Identification of one of the apurinic/apyrimidinic lyase active sites of topoisomerase v by structural and functional studies

Rakhi Rajan; Rajendra Prasad; Bhupesh Taneja; Samuel H. Wilson; Alfonso Mondragón

doi:10.1093/nar/gks1017

Identification of one of the apurinic/apyrimidinic lyase active sites of topoisomerase v by structural and functional studies

Rakhi Rajan, Rajendra Prasad, Bhupesh Taneja, Samuel H. Wilson, Alfonso Mondragón^*

^*Corresponding author for this work

Molecular Biosciences

Research output: Contribution to journal › Article › peer-review

7 Scopus citations

Abstract

Topoisomerase V (Topo-V) is the only member of a novel topoisomerase subtype. Topo-V is unique because it is a bifunctional enzyme carrying both topoisomerase and DNA repair lyase activities within the same protein. Previous studies had shown that the topoisomerase domain spans the N-terminus of the protein and is followed by 12 tandem helix-hairpin-helix [(HhH)₂] domains. There are at least two DNA repair lyase active sites for apurinic/apyrimidinic (AP) site processing, one within the N-terminal region and the second within the C-terminal domain of Topo-V, but their exact locations and characteristics are unknown. In the present study, the N-terminal 78-kDa fragment of Topo-V (Topo-78), containing the topoisomerase domain and one of the lyase DNA repair domains, was characterized by structural and biochemical studies. The results show that an N-terminal 69-kDa fragment is the minimal fragment with both topoisomerase and AP lyase activities. The lyase active site of Topo-78 is at the junction of the fifth and sixth (HhH)₂ domains. From the biochemical and structural data, it appears that Lys571 is the most probable nucleophile responsible for the lyase activity. Our experiments also suggest that Topo-V most likely acts as a Class I AP endonuclease in vivo.

Original language	English (US)
Pages (from-to)	657-666
Number of pages	10
Journal	Nucleic acids research
Volume	41
Issue number	1
DOIs	https://doi.org/10.1093/nar/gks1017
State	Published - Jan 2013

Funding

We acknowledge staff and instrumentation support from the Keck Biophysics Facility and the Center for Structural Biology at Northwestern University, and Life Science Collaborative Access Team station (LS-CAT) at the Advanced Photon Source (APS) at Argonne National Laboratory. LS-CAT was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor. Use of the APS is supported by the Department of Energy (DOE). Support from the R.H. Lurie Comprehensive Cancer Center of Northwestern University to the Structural Biology Facility is also acknowledged. We thank Alexei Slesarev for providing some of the Topoisomerase V plasmids. American Heart Association postdoctoral fellow [10POST2600325 to R.R.]. National Institutes of Health [R01GM51350 to A.M.]. Funding for open access charge: NIH.

ASJC Scopus subject areas

Genetics

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10.1093/nar/gks1017

Cite this

@article{b2df2fd1b5474c3b9d53e5a17d795d92,

title = "Identification of one of the apurinic/apyrimidinic lyase active sites of topoisomerase v by structural and functional studies",

abstract = "Topoisomerase V (Topo-V) is the only member of a novel topoisomerase subtype. Topo-V is unique because it is a bifunctional enzyme carrying both topoisomerase and DNA repair lyase activities within the same protein. Previous studies had shown that the topoisomerase domain spans the N-terminus of the protein and is followed by 12 tandem helix-hairpin-helix [(HhH)2] domains. There are at least two DNA repair lyase active sites for apurinic/apyrimidinic (AP) site processing, one within the N-terminal region and the second within the C-terminal domain of Topo-V, but their exact locations and characteristics are unknown. In the present study, the N-terminal 78-kDa fragment of Topo-V (Topo-78), containing the topoisomerase domain and one of the lyase DNA repair domains, was characterized by structural and biochemical studies. The results show that an N-terminal 69-kDa fragment is the minimal fragment with both topoisomerase and AP lyase activities. The lyase active site of Topo-78 is at the junction of the fifth and sixth (HhH)2 domains. From the biochemical and structural data, it appears that Lys571 is the most probable nucleophile responsible for the lyase activity. Our experiments also suggest that Topo-V most likely acts as a Class I AP endonuclease in vivo.",

author = "Rakhi Rajan and Rajendra Prasad and Bhupesh Taneja and Wilson, {Samuel H.} and Alfonso Mondrag{\'o}n",

note = "Funding Information: We acknowledge staff and instrumentation support from the Keck Biophysics Facility and the Center for Structural Biology at Northwestern University, and Life Science Collaborative Access Team station (LS-CAT) at the Advanced Photon Source (APS) at Argonne National Laboratory. LS-CAT was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor. Use of the APS is supported by the Department of Energy (DOE). Support from the R.H. Lurie Comprehensive Cancer Center of Northwestern University to the Structural Biology Facility is also acknowledged. We thank Alexei Slesarev for providing some of the Topoisomerase V plasmids. Funding Information: American Heart Association postdoctoral fellow [10POST2600325 to R.R.]. National Institutes of Health [R01GM51350 to A.M.]. Funding for open access charge: NIH.",

year = "2013",

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doi = "10.1093/nar/gks1017",

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T1 - Identification of one of the apurinic/apyrimidinic lyase active sites of topoisomerase v by structural and functional studies

AU - Rajan, Rakhi

AU - Prasad, Rajendra

AU - Taneja, Bhupesh

AU - Wilson, Samuel H.

AU - Mondragón, Alfonso

N1 - Funding Information: We acknowledge staff and instrumentation support from the Keck Biophysics Facility and the Center for Structural Biology at Northwestern University, and Life Science Collaborative Access Team station (LS-CAT) at the Advanced Photon Source (APS) at Argonne National Laboratory. LS-CAT was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor. Use of the APS is supported by the Department of Energy (DOE). Support from the R.H. Lurie Comprehensive Cancer Center of Northwestern University to the Structural Biology Facility is also acknowledged. We thank Alexei Slesarev for providing some of the Topoisomerase V plasmids. Funding Information: American Heart Association postdoctoral fellow [10POST2600325 to R.R.]. National Institutes of Health [R01GM51350 to A.M.]. Funding for open access charge: NIH.

PY - 2013/1

Y1 - 2013/1

N2 - Topoisomerase V (Topo-V) is the only member of a novel topoisomerase subtype. Topo-V is unique because it is a bifunctional enzyme carrying both topoisomerase and DNA repair lyase activities within the same protein. Previous studies had shown that the topoisomerase domain spans the N-terminus of the protein and is followed by 12 tandem helix-hairpin-helix [(HhH)2] domains. There are at least two DNA repair lyase active sites for apurinic/apyrimidinic (AP) site processing, one within the N-terminal region and the second within the C-terminal domain of Topo-V, but their exact locations and characteristics are unknown. In the present study, the N-terminal 78-kDa fragment of Topo-V (Topo-78), containing the topoisomerase domain and one of the lyase DNA repair domains, was characterized by structural and biochemical studies. The results show that an N-terminal 69-kDa fragment is the minimal fragment with both topoisomerase and AP lyase activities. The lyase active site of Topo-78 is at the junction of the fifth and sixth (HhH)2 domains. From the biochemical and structural data, it appears that Lys571 is the most probable nucleophile responsible for the lyase activity. Our experiments also suggest that Topo-V most likely acts as a Class I AP endonuclease in vivo.

AB - Topoisomerase V (Topo-V) is the only member of a novel topoisomerase subtype. Topo-V is unique because it is a bifunctional enzyme carrying both topoisomerase and DNA repair lyase activities within the same protein. Previous studies had shown that the topoisomerase domain spans the N-terminus of the protein and is followed by 12 tandem helix-hairpin-helix [(HhH)2] domains. There are at least two DNA repair lyase active sites for apurinic/apyrimidinic (AP) site processing, one within the N-terminal region and the second within the C-terminal domain of Topo-V, but their exact locations and characteristics are unknown. In the present study, the N-terminal 78-kDa fragment of Topo-V (Topo-78), containing the topoisomerase domain and one of the lyase DNA repair domains, was characterized by structural and biochemical studies. The results show that an N-terminal 69-kDa fragment is the minimal fragment with both topoisomerase and AP lyase activities. The lyase active site of Topo-78 is at the junction of the fifth and sixth (HhH)2 domains. From the biochemical and structural data, it appears that Lys571 is the most probable nucleophile responsible for the lyase activity. Our experiments also suggest that Topo-V most likely acts as a Class I AP endonuclease in vivo.

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DO - 10.1093/nar/gks1017

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JF - Nucleic acids research

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Identification of one of the apurinic/apyrimidinic lyase active sites of topoisomerase v by structural and functional studies

Abstract

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ASJC Scopus subject areas

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Crystal structure of Topo-78, an N-terminal 78kDa fragment of topoisomerase V

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Identification of one of the apurinic/apyrimidinic lyase active sites of topoisomerase v by structural and functional studies

Abstract

Funding

ASJC Scopus subject areas

Access to Document

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Datasets

Crystal structure of Topo-78, an N-terminal 78kDa fragment of topoisomerase V

Cite this