Mechanistic insights from structure of Mycobacterium smegmatis topoisomerase I with ssDNA bound to both N- and C-terminal domains.

Xiaobing Zuo,Nan Cao,Thirunavukkarasu Annamalai,Yuk-Ching Tse-Dinh,Kemin Tan

doi:10.1093/nar/gkaa201

Xiaobing Zuo, Nan Cao + Show 3 more

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https://doi.org/10.1093/nar/gkaa201

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Abstract

Type IA topoisomerases interact with G-strand and T-strand ssDNA to regulate DNA topology. However, simultaneous binding of two ssDNA segments to a type IA topoisomerase has not been observed previously. We report here the crystal structure of a type IA topoisomerase with ssDNA segments bound in opposite polarity to the N- and C-terminal domains. Titration of small ssDNA oligonucleotides to Mycobacterium smegmatis topoisomerase I with progressive C-terminal deletions showed that the C-terminal region has higher affinity for ssDNA than the N-terminal active site. This allows the C-terminal domains to capture one strand of underwound negatively supercoiled DNA substrate first and position the N-terminal domains to bind and cleave the opposite strand in the relaxation reaction. Efficiency of negative supercoiling relaxation increases with the number of domains that bind ssDNA primarily with conserved aromatic residues and possibly with assistance from polar/basic residues. A comparison of bacterial topoisomerase I structures showed that a conserved transesterification unit (N-terminal toroid structure) for cutting and rejoining of a ssDNA strand can be combined with two different types of C-terminal ssDNA binding domains to form diverse bacterial topoisomerase I enzymes that are highly efficient in their physiological role of preventing excess negative supercoiling in the genome.

Highlights

DNA topoisomerases are required for regulating DNA supercoiling and resolving topological entanglements that arise during essential cellular processes including replication, transcription, recombination and repair [1,2,3]
After determining the crystal structures of MtbTOP1-704t in the absence and in the presence of single-stranded DNA (ssDNA) [23,26], we have tried to crystallize full-length mycobacterial topoisomerases in order to further verify our earlier prediction of the C-terminal domain organization of these enzymes and, most importantly, to understand how these distinct C-terminal domains interact with DNA
Considering a possible 13-residue flexible link between domain 7 (D7) and domain 8 (D8) of these two mycobacterial topoisomerases (Figure 1), we made two truncated forms terminating after D7, MsmTOP1-839t and MtbTOP1-840t

Summary

Introduction

DNA topoisomerases are required for regulating DNA supercoiling and resolving topological entanglements that arise during essential cellular processes including replication, transcription, recombination and repair [1,2,3]. To carry out these catalytic functions, topoisomerases utilize an active site tyrosine nucleophile [4] to break the DNA phosphodiester linkage, form a covalent topoisomeraseDNA intermediate, and allow DNA passage through the break (called the DNA ‘gate’) before the active site tyrosine is regenerated in the DNA rejoining step. A variety of type IA topoisomerases from every taxonomic kingdom have been shown to possess RNA topoisomerase activities that may be required for promoting mRNA translation [9,10] and RNAi-induced gene silencing [11]

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