Mycobacteriophage exploit NHEJ to facilitate genome circularization
Authors: Graham F Hatfull
James M Daley
Jaroslaw Dziadek
Thomas E Wilson
Andrew J Green
Tricia L Velting
Phillip L Palmbos
Louise M Tonkin
Robert S Pitcher
Steve Cresawn
Malgorzata Korycka-Machala
Anna Brzostek
Aidan J Doherty
Journal: Molecular Cell (2006)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: Ku-dependent nonhomologous end joining; mycobacteriophages; DNA ligase; genome circularization; viral infections
Affiliations: Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton BN1 9RQ, UK.

Abstract:  Ku-dependent nonhomologous end joining (NHEJ) is a double-strand break repair process conserved in all branches of cellular life but has not previously been implicated in the DNA metabolic processes of viruses. We identified Ku homologs in Corndog and Omega, two related mycobacteriophages of Mycobacterium smegmatis. These proteins formed homodimers and bound DNA ends in a manner identical to other Ku's and stimulated joining of ends by the host NHEJ DNA ligase (LigD). Omega and Corndog are unusual in having short 4 base cos ends that would not be expected to self-anneal and would therefore require NHEJ during phage genome circularization. Consistently, M. smegmatis LigD null strains are entirely and selectively unable to support infection by Corndog or Omega, with concomitant failure of genome circularization. These results establish a new paradigm for sequestration of the host cell NHEJ process by bacteriophage and provide a framework for understanding similar transactions in eukaryotic viral infections. 

Retracted: No

PubPeer comments:
 **Summary:** The key to understanding both this 2014 correction (1) and the original 2012 paper (2) is identifying equivocation, and the key to identifying equivocation is discovering the gaps between these separate but dependent historical records. A reconciliation of the two documents shows that the original paper printed u201c6u201d as the key cutoff point for the results, while 5 was actually used off-text to get those results. The equivocal presentation of this Corrigendum obscures this fact. **Premises and Conclusion:** **2012 Copenhagen familial hypercholesterolemia (FH) prevalence study (2):** -tThe text shows a key cutoff point within a diagnostic scoring system to be u201c6.u201d -tThe paper then classifies those with passing scores as FH patients, arriving at a total of 502. **2014 Corrigendum** (1): Later a correction is published. Table 1 illustrates the following. -tThe original document used a cutoff point of 5. This resulted in the identification of 502 FH patients. -tThe original document should have used 6 as the cutoff point. Using such, the FH scoring system now identifies 309 participants as having FH. **Premises from both documents for a readeru2019s reconciliation:** The reader can clearly see and directly verify the original document. The Corrigendum incorrectly labels key *cutoff* points. -tu201c6u201d was actually printed as the key cutoff point in the original document. It is the true original version and is already correct. Nonetheless, it is labeled u201cCorrected Versionu201d in the Corrigendum. -t5 as the key cutoff point was not printed under diagnostic criteria in the original document. Nonetheless, it is labeled u201cOriginal Versionu201d in the Corrigendum. If the *results* in the correcting document are accepted as correct, then we add the following premises. -tThe correcting document shows that 502 is an erroneous result in that original document. -tThe correcting document shows that the result of 502 in the original version was derived from the cutoff point of 5. Conclusion from the above premises. -Therefore, arriving at 502 in the original document must have been due to using 5 off-text as the key cutoff point, while u201c6u201d was merely printed and not actually used for the results. A full understanding of the nonstandard cutoff used off-text in the original version is obscured by the equivocal presentation of the Corrigendum. **Detailed presentation:** The 2014 Corrigendum (1) refers to the 2012 Copenhagen study (2). [See my [comments] on the 2016 Copenhagen FH prevalence study. (3)] In the 2012 paper, the authors employed the Dutch Lipid Clinic Network criteria (DLCN) to classify participants according to their probability of suffering from FH. The authors declared that participants classified as u201cdefinite or probable FH combinedu201d were counted for the FH prevalence estimate. Therefore, the cutoff point for Probable FH drove the paperu2019s results. That key cutoff point was clearly printed as u201c6u201d under u201cDiagnostic Criteria,u201d as u201c 5u201d under u201cResults,u201d and as u201c6u201d in Supplementary Table 1. In all, there are at least seven references, explicit or implicit, to using the number 6 as the threshold for Probable FH. This cutoff point was already established in FH literature. (4) The paper claimed 502 participants, resulting in a prevalence of 1:137. As the authors put it, u201cThis is more frequent than the commonly reported prevalence of one in 500 for heterozygous FH.u201d This was later declared to be an error. As a consequence, the authors now present two versions of the results, an u201cOriginal Versionu201d and a u201cCorrected Version.u201d However, the presentation of the u201cOriginal Versionu201d and u201cCorrected Versionu201d in the Corrigendum is equivocal. From the Corrigendum: u201cIn the paper participants were originally classified as shown in the table below in the columns labeled u2018Original version,u2019 but should have been classified as shown in the column labeled u2018Corrected version.u2019 u201c In the most responsible reading, the phrase u201coriginal versionu201d is anchored to the phrase u201cin the paper.u201d But the diagnostic criteria under the Corrigendumu2019s column u201coriginal versionu201d were not u201cin the paper.u201d And the u201ccorrected versionu201d of the u201cdiagnostic criteriau201d were in fact already u201cin the paper.u201d  When the Corrigendum employs the words u201cin the paperu201d and u201coriginal versionu201d within the context of the words, u201coriginally classified,u201d it appears that u201c5u201d was first disclosed as the cutoff for the Probable FH category and that it was then changed to u201c6.u201d However, u201c6u201d was already printed in the original version. There is no change with the original diagnostic criteria, nor is there a real correction in the Corrigendum. Nonetheless, the published *results* changed:  So, reconciling the numbers provided in the Corrigendum with those in the original document, it is clear that the original documentu2019s *printed result* was later corrected but not its *printed criteria*. What must have actually happened to that original version? A deviation from the declared criteria must have been employed off-text. 6 is the well-known cutoff point for Probable FH, and u201c6u201d was printed in the original version. However, the Corrigendum tells us that the original result of 502 was derived from a threshold of 5, not 6. And in the original version, u201c5u201d was not printed under diagnostic criteria as the cutoff point, u201c6u201d was. Thus, 5 was used but not disclosed. u201c6u201d was printed but not used. In the Corrigendum, the labels in Table 1 under u201cDiagnostic Criteriau201d -- u201cOriginal Versionu201d and u201cCorrected Versionu201d -- are each presented in the otheru2019s stead. u201c6u201d as the cutoff point was the original version and is not actually a corrected version. u201c5u201d as the cutoff was not in the original versionu2019s diagnostic criteria. **Disclosing the undisclosed use of 5 as the real cutoff point belongs to a fully corrected version. The equivocal presentation of the Corrigendum however dodges this revelation.** The Corrigendum is equivocal because it begs the question of what was actually done off-text. But after we reconcile the original and correcting documents, we can expose the gaps, fill them with products of deduction, and present a less ambiguous correction. Here is a reconciliation modeled on the authorsu2019 Table 1 of the Corrigendum. (Because the cutoff point for the u201cProbable FHu201d category drives the results, Iu2019ve enlarged that number and highlighted it in yellow. Iu2019ve colored the cells which are key to unpacking the equivocation in red.)  From the Corrigendum: u201cThe consequence of this misclassification is a slight overestimation u2026u201d One difference between a harmless typographical error and a critical flaw is whether or not the results change significantly. How far did the results stray? Measuring from the correct result, 309 FH, to the incorrect result, 502 FH, the difference is over 60%. Printing u201c6u201d while using 5 off-text does not appear to be a problem of typographical error. There were at least seven references to the number 6 in the original version. (See supplementary material below.) And by definition, the fact that 6 was indeed the correct number makes an argument for u201c6u201d as a typographical error impossible. Under reconciliation, the two documents become a tacit admission of off-text deviation from the well-established DLCN criteria, and using the result in the original version created the illusion that the number 6 had been employed as the cutoff point. From the Corrigendum: u201cThis does not change the main conclusion of the paper ...u201d It would be more accurate to say that the change did not *flip* the conclusion, but the results did change. By definition, the lower the bar the more participants one will count. And because the DLCN scoring system assigns participants a probability of bearing an FH mutation, with each tick lower of that threshold, the more questionable the results become. The error had tripled the well-established FH prevalence estimate of 1 in 500. (5)  This leaves us with an important question about the Corrigendum: If it were explicitly admitted from the outset that an undisclosed and nonstandard cutoff point was used to reach the original results, would this paper survive retraction considerations? However, as it is, this Corrigendum itself is inconspicuous. One can sift through Google scholar and find other papers which continue to cite the error and not the correction. https: scholar.google.com scholar?hl=en as_sdt=5%2C48 sciodt=0%2C48 as_ylo=2015 cites=2557072895858201649 scipsc=1 q=%221%3A137%22 btnG= And, https: scholar.google.com scholar?start=10 q=%221+in+137%22 hl=en as_sdt=5,48 sciodt=0,48 as_ylo=2015 cites=2557072895858201649 scipsc=1 **Concluding Q A:** The lower limit of Probable FH was used in the 2012 Copenhagen paper to reach the prevalence results. According to the authorsu2019 text and its reference to the World Health Organization (4), what DLCN cutoff point for Probable FH is standard? -t6 In the 2012 Copenhagen paper, what number was originally published under u201cDiagnostic Criteriau201d as the lower limit to Probable FH? -tu201c6u201d In the 2012 Copenhagen paper, was the number u201c5u201d printed under u201cDiagnostic Criteriau201d as the lower limit to Probable FH? -tNo. Given the one-way passage of time, is it possible for the cutoff point for Probable FH in the earlier 2012 document to be changed by the later 2014 Corrigendum? -tNo. In the 2012 Copenhagen paper, was the result for Probable and Definite FH printed as u201c502u201d? -tYes. Did the 2014 Corrigendum show that 502 is derived from a cutoff point of 5? -tYes. Therefore, was 5 the actual and off-text cutoff point used for the original versionu2019s prevalence result? -tYes. Did *using* u201c5u201d as the off-text cutoff point while *printing* u201c6u201d create an illusion that FH prevalence had tripled? -tYes.  u2003 **Supplementary:** **Counting the number of times six and five were used to classify Probable FH:** In the 2012 paper, there were at least seven references to the number 6, explicit and implicit. u201c5u201d was printed once for Probable FH, and it was immediately contradicted in the same sentence. **First 6:** On page 1, we read: u201cMain Outcome Measures: FH (definite probable) was defined as a Dutch Lipid Clinic Network score higher than 5.u201d u201cHigher than 5u201d begins with the number 6, and 6 is the established cutoff point. This is the first reference to the number 6 as the floor for the u201cProbable FHu201d category. **Second and third 6:** On page 2, under the heading, u201cDiagnostic criteria for fFH [sic],u201d (screenshot below), the cutoff point for the u201cProbable FHu201d category was printed as u201c6.u201d We see that the category for u201cPossible FHu201d already has u201c5u201d printed as its ceiling, and this clearly implies that the next category up u2013 Probable FH -- must begin with the number 6. These are the second and third references to the number 6 as the floor for u201cProbable FH,u201d one explicit and one implicit.  **Fourth and Fifth 6:** Here is a screenshot from Supplementary Table 1. It also prints the cutoff for u201cProbable FHu201d as u201c6.u201d And u201cPossible FH,u201d again, is printed with the ceiling of u201c5,u201d implying the number 6 as the floor for the next category up. These are the fourth and fifth references to the number 6, explicit and implicit.  Iu2019ve saved the contradictory passage for last. **First 5 (screenshot below):** Straddling pages 3 and 4, in the u201cResults,u201d u201cProbable FH (5-8)u201d is the first and only printing of u201c5u201d as the cutoff, and it is immediately contradicted within the same sentence with u201c 5.u201d  **Sixth 6 (screenshot above):** It is explicitly stated that u201c 5u201d was the cutoff for the prevalence result of 1 in 137. Printing u201c 5u201d means the number 6 and above, and we would have to see u201cu22655u201d to interpret this cutoff as 5 and above. u201c 5u201d is the sixth reference for the number 6 as the floor for u201cProbable FH.u201d **Seventh 6 (screenshot above):** Again, the range for the next lower category, u201cPossible FH,u201d is explicitly recorded as u201c3 - 5 points.u201d By printing the upper limit of u201cPossible FHu201d as u201c5,u201d the lower limit of u201cProbable FHu201d must be the number 6. This is the seventh reference to the number 6. Again, given the seven references to the number 6, printing u201c6u201d while using 5 off-text does not appear to be a problem of typographical error. And the fact that 6 was indeed the correct number, makes an argument for u201c6u201d as a typographical error impossible. References 1. Corrigendum: Familial Hypercholesterolemia in the Danish General Population: Prevalence, Coronary Artery Disease, and Cholesterol-Lowering Medication. Benn, Marianne, et al. 12, s.l. : J Clin Endocrinol Metab, 2014, Vol. 99. doi: 10.1210 jc.2014-3926. 2. Familial Hypercholesterolemia in the Danish General Population: Prevalence, Coronary Artery Disease, and Cholesterol-Lowering Medication. Benn, Marianne, et al. 11, s.l. : J Clin Endocrinol Meta, 2012, Vol. 97. doi:10.1210 jc.2012-1563. 3. Mutations causative of familial hypercholesterolaemia: screening of 98 098 individuals from the Copenhagen General Population Study estimated a prevalence of 1 in 217. Benn, Marianne, et al. 17, s.l. : European Heart Journal, 2016, European Heart Journal , Vol. 37, pp. 1384 - 1394. doi:10.1093 eurheartj ehw028. 4. Familial Hypercholesterolemia (FH). Report of a Second WHO Consultation. WHO. Geneva : World Health Organization, 1999. 5. The LDL Receptor. Goldstein, Joseph L. and Brown, Michael S. 4, s.l. : Arterioscler Thromb Vasc Biol. , 2009, Arterioscler Thromb Vasc Biol. , Vol. 29, pp. 431 - 438. doi: 10.1161 ATVBAHA.108.179564. ,