1. XLF AND XRCC4 INTERACT WITH TELOMERIC PROTEINS Anderson, Spencer 2 ; Tran, Phung 2 ; Luna, Paloma 2, Anderson, Chris 1 ; Baidon, Mario 1 ; Nunez, Tatiana 1 ; Bui, Daniel 1 ; Jain, Renu 2 ; Jabbur, James 2 ; Ribes-Zamora, Albert 1 1 Biology Department, University of St. Thomas, Houston, TX 2 Department of Biology and Physical Sciences, Houston Community College (Central), Houston, TX 1. INTRODUCTION Non-homologous end-joining (NHEJ) is the main repair pathway for processing double-strand DNA breaks (DSBs) and is mediated by a complex of proteins comprised of the Ku subunits (Ku70/80), Ligase4, XRCC4, Aprataxin-and-PNK-like factor (APLF), 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, Ku is present at telomeres through interactions with resident telomeric proteins like TRF1, TRF2 and RAP1, as is the case for DNA-PKcs. Currently, the telomeric status of several NHEJ proteins is still unknown. XLF and XRCC4 are both homodimers that can form not only high order multimer structures but also filaments composed of alternating XRCC4 and XLF molecules. Both proteins have been shown to act at different steps of the NHEJ pathway, including end processing and recruitment of DNA Ligase IV to sites of DNA damage. Hypothesis: In this study we are investigating whether XLF and XRCC4 can be found localized at telomeres, similar to other NHEJ proteins, through interactions with telomeric proteins such as TRF1, TRF2 and RAP1. Protein-Fragment Complementation Assay (PCA) is a technique that uses fluorescence to visualize protein-protein interactions. A fluorescent protein (Venus-YPF) is split into two fragments (N-terminal or V1 and C- terminal or V2) that are then fused to the proteins of interest and whose interaction reconstitutes 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, TRF1, TRF2 and RAP1 were fused to V1 and V2. RAD21 was used as a 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 assay 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 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 the reverse order of Venus fragments. Figure 3. Interaction localization confirmed by PFC assay. (A) Fluorescence microscopy of mammalian cells transfected with XRCC4 and RAP1 constructs. Fluorescence was observed in individual cells 48 hours post- transfection. Images of XRCC4-RAP1 interaction show nuclear localization but absence of punctate florescence as in Figure 2A and 2B. (B) Same as in (A) but employing transfection with XLF and RAP1 constructs. Davis, A.J. and Chen, D.J. (2013). DNA double strand break repair via non-homologous end joining. Transl. Cancer Res. 2(3):130-143. 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. 6. CONCLUSIONS AND FUTURE DIRECTIONS 2. METHODS: PROTEIN FRAGMENT COMPLEMENTATION ASSAY 4. XLF AND XRCC4 INTERACT WITH RAP1 Figure 1. Experimental set up. (Left) Representation of the Protein Fragment Complementation Assay, or PCA. (Right) Constructs used in this study. PCA measurements and co-localization studies with TRF2 demonstrates that XLF and XRCC4 interact with TRF1 at telomeres. We have also detected and interaction between RAP1 with either XRCC4 and XLF throughout the nucleus Future Directions: Validation of our PCA results with Western assay; to confirm equivalent protein co-expression from transfected plasmid DNA. Validation of PCA results with an additional cell line; demonstrates conservation of the mechanism. Interactome mapping; characterizing important amino acid residues on XLF and XRCC4 employing site-directed mutagenesis. Supported by the UST/HCCS STEM Scholars Program (P031C110128-12) from the US Dept. of Education Fluorescence DAPIYFP YFP-DAPI Merge V1-XRCC4 V2-RAP1 V1-XLF V2-RAP1 YFP-DAPI Contrast Merge V2 CMV V1 TRF1 CMVV1 TRF2 CMVV1 XLF CMV V1 XRCC4 RAD21 CMVV1 CMV TRF1 CMVV2 TRF2 CMVV2 XLF CMVV2 XRCC4 RAD21 CMVV2 RAP1 CMV A B 3. XLF AND XRCC4 INTERACTION WITH TRF1 CO-LOCALIZES WITH TRF2 DAPI dsRed YFP YFP-dsRed Merge YFP-dsRed-DAPI Merge V1-TRF1 V2-XLF dsRed-TRF2 B YFPdsRed YFP-dsRed-DAPI Merge YFP-dsRed Merge V1-XLF V2-TRF1 dsRed-TRF2 C REFERENCES YFP-DAPI Contrast Merge YFP-DAPI MergeDAPIYFP 5. WESTERN ASSAY DEVELOPMENT CONFIRMS PROTEIN COEXPRESSION IN TRANSFECTED CELLS V2-RAD21 V1-RAD21 V1-RAP1 V1-XRCC4 and V2-RAP1 V1-XRCC4 + V2-RAD21 V1-XRCC4 + V2-TRF2 V1-XRCC4 + V2-RAP1 V1-RAP1 + V2-XRCC4 V2-XRCC4 V1-RAD21 + V2-RAP1 V1-RAP1 + V2-RAD21 (A) Fluorescence quantification of Protein Complementation Assays (PCA) for mammalian cells transfected with indicated plasmid pairs. (A) Each bar represents the average of three different measurements for three different transfections for each indicated plasmid pair. Rad21 was used as a nonspecific control for interaction. The error bars represent standard deviation of the mean. (B) Protein levels resulting from transfections in (A) detected by immunoblot analysis of equal amounts of WCE using a GFP antibody. A B