XLF AND XRCC4 INTERACT WITH RAP1 Pham, Q. 1, Baidon, M. 1 , Nunez, T XLF AND XRCC4 INTERACT WITH RAP1 Pham, Q. 1, Baidon, M. 1 , Nunez, T. 1, Bui, D. 1 Anderson, C. 1, Alsina, K.1 Le, T. 2, Abbas, A. 2, Behe, F. 2, Jain, R. 2, Jabbur, J. 2, Ribes-Zamora, A. 1 1Biology Department, University of St Thomas, Houston, TX 2Department of Biology, Houston Community College, Houston, TX fluorescence machine infinity 200 pro each bar represents 3 different measurements for 3 different transfections for a total of 9 1. INTRODUCTION 3. XLF AND XRCC4 INTERACTION WITH TRF1 CO-LOCALIZES WITH TRF2 6. FUTURE DIRECTIONS Non-homologous end-joining (NHEJ) is the main repair pathway for processing double- strand DNA breaks (DSBs) in Eukarya. In mammals, NHEJ is mediated by a complex of proteins comprised of the Ku subunits (Ku70 and Ku86), Lig4, XRCC4, Artemis, DNA-PKcs and XLF, also known as Cernunnos. Paradoxically, several of these NHEJ proteins are also found at telomeres despite the fact that one of the main functions of the complex structure at telomeres is to provide a protective cap that prevents NHEJ proteins from recognizing the natural ends of chromosomes as DSBs. For instance, NHEJ proteins Ku and DNA-PKs present at telomeres through interactions with resident telomeric proteins like TRF1 and TRF2. XLF and XRCC4 are both homodimers that form both high order multimer structures and filaments composed of alternating XRCC4 and XLF molecules. Mammalian Rap1 is one of the six protein involved in forming the telomere Shelterin complex, interacting with TRF2, assisting in telomere folding and structure. However, Rap1 is also involved in gene expression and other cellular functions, thus is not limited to telomeric localization. A A B DAPI dsRed YFP YFP-dsRed Merge YFP-dsRed-DAPI V1-TRF1 V2-XLF dsRed-TRF2 To perform western blot analysis to rule out differences in expression as a possible alternative explanation for our results and to confirm interactions by co-IP analysis. Methodology: TRF1 is marked with Flag. XLF and XRCC4 are respectively marked with Myc. CO-IP is conducted using magnetic protein G beads with capture antibody (Anti-FLAG / Anti-Myc). Instead of using centrifugation in order to precipitate the beads, beads are magnetized so that the target proteins can be collected. Western blot: Collected purified proteins are then ran through a western blot gel. A set of primary rat antibodies is used against Flag and Myc. Rat Anti-FLAG (for Flag-TRF1) and Rat Anti-C-Myc (for Myc-XLF/XRCC4).A secondary antibody (rabbit anti-rat) is then combined with a visualization system (HRP - enzyme horseradish peroxidase). Expected results: Presence of 2 bands corresponding to XLF or XRCC4 and TRF1 that confirms the interaction between these proteins. C YFP dsRed YFP-dsRed-DAPI Merge YFP-dsRed V1-XLF V2-TRF1 dsRed-TRF2 V1-XLF V2-Rad21 V1-XLF V2-TRF1 V1-XLF V2TRF2 V1-XRCC4 V2-Rad21 V1-XRCC4 V2-TRF1 V1-XRCC4 V2-TRF2 Figure 2. PCA assays measurements and interaction localization. (A) Fluorescence measurements of mammalian cells transfected with indicated constructs indicating that XLF interacts with both TRF1 and TRF2 while XRCC4 only interacts with TRF1. Fluorescence was measured in 5 x 104 cells 48 hours after transfection using Tecan Infinity200Pro plate reader. Each bar represents three different measurements for three different transfections for a total of 9 measurements for each different V1/V2 combination. (B) De-convoluted images of XLF-TRF1 interaction showing its co-localization with TRF2, suggesting that this interaction occurs at telomeres. (C) Same as in (B) but changing the order of Venus fragments. Hypothesis: Since some NHEJ proteins are found localized at the end of natural chromosomes, we hypothesize that XLF and XRCC4 may also be found at telomeres through interactions with telomeric proteins like TRF1, TRF2 or RAP1. Rap1 TIN2 TPP1 Pot1 TRF1 TRF2 T-Loop D-Loop Myc 4. XLF AND XRCC4 INTERACT WITH RAP1 XRCC4 TRF1 Flag Myc TRF1 Flag XRCC4 B A DAPI YFP YFP-DAPI Merge TRF1 Fluorescence 2. METHODS: PROTEIN FRAGMENT COMPLEMENTATION Myc TRF1 Myc XLF or XRCC4 interacts with TRF1 XLF or XRCC4 does not interact with TRF1 Figure 4. XLF Interacts with RAP1. (A) mammalian cells were transfected with indicated constructs. Pictures were taken 48 hr after transfection, showing staining consistent with interaction within nucleus between XLF and RAP1 (B) Fluorescence measurements in same transfected cells indicate the intensity of interaction between XLF and RAP1. Each bar represents three different measurements for three different transfections for a total of 9 measurements for each different V1/V2 combination. Similar procedure is then used to test the interaction between XRCC4 and RAP1. Results matched our expectations. Protein-Fragment Complementation Assay (PCA) is a technique that uses fluorescence to visualize protein-protein interactions. A fluorescent protein (Venus-YPF) is split in two fragments (N-terminal or V1 and C-terminal or V2) that are fused to the proteins of interest whose interaction reconstitute the fluorophore. Previous studies have shown that V1 and V2 fragments can not reform Venus-YFP unless the proteins of interest are interacting. To test our hypothesis, XRCC4, XLF, RAP1, TRF1, and TRF2 were fused to V1 and V2 and Rad21 was used as non-specific control. Some of the advantages of using PCA over other protein binding detection techniques are that it can be done in live cells and that it provides information about the localization of the interaction . 7. REFERENCES V1-XLF V2-Rad21 V1-XLF V2-TRF1 V1-XLF V2-TRF2 V1-XLF V2-RAP1 V1-RAP1 V2-XLF V1-Rad21 V2-RAP1 V1 V2 CMV TRF1 TRF2 XLF XRCC4 RAD21 RAP1 Lee, K.J., Jovanovic ,M., Udayakumar, D., Bladen, C.L., Dynan, W.S. (2004). Identification of DNA-PKcs phosphorylation sites and effects of mutation of these sites on DNA end joining in a cell-free system. DNA Repair (Amst). 4;3(3):267-76. Lieber M.R. (2010). The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway. Annu. Rev. Biochem. 79:181–211.  Palm, W., and de Lange, T. (2008). How shelterin protects mammalian telomeres. Annu. Rev. Genet. 42: 301-334. Remy, I., and Michnick, S.W. (2007). Application of protein-fragment complementation assays in cell biology. Biotechniques 42, 137, 139, 141 passim Riha, K., Heacock, M. L., Shippen, D. E. (2006). The role of the nonhomologous end-joining DNA double-strand break repair pathway in telomere biology. Annu. Rev. Genet. 40: 237–277. 5. CONCLUSIONS PCA and co-localization studies demonstrate that XLF interacts with TRF1 at telomeres. XRCC4 also interacts with TRF1 in a punctuate manner consistent with telomere localization. Finally, our results support the hypothesis that XLF and XRCC4 interacts with RAP1. Supported by the UST/HCCS STEM Scholars Program (P031C110128-12) from the US Department of Education and the University of St. Thomas Cullen Smith Chair of Biology   Figure 1. Experimental set up. (Left) Representation of the Protein Fragment Complementation Assay, or PCA. (Right) Constructs used in this study.