Functional dissection of the C-terminal domain of type II DNA topoisomerase from the kinetoplastid hemoflagellate Leishmania donovani
Authors: Chhabinath Mandal
Hemanta K Majumder
Tanushri Sengupta
Mandira Mukherjee
Aditi Das
Journal: Nucleic Acids Research (2003)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: amino acid sequences; C-terminal domain; topoisomerases; Leishmania donovani; deletion mutants
Affiliations: Department of Molecular Parasitology, Indian Institute of Chemical Biology, Kolkata 700032, India.

Abstract:  The amino acid sequences of the C-terminal domain (CTD) of the type II DNA topoisomerases are divergent and species specific as compared with the highly conserved N-terminal and central domains. A set of C-terminal deletion mutants of Leishmania donovani topoisomerase II was constructed. Removal of more than 178 amino acids out of 1236 amino acid residues from the C-terminus inactivates the enzyme, whereas removal of 118 amino acids or less has no apparent effect on the ability of the parasite enzyme to complement a temperature-sensitive mutation of the Saccharomyces cerevisiae topoisomerase II gene. Deletion analysis revealed a potent nuclear localization signal (NLS) within the amino acid residues 998-1058. Immunomicroscopy results suggest that the removal of an NLS in the CTD is likely to contribute to the physiological dysfunction of these proteins. Modeling of the LdTOP2 based on the crystal structure of the yeast type II DNA topoisomerase showed that the parasite protein assumes a structure similar to its yeast counterpart harboring all the conserved residues in a structurally similar position. However, a marked difference in electrostatic potential was found in a span of 60 amino acid residues (998-1058), which also do not have any homology with topoisomerase II sequences. Such significant differences can be exploited by the structure-based design of selective inhibitors using the structure of the Leishmania enzyme as a template. 

Retracted: No

PubPeer comments:
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 How to explain the quintuplication of the colony morphology at 22 degree Celsius, and also the same colony morphology at 22 and 37 degrre Celsius in figure 2B? , 
 The mentioned figure refers to complementation assay that was done in S. cerevisiae strain RS192, a temperature-sensitive mutant for topoisomerase II. The figure consists of three parts: 1. A schematic diagram indicating the placement of the topoisomerase II mutants on the petriplates. 2. Two petriplates that were streaked with RS192 strain containing either the wild type or mutant topoisomerase constructs. The petriplates were then incubated at 22C (permissive) or 37C (non-permissive) temperatures. 3. Our findings indicate that the full length and some of the mutants were able to complement the temperature-sensitive topo II mutation of RS192 and while longer deletions failed to complement the temperature sensitivity. We disapprove the suggestion of quintuplication or fabrication. Similar streaking patterns can be attributed to the cultures done by the same person. The experiments were performed long back (almost 17 years back). However, the results were repeated several times and were highly reproducible. Additionally, we donu2019t think such colony morphologies can be fabricated. Thanks, H. K. Majumder , 
 No, the streaks are far too similar to be different. It s pretty obvious. Upon zooming in the plate on the left is significantly blurred compared to the plate on the right and there is some haloing around the colonies; I d suspect only the right one is legit, if either is. Sorry, these colonies can definitely be fabricated, we ve seen this sort of thing many times before. Perhaps the findings have held up, but that doesn t change the fact this image data looks really, really questionable. The gel image is poor quality, but there are obvious splice lines and duplications. Where there s smoke, there s fire. This needs to be corrected, doesn t really matter how old it is. , 
 #5, Dr. Majumder, as explained by Lyophyllum in comment #6, it is exceeding obvious that there are multiple copies of the same streak-out in Figure 2B. Your claim that this degree of identity could occur because they were  done by the same person is simply not credible. They are obviously identical, not just somewhat similar. You are denying what is readily apparent for everyone to see. Moreover, Figure 3 clearly has multiple duplicated lanes, as illustrated above in comment #2. By enhancing contrast, etc., it is easy to see how several lanes are spliced  strips . An example is posted below. These irregularities seriously compromise the manuscript. A correction, at the very least, is in order.  , 
 #6 I didn t understand the concern fully. Are you suggesting that the triangular sectors of yeast growth are spliced together to make a circular agar plate? , 
 #8 RE: * Are you suggesting that the triangular sectors of yeast growth are spliced together to make a circular agar plate? * Yes, that is exactly what is being suggested, and it is annotated clearly in comments #1 and #3 above. , 
 #9 Sorry for my oversight. I should have first seen a magnified version of the figure from the manuscript. I agree, the similarities are too compelling to be a coincidence. , 
 Dear PubPeer: As we have said before, we continue to maintain that we did not do any data fabrication. Due to the fact that the work was done 17 to 18 years back, we are unable to produce the original pictographs. At the time this work was done we were unaware of servers or cloud systems where perhaps all data could have been stored indefinitely. With the pictographs and laboratory notebooks changing hands through the generations of graduate students and post-docs, we are unable to produce the original data. Below we respond to the comments. Comment 6 : The commenter thinks that the figure on the right is perhaps the legitimate image and the one on the left is fabricated. I would like to point out that the left image is of the strain RS192 grown at 22C with various LdTOP2 mutants. RS192 will produce colonies at 22C anyway without the help from (LdTOP2). Moreover, the work has been cited by many authors (KL Gilroy et. al., Nucleic Acids Res, 2006; SO Obado et. al., Nucleic Acids Res, 2011, JS Dickey et. al., Biochem, 2005 and many others). One wonders why it is being thought that the data for such a simple experiment (streaking) would be fabricated. After all it would have been much easier for us to do some re-streaking if the quality of the plate was not up to our liking, than splicing out triangular sectors and then re-assembling them with such surgical precision that the splice lines do not show up. Furthermore, one can see the original and unbroken rim of the plastic ware used for the culture through the image. Figure #3 : In this figure we presented the results of a decatenation assay where minicircles are released from the kDNA network in the presence TOP2. This image was taken in one of the first generation Biorad Gel Doc systems and when seen in current time we agree that image is definitely not of high quality. But at that time that was the best system which was available to us. As is evident from the image that the gel was running slightly concave and there was a little smearing from the crude extract-DNA interaction. We did not make any attempt to correct these two things. Moreover, has it been thought why we should have morphed the lanes that did not even show the release of minicircles, what was the need for us to do so. We would like to conclude by stating in good faith that we did not indulge in any wrong doing. Nevertheless we are open to further discussion on the scientific aspects of this work if needed. Kind regards, Hemanta K. Majumder , 
 #11 Without original data, none of what you claim (beyond the citations) can be verified. The file size of these images is not inordinately large; it s somewhat puzzling that one would assume they would have required access to a  cloud system in 2001 to preserve them. Old data does occasionally go missing, but if you re expecting an  oh well, in light of the image irregularities identified above, I m not sure what to tell you. It s incumbent upon you to support your results whenever they are questioned, and unfortunately you are unable to do so in this instance, leaving us with a  preponderance of the evidence standard to question credibility. Based on years of examining images shared by me and many of the other commenters here, these identified images are quite characteristic of inappropriate manipulation as seen time and time again in these threads. I would recommend you examine some of the other threads on PubPeer and see for yourself the numerous examples of  too similar to be different image features, often lacking clear splice boundaries, not reflective of normal variation, etc. What s shown here is another textbook example. If it is normal in your laboratory for notebooks to  change hands without adequate oversight and maintenance, such that they would go missing, that s another problem that needs to be addressed. Lab notebooks should remain in your possession after individuals leave the group, and there should be a chain of custody if they are ever given to someone else. Principal Investigators should take especially careful attention to identifying and locating the storage locations for data and records underlying published reports before they are submitted, and for the entirety of an individual s work product prior to their separation. I hope that your lab management processes have improved during the time between the publication of this paper and now. , 
 #11 First, citations are never an endorsement of the work in totality. And citing authors might not have noticed above issues with the figures. Second, it does not require any special skills to copy an area with yeast growth from one plate and morph onto another plate. There can be many reasons for doing this even for simple growth assays. , 
 8th March 2021 Editorial Expression of Concern. https: academic.oup.com nar advance-article doi 10.1093 nar gkab171 6163095 Editorial Expression of Concern on article u2018Functional dissection of the C-terminal domain of type II DNA topoisomerase from the kinetoplastid hemoflagellate Leishmania donovaniu2019 Nucleic Acids Research, gkab171, https: doi.org 10.1093 nar gkab171 Published: 08 March 2021 On 15 September 2003, NAR published the article u2018Functional dissection of the C-terminal domain of type II DNA topoisomerase from the kinetoplastid hemoflagellate Leishmania donovaniu2019 by Tanushri Sengupta, Mandira Mukherjee, Chhabinath Mandal, Aditi Das, and Hemanta K. Majumder (1). The Editors were recently alerted by a reader that the bacterial colonies depicted in Figure 2B have been duplicated within and across the figure panels despite different using different clones and incubation conditions. In Figure 3C, multiple lanes have also clearly been spliced in. Details of the duplications and splicing have been published in PubPeer: https: pubpeer.com publications 34E9446142CBE8FCD55311F1EB9C1C The Authors are unable to provide the original data or a reasonable explanation for the image manipulations. Pending the results of a full investigation at the Authorsu2019 institution, the Editors advise Readers to examine the details of this study with care. Keith R. Fox, Barry L. Stoddard Senior Executive Editors REFERENCES 1.tSengupta T., Mukherjee M., Mandal C., Das A., Majumder H.K. Functional dissection of the C-terminal domain of type II DNA topoisomerase from the kinetoplastid hemoflagellate Leishmaniadonovani. Nucleic. Acids. Res. 2003; 31:5305u20135316. , 
 Retracted [4 June 2021] . -------------------------------------------------- In August 2020, the Editors were alerted by a reader that the bacterial colonies depicted in Figure 2B of the above article had been duplicated within and across the figure panels despite using different clones and incubation conditions (1). In Figure 3C, multiple lanes have also clearly been spliced in. Details of the duplications and splicing have been published in PubPeer: https: pubpeer.com publications 34E9446142CBE8FCD55311F1EB9C1C The Authorsu2019 institution (Indian Institute of Chemical Biology) had already investigated in 2019 and concluded that there was strong evidence of image manipulation. They also recommended that the raw data should be examined. However, the Authors are unable to provide the original data or a reasonable explanation for the image manipulations. The Editors and Authors are therefore jointly retracting the article from *Nucleic Acids Research*. ,