Transcript xlf and xrcc4 interact with telomeric proteins

XLF AND XRCC4 INTERACT WITH TELOMERIC PROTEINS
Anderson, Spencer2; Tran, Phung2; Luna, Paloma2 , Anderson, Chris1; Baidon, Mario1; Nunez, Tatiana1; Bui, Daniel1; Jain, Renu2; Jabbur, James2; Ribes-Zamora, Albert1
1Biology Department, University of St. Thomas, Houston, TX
2Department of Biology and Physical Sciences, Houston Community College (Central), Houston, TX
3. XLF AND XRCC4 INTERACTION WITH TRF1 CO-LOCALIZES WITH TRF2
5. WESTERN ASSAY DEVELOPMENT CONFIRMS PROTEIN
COEXPRESSION IN TRANSFECTED CELLS
1. INTRODUCTION
A
Non-homologous end-joining (NHEJ) is the main repair pathway for processing
A
B
Fluorescence
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), DNAPKcs and XLF, also known as Cernunnos. Paradoxically, several of these NHEJ proteins are
V1-RAD21
also found at telomeres, despite the fact that one of the main functions of the complex
V2-RAD21
structure at telomeres is to provide a protective cap that prevents NHEJ proteins from
V1-RAP1
recognizing the natural ends of chromosomes as DSBs. For instance, Ku is present at
V1-XRCC4 and
V2-RAP1
V2-XRCC4
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
V1-TRF1 V2-XLF dsRed-TRF2
is still unknown. XLF and XRCC4 are both homodimers that can form not only high order
B
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.
dsRed
DAPI
Hypothesis: In this study we are investigating whether XLF and XRCC4 can be found
YFP-dsRed
Merge
YFP
YFP-dsRed-DAPI
Merge
V1-XLF V2-TRF1 dsRed-TRF2
localized at telomeres, similar to other NHEJ proteins, through interactions with telomeric
C
proteins such as TRF1, TRF2 and RAP1.
(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.
6. CONCLUSIONS AND FUTURE DIRECTIONS
2. METHODS: PROTEIN FRAGMENT
COMPLEMENTATION ASSAY
• PCA measurements and co-localization studies with TRF2 demonstrates that XLF and XRCC4
YFP
• 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 Cterminal 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
dsRed
YFP-dsRed
Merge
YFP-dsRed-DAPI
Merge
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 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 the reverse order of Venus fragments.
can not reform Venus-YFP unless the proteins of interest are interacting.
4. XLF AND XRCC4 INTERACT WITH RAP1
• To test our hypothesis, XRCC4, XLF, TRF1, TRF2 and RAP1 were fused to V1 and V2.
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.
V1-XRCC4 V2-RAP1
RAD21 was used as a non-specific control.
• Some of the advantages of using PCA over other protein binding detection techniques
A
REFERENCES
are that it can be done in live cells and that it provides information about the
localization of the interaction .
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.
CMV
CMV
CMV
CMV
CMV
CMV
CMV
CMV
CMV
CMV
CMV
V1
V2
V1
V2
V1
V2
V1
V2
V1
V2
V2
TRF1
TRF1
TRF2
TRF2
XLF
XLF
XRCC4
XRCC4
RAD21
RAD21
RAP1
Figure 1. Experimental set up. (Left) Representation of the Protein Fragment
Complementation Assay, or PCA. (Right) Constructs used in this study.
DAPI
YFP
YFP-DAPI Merge
YFP-DAPI
Contrast Merge
V1-XLF V2-RAP1
B
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.
DAPI
YFP
YFP-DAPI Merge
YFP-DAPI
Contrast Merge
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 posttransfection. 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.
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.
Supported by the UST/HCCS STEM Scholars Program (P031C110128-12) from the US Dept. of Education