Identification of a novel motif in DNA ligases exemplified by DNA ligase IV

Abstract

Penny (2006) Identification of a novel motif in DNA ligases exemplified by DNA ligase IV. DNA Repair, 5 (7). pp. 788-798. ISSN 1568-7864 This version is available from Sussex Research Online: http://sro.sussex.ac.uk/17805/ This document is made available in accordance with publisher policies and may differ from the published version or from the version of record. If you wish to cite this item you are advised to consult the publisher's version. Please see the URL above for details on accessing the published version. Copyright and reuse:Sussex Research Online is a digital repository of the research output of the University.Copyright and all moral rights to the version of the paper presented here belong to the individual author(s) and/or other copyright owners. To the extent reasonable and practicable, the material made available in SRO has been checked for eligibility before being made available.Copies of full text items generally can be reproduced, displayed or performed and given to third parties in any format or medium for personal research or study, educational, or not-for-profit purposes without prior permission or charge, provided that the authors, title and full bibliographic details are credited, a hyperlink and/or URL is given for the original metadata page and the content is not changed in any way. motif Va, present in eukaryotic DNA ligases. We carried out mutational analysis of residues within motif Va examining the impact on adenylation, double-stranded ligation, and DNA binding. We interpret our results using the DNA ligase I:DNA crystal structure. Substitution of the glycine at position 468 with an alanine or glutamic acid severely compromises protein activity and stability. Substitution of G469 with an alanine or glutamic acid is better tolerated but still impacts upon activity and protein stability. These finding suggest that G468 and G469 are important for protein stability and provide insight into the hypomorphic nature of the G469E mutation identified in a LIG4 syndrome patient. In contrast, residues 470, 473 and 476 within motif Va can be changed to alanine residues without any impact on DNA binding or adenylation activity. Importantly however, such mutational changes do impact upon double stranded ligation activity. Considered in light of the DNA ligase I:DNA crystal structure, our findings suggests that motif Va functions as part of a molecular pincer that maintains the DNA in a conformation that is required for ligation.3

Loss of DNA ligase IV prevents recognition of DNA by double-strand break repair proteins XRCC4 and XLF

“…Other ligases are capable of DNA recognition regardless of adenylation status. Recombinant ligase IV/XRCC4 exhibits a weak DNA-binding activity on short DNA substrates and it has been reported that formation of the ligase IV-adenylate is necessary for Ku-mediated binding of recombinant ligase IV/XRCC4 to short DNA substrates (47,48). Along these lines, we found that XRCC4 in cell line 180BRM, which expresses a mutant ligase IV with reduced ability to form the ligase IV-adenylate, fails to bind DNA (data not shown).…”

High-Content Analysis Provides Mechanistic Insights into the Testicular Toxicity of Bisphenol A and Selected Analogues in Mouse Spermatogonial Cells

“…Several assays can measure and quantify cellular responses to DNA double-strand breaks. The proteins, such as c-H2AX, ATM, CDKN1A, and TP53, involved in the early steps of the cellular response in sensing DNA damage and controlling progression, have been proposed as the top group of candidate biomarkers (Marchetti et al, 2006). Due to the signal amplification of c-H2AX foci at the site of DNA damage, the detection of c-H2AX has been identified as an early sensitive cellular biomarker of DNA damage (Nikolova et al, 2014;Rogakou et al, 1998).…”

The spatial organization of non-homologous end joining: From bridging to end joining

“…Since the ATP-binding pocket lies between the subdomains, it is likely that M249V, R278H, Q280R and H282L lead to instability or conformational change in the ATP-binding pocket, resulting in a large reduction of the adenylation efficiency as reported for R278H [149]. G469, a residue in motif Va, which is important for the adenylation of LigIV [103], is completely buried and surrounded by large hydrophobic residues. Therefore, it is likely that G469E leads to disruption of the conformation of OBD.…”