E. coli RecQ protein is a multifunctional helicase with homologs that include the S. cerevisiae Sgs1 helicase and the H. sapiens Wrn and Blm helicases. Here we show that RecQ helicase unwinds a covalently closed double-stranded DNA (dsDNA) substrate and that this activity specifically stimulates E. coli topoisomerase III (Topo III) to fully catenate dsDNA molecules. We propose that these proteins functionally interact and that their shared activity is responsible for control of DNA recombination. RecQ helicase has a comparable effect on the Topo III homolog of S. cerevisiae, consistent with other RecQ and Topo III homologs acting together in a similar capacity. These findings highlight a novel, conserved activity that offers insight into the function of the other RecQ-like helicases.
A variety of cellular processes, including DNA replication, transcription, and chromosome condensation, require enzymes that can regulate the ensuing topological changes occurring in DNA. Such enzymes-DNA topoisomerases-alter DNA topology by catalysing the cleavage of single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA), the passage of DNA through the resulting break, and the rejoining of the broken phosphodiester backbone. DNA topoisomerase III from Escherichia coli belongs to the type IA family of DNA topoisomerases, which transiently cleave ssDNA via formation of a covalent 5' phosphotyrosine intermediate. Here we report the crystal structure, at 2.05 A resolution, of an inactive mutant of E. coli DNA topoisomerase III in a non-covalent complex with an 8-base ssDNA molecule. The enzyme undergoes a conformational change that allows the oligonucleotide to bind within a groove leading to the active site. We note that the ssDNA molecule adopts a conformation like that of B-DNA while bound to the enzyme. The position of the DNA within the realigned active site provides insight into the role of several highly conserved residues during catalysis. These findings confirm various aspects of the type IA topoisomerase mechanism while suggesting functional implications for other topoisomerases and proteins that perform DNA rearrangements.
Type I DNA topoisomerases are ubiquitous enzymes involved in many aspects of DNA metabolism. Escherichia coli possesses two type I topoisomerase activities, DNA topoisomerase I (Topo I) and III (Topo III). The gene encoding Topo III (topB) can be deleted without affecting cell viability. Cells possessing a deletion of the gene encoding Topo I (topA) are only viable in the presence of an additional compensatory mutation. In the presence of compensatory mutations, Topo I deletion strains grow normally; however, if Topo III activity is repressed in these cells, they filament extensively and possess an abnormal nucleoid structure. These defects can be suppressed by the deletion of the recA gene, suggesting that these enzymes may be involved in RecA-mediated recombination and may specifically resolve recombination intermediates before partitioning.
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