doi: 10.1093/nar/gkw122. Epub 2016 Feb 22.
Methanopyrus kandleri topoisomerase V contains three distinct AP lyase active sites in addition to the topoisomerase active site
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- PMID:26908655
- PMCID: PMC4838376
- DOI: 10.1093/nar/gkw122
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Methanopyrus kandleri topoisomerase V contains three distinct AP lyase active sites in addition to the topoisomerase active site
Rakhi Rajan et al. Nucleic Acids Res..
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Abstract
Topoisomerase V (Topo-V) is the only topoisomerase with both topoisomerase and DNA repair activities. The topoisomerase activity is conferred by a small alpha-helical domain, whereas the AP lyase activity is found in a region formed by 12 tandem helix-hairpin-helix ((HhH)2) domains. Although it was known that Topo-V has multiple repair sites, only one had been mapped. Here, we show that Topo-V has three AP lyase sites. The atomic structure and Small Angle X-ray Scattering studies of a 97 kDa fragment spanning the topoisomerase and 10 (HhH)2 domains reveal that the (HhH)2 domains extend away from the topoisomerase domain. A combination of biochemical and structural observations allow the mapping of the second repair site to the junction of the 9th and 10th (HhH)2 domains. The second site is structurally similar to the first one and to the sites found in other AP lyases. The 3rd AP lyase site is located in the 12th (HhH)2 domain. The results show that Topo-V is an unusual protein: it is the only known protein with more than one (HhH)2 domain, the only known topoisomerase with dual activities and is also unique by having three AP lyase repair sites in the same polypeptide.
© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.
Figures
Identification of a second AP lyase active site in Topo-V. (A) Schematic representation of the Topo-V protein and the various fragments studied. The N-terminus of the protein corresponds to the topoisomerase domain (red) and is followed by 12 tandem (HhH)2 domains (small boxes). The fragments studied were made by removing (HhH)2 domains from the C-terminus of the protein. (B) Quantification of the AP lyase activity for full-length Topo-V and different Topo-V fragments (3 replications). All fragments were made in a backbone where the first AP lyase active site was inactivated (ΔRS1). All fragments larger than 91 kDa exhibit AP lyase activity, showing that the second repair site is located in the Topo-97 fragment. The wild-type Topo-78 fragment was used as a positive control for AP lyase activity as it contains only one well-characterized repair active site. The partial cleavage of control DNA is due to the inherent instability of abasic DNA at the temperature used for the experiments. In all cases the level of activity reported is based on three replications and the error bars correspond to the standard error. (C) Gel illustrating experiments to locate the second AP lyase active site. The upper band corresponds to uncleaved DNA (S) while the lower band on the gel shows the product after cleavage of the DNA at the abasic site (P). Topo-78: wild-type 78 kDa fragment; Topo-78(ΔRS1): 78 kDa fragment with K566A/K570A/K571A mutations to inactivate the first repair site; Topo-V(ΔRS1): full-length Topo-V with the first repair site inactivated.
Identification of a third AP lyase active site in Topo-V. (Top) Schematic representation of the Topo-V fragment used to identify the third AP lyase active site. In the fragment, Topo-103, the last (HhH)2 domain has been removed (shown shaded). (Bottom) Quantification of the AP lyase activity for full length Topo-V and the Topo-103 fragment. The wild-type protein with the first two active sites inactivated (Topo-V(ΔRS1ΔRS2)) retained partial activity, indicating the presence of a third repair site. The Topo-103(ΔRS1ΔRS2) fragment, corresponding to the Topo-103 fragment with the first two AP lyase active site mutated, showed no AP lyase activity, consistent with the existence of a third active site in the last (HhH)2 domain. The wild-type Topo-78 fragment was used as a positive control for AP lyase activity as it contains only one well-characterized repair active site. Topo-78: wild-type 78 kDa fragment; Topo-78(ΔRS1): 78 kDa fragment with three mutations to inactivate the first repair site. The partial cleavage of control DNA is due to the inherent instability of abasic DNA at the temperature used for the experiments. In all cases the level of activity reported is based on three replications and the error bars correspond to the standard error. The red dashed line corresponds to the level of cleavage of the control DNA and serves to indicate baseline activity.
Crystal structure of the Topo-97 fragment. (A) Schematic diagram of the Topo-V protein. The Topo-97 fragment consists of the topoisomerase and 10 tandem (HhH)2 domains. The last two (HhH)2 domains in the full length protein are not part of the fragment and are shown for reference only. (B) The diagram shows a model of the Topo-97 fragment in two orthogonal views. The seventh putative (HhH)2 domain is not visible in the structure and may represent a hinge point. For reference, the coloring of the (HhH)2 domains is similar to the one in A. (C) Electrostatic surface representations of the Topo-97 fragment of topoisomerase V show that there is a positively charged region along one edge of the (HhH)2 domains, which may represent binding areas for DNA. The electrostatic potential was calculated with the program APBS (37). The surface is colored with a blue to red gradient from +8 to −8 KbT/ec.
Similarity amongst the (HhH)2 domains in Topo-V. (A) Schematic diagram illustrating the general architecture of an (HhH)2 domain. Each domain is formed by two HhH motifs (green and red) linked by a short helix (light blue). Topo-V is unique as it has 12 tandem (HhH)2 domains. (B) Superposition of the nine (HhH)2 domains observed in the Topo-97 structure. The (HhH)2 domains were superposed using an SSM alignment. The domains superpose well and in general have a similar structure. In general, adjacent (HhH)2 domains are connected by a short loop, except for domains 2 and 3, where the last helix of domain 2 extends to form the first helix of domain 3. (C) Structure-based sequence alignment of the (HhH)2 domains. The sequences of the 9 ordered (HhH)2 domains were aligned based on the SSM superposition whereas domains 7, 11 and 12 were manually aligned based only on sequence similarity. The alignment shows the presence of a few strictly conserved residues (red) in the first HhH motif and some highly conserved residues (cyan) in both motifs. The catalytic lysines for the first and second AP lyase sites are marked by stars, lysines that were mutated to quantitate activity are shown by circles. Yellow shaded areas correspond to regions whose structure is unknown. In the case of domain 6, the green shaded region represents an area that is ordered in the Topo-78 (green), but disordered in the Topo-97 structure. Secondary structure elements are shown above as a reference and using the same coloring as in panel A.
Structure of the second AP lyase active site in Topo-V. (A) Schematic diagram of the structure of the first (left, yellow) and second (right, orange) AP lyase active sites in Topo-V. The two active sites are located in the (HhH)2 junctions of domains 5 and 6 and domains 9 and 10, respectively. For simplicity, only some of the side chains are drawn. (B) Superposition of the first and second AP lyase active sites. The two regions are structurally similar, although the loop connecting the two helices is different due to the absence of one amino acid in the second repair site. The catalytic lysine in the first repair site is K571, but there is no direct equivalent to this residue in the second site. Instead, K809 occupies a similar spatial position. Coloring as in A. (C) Superposition of the second repair active site of Topo-V (orange), GS Endo III (pink) and rat Pol β (blue). The overall structure of the three active sites is similar. Note that K809 in Topo-V, K121 in GS Endo III, and K72 in rat Pol β are all in a similar spatial position. K121 and K72 are the catalytic lysines in Endo III and polymerase β, respectively, whereas K809 is the likely active site lysine of the second repair site in Topo-V. (D) Sequence comparison of four different AP lyase repair active sites. The structural, sequence and biochemical analysis strongly suggest that K809 (star) is the nucleophile in the second repair active site of Topo-V.
SAXS envelope of Topo-97 shows an elongated molecule. Two orthogonal views of the SAXS envelope with a model of Topo-97 manually fit into the envelope. The SAXS envelope shows an elongated molecule that can accommodate the Topo-97 fragment. The model of Topo-97 based on the crystal structure was manually fit by breaking it into two parts, one comprising the topoisomerase domain and the first six (HhH)2 domains and the second comprising (HhH)2 domains 8–10.
References
- Chen S.H., Chan N.L., Hsieh T.S. New mechanistic and functional insights into DNA topoisomerases. Annu. Rev. Biochem. 2013;82:139–170. - PubMed
- Schoeffler A.J., Berger J.M. DNA topoisomerases: harnessing and constraining energy to govern chromosome topology. Q. Rev. Biophys. 2008;41:41–101. - PubMed
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