Abstract
Topoisomerase IIIα (Top3α) is an essential component of the double Holliday junction (dHJ) dissolvasome complex in metazoans, along with Blm and Rmi1/2. This important anti-recombinogenic function cannot be performed by Top3β, the other type IA topoisomerase present in metazoans. The two share a catalytic core but diverge in their tail regions. To understand this difference in function, we investigated the role of the unique C terminus of Top3α. The Drosophila C terminus contains an insert region not conserved among metazoans. This insert contributes an independent interaction with Blm, which may account for the absence of Rmi1 in Drosophila. Mutant Top3α lacking this insert maintains the ability to perform dHJ dissolution but only partially rescues a top3α null fly line, indicating an in vivo role for the insert. Truncation of the C terminus has a minimal effect on the type IA relaxation activity of Top3α; however, dHJ dissolution is greatly reduced. The Top3α C terminus was found to strongly interact with both Blm and DNA, which are critical to the dissolution reaction; these interactions are greatly reduced in the truncated enzyme. The truncation mutant also cannot rescue the viability of top3α null flies, indicating an essential in vivo role. Our data therefore suggest that the Top3α C terminus has an important role in dHJ dissolution (by providing an interaction interface for Blm and DNA) and an essential function in vivo.
Highlights
Topoisomerase III␣ (Top3␣) and Bloom helicase (Blm) dissolve Holliday junctions into non-crossover products
There are a pair of type IA topoisomerases: Top3␣ and Top3. Both can perform relaxation on topologically constrained DNA with single-stranded regions (16 –19), Top3 cannot substitute for Top3␣ in double Holliday junction (dHJ) dissolution [2]
Top3␣ C Terminus Is Required for dHJ Dissolution—Because Top3␣ and Top3 have distinct cellular and biochemical functions, and the major structural difference resides in their C termini (Fig. 1A), we examined the properties of the Top3␣ C terminus
Summary
Topoisomerase III␣ (Top3␣) and Blm dissolve Holliday junctions into non-crossover products. Our data suggest that the Top3␣ C terminus has an important role in dHJ dissolution (by providing an interaction interface for Blm and DNA) and an essential function in vivo. The complex of Bloom helicase (Blm) and topoisomerase III␣ (Top3␣) has been shown to convergently migrate double Holliday junctions (dHJs), an intermediate of double-strand break repair, into solely non-crossover products [1, 2] The significance of this anti-recombinogenic function can be seen in the phenotype of. In addition to the conserved Top3␣ C terminus, the Drosophila enzyme contains an inserted region that can independently bind to Blm, perhaps compensating for the absence of the Rmi structural proteins. These data reveal the essential role for the Top3␣ C terminus in recombinational repair
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