Fluorescenc e B XLF AND XRCC4 INTERACT WITH TELOMERIC PROTEINS Nunez, T. 1, Bui, D. 1 Baidon, M. 1, Anderson, C. 1, Alsina, K. 1 Le, T. 2, Abbas, A. 2, Behe, F. 2, Jain, R. 2, Jabbur, J. 2, Sen, P. 2, Ribes-Zamora, A. 1 1 Biology Department, University of St Thomas, Houston, TX 2 Department of Biology, Houston Community College, Houston, TX 1. INTRODUCTION 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. 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. A Figure 2. PFC assays measurements and interaction localization. (A) Fluorescence measurements of HEK293T 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 10 4 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. Figure 4. XLF Interacts with RAP1. (A) HEK293T 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. Figure 5. XRCC4 Interacts with RAP1. (A) HEK293T cells were transfected with indicated constructs. Pictures were taken 48 hr after transfection showing staining consistent with interaction within nucleus between XRCC4 and RAP1 (B) Fluorescence measurements in same transfected cells indicating intesity of the interaction between XRCC4 and RAP1. Each bar represents three different measurements for three different transfections for a total of 9 measurements for each different V1/V2 combination. A 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): 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: 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– XLF AND XRCC4 INTERACTION WITH TRF1 CO-LOCALIZES WITH TRF2 2. METHODS: PROTEIN FRAGMENT COMPLEMENTATION DAPI dsR ed YFP YFP-dsRed Merge YFP-dsRed- DAPI Merge V1-TRF1 V2-XLF dsRed-TRF2 YFPdsR ed YFP-dsRed- DAPI Merge YFP-dsRed Merge V1-XLF V2-TRF1 dsRed-TRF2 4. XLF INTERACTS WITH RAP1 5. XRCC4 INTERACTS WITH RAP1 Figure 1. Experimental set up. (Left) Representation of the Protein Fragment Complementation Assay, or PCA. (Right) Constructs used in this study. BC 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. Future Directions: To perfom 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. Supported by the UST/HCCS STEM Scholars Program (P031C ) from the US Department of Education A DAPIYFP YFP-DAPI Merge YFP-DAPI-Contrast Merge Cell Contrast YFPDAPI YFP-DAPI Merge YFP-DAPI-Contrast Merge Cell Contrast V1 V2 CMVCMV CMVCMV V1 TRF1 CMVCMV V1 TRF2 CMVCMV V1 XLF CMVCMV V1 XRCC4 RAD21 CMVCMV V1 CMVCMV TRF1 CMVCMV V2 TRF2 CMVCMV V2 XLF CMVCMV V2 XRCC4 RAD21 CMVCMV V2 CMVCMV RAP1 6. CONCLUSIONS AND FUTURE DIRECTIONS Rap1Rap1 TIN2TIN2 TPP1TPP1 Pot1Pot1 TRF1 TRF1 TRF2TRF2 T-LoopT-Loop D-LoopD-Loop Hypothesis: Since of several NHEJ are found localized at the end of natural chromosomes, we hypothesize that XLF and XRCC4 may also be found at telomeres thorugh interactions with telomeric proteins like TRF1, TRF2 or RAP1. 7. REFERENCES B Fluorescen ce