacTopoIIIa is promiscuous A fundamental property of type IA topoisomerases is the absolute requirement of Mg2+ for the large conformational change required during DNA relaxation, but other divalent cations can substitute [5,32]
Recent publications have proposed a two metal mechanism for type IA and type II topoisomerases wherein one may bind acidic residues within the TOPRIM motif causing the necessary large conformational changes while the other may play a transient role in stabilizing the transition state in preparation for attack on the scissile phosphate [33,34]
Summary
Topoisomerases manage DNA supercoiling and are classified according to the mechanism of action employed [1,2,3,4]. Topoisomerases form a phosphotyrosine intermediate while cleaving one strand (type I or odd-numbered) or two strands (type II or even-numbered) of DNA. Before the initial transesterification is reversed and a ligated DNA backbone regenerated, type I and II enzymes change the DNA linking number (Lk) by 1 and 2, respectively. Type I topoisomerases are ascribed further distinction based on the polarity of the covalent intermediate formed. Type IA (like Type II) forms a 59-phosphotyrosine adduct while type IB forms 39-phosphotyrosine covalent adduct. The structure and mechanism of each class are distinct
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