Structures of topoisomerase V in complex with DNA reveal unusual DNA-binding mode and novel relaxation mechanism
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Evaluation Summary:
Osterman and Modragon report the first crystal structure of topoisomerase V in complex with DNA. Topoisomerase V is an unusual protein in that homologs have only been found in the archaeal Methanopyrus genus. The paper is likely of interest to those studying topoisomerase biology and biochemistry, and the wider audience interested in DNA replication and repair transactions.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Abstract
Topoisomerase V is a unique topoisomerase that combines DNA repair and topoisomerase activities. The enzyme has an unusual arrangement, with a small topoisomerase domain followed by 12 tandem (HhH) 2 domains, which include 3 AP lyase repair domains. The uncommon architecture of this enzyme bears no resemblance to any other known topoisomerase. Here, we present structures of topoisomerase V in complex with DNA. The structures show that the (HhH) 2 domains wrap around the DNA and in this manner appear to act as a processivity factor. There is a conformational change in the protein to expose the topoisomerase active site. The DNA bends sharply to enter the active site, which melts the DNA and probably facilitates relaxation. The structures show a DNA-binding mode not observed before and provide information on the way this atypical topoisomerase relaxes DNA. In common with type IB enzymes, topoisomerase V relaxes DNA using a controlled rotation mechanism, but the structures show that topoisomerase V accomplishes this in different manner. Overall, the structures firmly establish that type IC topoisomerases form a distinct type of topoisomerases, with no similarities to other types at the sequence, structural, or mechanistic level. They represent a completely different solution to DNA relaxation.
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Evaluation Summary:
Osterman and Modragon report the first crystal structure of topoisomerase V in complex with DNA. Topoisomerase V is an unusual protein in that homologs have only been found in the archaeal Methanopyrus genus. The paper is likely of interest to those studying topoisomerase biology and biochemistry, and the wider audience interested in DNA replication and repair transactions.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
Overall the paper documents a challenging structure determination of two Topoisomerase V DNA complexes using a combination of molecular replacement and heavy atom phasing. The crystal structure of Methanopyrus kandleri Topoisomerase V DNA complexes reveal two important unique features of the topoisomerase V DNA binding mechanism. The first is that the active site toggles between opened DNA accessible state to a closed state where the active site cleft is blocked and inaccessible to nucleic acid. A second striking feature is the assembly of an array of helix-hairpin-helix motifs that wrap the duplex DNA. The authors propose the DNA wrapping facilitates a possible function of the HhH domains as a processivity factor.
This protein harbors both topoisomerase and AP-lyase activities. A caveat of the work is that …
Reviewer #1 (Public Review):
Overall the paper documents a challenging structure determination of two Topoisomerase V DNA complexes using a combination of molecular replacement and heavy atom phasing. The crystal structure of Methanopyrus kandleri Topoisomerase V DNA complexes reveal two important unique features of the topoisomerase V DNA binding mechanism. The first is that the active site toggles between opened DNA accessible state to a closed state where the active site cleft is blocked and inaccessible to nucleic acid. A second striking feature is the assembly of an array of helix-hairpin-helix motifs that wrap the duplex DNA. The authors propose the DNA wrapping facilitates a possible function of the HhH domains as a processivity factor.
This protein harbors both topoisomerase and AP-lyase activities. A caveat of the work is that the DNA bound form of the protein does not reach either the Ap- lyase or topoisomerase active sites, so insights into the catalytic mechanism(s) of the protein is limited.
The major limitation of the study is that none of the novel aspects of the Topoisomerase V DNA binding and Topoisomerase mechanism proposed herein are evalulated experimentally. In this sense, the study is quite preliminary and hypotheses stemming from the structural work remain untested.
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Reviewer #2 (Public Review):
The DNA topoisomerases are key enzymes in DNA replication and transcription in all cells. They are broadly classified into two types, I and II, depending on whether they catalyse reactions involving transient single- or double-stranded breaks. Topoisomerase V belongs to type IC, which is perhaps the least well-understood of these enzymes. Topo V, which has so far been only found in the archaeal genus Methanopyrus, has both AP lyase and topoisomerase activities. Although protein structures of topo V exist, there are currently no structures of topo V-DNA complexes.
The authors co-crystallised a truncated version of topo V with several short (~40 bp) DNA fragments containing abasic sites, to emulate natural substrates; both symmetric and asymmetric DNAs were utilised. Several crystal structures were solved and …
Reviewer #2 (Public Review):
The DNA topoisomerases are key enzymes in DNA replication and transcription in all cells. They are broadly classified into two types, I and II, depending on whether they catalyse reactions involving transient single- or double-stranded breaks. Topoisomerase V belongs to type IC, which is perhaps the least well-understood of these enzymes. Topo V, which has so far been only found in the archaeal genus Methanopyrus, has both AP lyase and topoisomerase activities. Although protein structures of topo V exist, there are currently no structures of topo V-DNA complexes.
The authors co-crystallised a truncated version of topo V with several short (~40 bp) DNA fragments containing abasic sites, to emulate natural substrates; both symmetric and asymmetric DNAs were utilised. Several crystal structures were solved and compared, enabling a description of the structure of the bound DNA and the configuration of the active site. Perhaps the most notable feature of the structures is that the DNA is sharply bent.
The strength of this manuscript is the novelty of seeing a type IC topoisomerase-DNA structure and getting insight into how the DNA is bound by the enzyme. However, the resolution of the structures is limited, and some important mechanistic aspects are not resolved, e.g. how topo V recognises DNA lesions. The data are all from X-ray crystallography and it is a pity that there is not corroborating data from biophysical/biochemical approaches, which may have enabled further insight into the mechanism etc. Thus the outcomes of the study are a little disappointing and more work is needed to provide key insights into topo V mechanism.
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