The Proximity Ligation Assay Reveals That at DNA Double-strand Breaks WRAP53β Associates with γH2AX and Controls Interactions Between RNF8 and MDC1

Overview

Journal Nucleus

Date 2016 Jan 7

PMID 26734725

Citations 13

Authors

Hanif Rassoolzadeh

Christos Coucoravas

Marianne Farnebo

Affiliations

Soon will be listed here.

Abstract

We recently demonstrated that WRAP53β acts as a key regulator of ubiquitin-dependent repair of DNA double-strand breaks. Here, we applied the proximity ligation assay (PLA) to show that at such breaks WRAP53β accumulates in close proximity to γH2AX and, furthermore as demonstrated by their co-immunoprecipitation (IP) binds to γH2AX, in a manner dependent on the ATM and ATR kinases. Moreover, formation of complexes between MDC1 and both its partners RNF8 and phosphorylated ATM was visualized. The interaction of MDC1 with RNF8, but not with ATM requires WRAP53β, suggesting that WRAP53β facilitates the former interaction without altering phosphorylation of MDC1 by ATM. Furthermore, our findings highlight PLA as a more sensitive method for the analysis of recruitment of repair factors and complex formation at DNA breaks that are difficult to detect using conventional immunofluorescence.

Citing Articles

The Role of WRAP53 in Cell Homeostasis and Carcinogenesis Onset.

Gadelha R, Machado C, DE Pinho Pessoa F, da Costa Pantoja L, Barreto I, Ribeiro R Curr Issues Mol Biol. 2022; 44(11):5498-5515.

PMID: 36354684 PMC: 9688736. DOI: 10.3390/cimb44110372.

DNA double-strand break-derived RNA drives TIRR/53BP1 complex dissociation.

Ketley R, Battistini F, Alagia A, Mondielli C, Iehl F, Balikci E Cell Rep. 2022; 41(4):111526.

PMID: 36288694 PMC: 9638026. DOI: 10.1016/j.celrep.2022.111526.

[Effect of 53 Targeted Co-Inhibitory Pathways Based on Comprehensive Bioinformatics Analysis in Treating Squamous Cell Carcinoma of the Head and Neck].

Yao Y, Liu W, Zhou M, Qiu T, Wang K Sichuan Da Xue Xue Bao Yi Xue Ban. 2022; 53(3):457-465.

PMID: 35642155 PMC: 10409416. DOI: 10.12182/20220560208.

Small Cajal body-associated RNA 2 (scaRNA2) regulates DNA repair pathway choice by inhibiting DNA-PK.

Bergstrand S, OBrien E, Coucoravas C, Hrossova D, Peirasmaki D, Schmidli S Nat Commun. 2022; 13(1):1015.

PMID: 35197472 PMC: 8866460. DOI: 10.1038/s41467-022-28646-5.

Immediate-Early, Early, and Late Responses to DNA Double Stranded Breaks.

Kieffer S, Lowndes N Front Genet. 2022; 13:793884.

PMID: 35173769 PMC: 8841529. DOI: 10.3389/fgene.2022.793884.

References

Soderberg O, Gullberg M, Jarvius M, Ridderstrale K, Leuchowius K, Jarvius J . Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nat Methods. 2006; 3(12):995-1000. DOI: 10.1038/nmeth947. View

Lord C, Ashworth A . The DNA damage response and cancer therapy. Nature. 2012; 481(7381):287-94. DOI: 10.1038/nature10760. View

Garcia-Closas M, Kristensen V, Langerod A, Qi Y, Yeager M, Burdett L . Common genetic variation in TP53 and its flanking genes, WDR79 and ATP1B2, and susceptibility to breast cancer. Int J Cancer. 2007; 121(11):2532-8. DOI: 10.1002/ijc.22985. View

Stucki M, Clapperton J, Mohammad D, Yaffe M, Smerdon S, Jackson S . MDC1 directly binds phosphorylated histone H2AX to regulate cellular responses to DNA double-strand breaks. Cell. 2005; 123(7):1213-26. DOI: 10.1016/j.cell.2005.09.038. View

Mahmoudi S, Henriksson S, Corcoran M, Mendez-Vidal C, Wiman K, Farnebo M . Wrap53, a natural p53 antisense transcript required for p53 induction upon DNA damage. Mol Cell. 2009; 33(4):462-71. DOI: 10.1016/j.molcel.2009.01.028. View

Medrek K, Magnowski P, Masojc B, Chudecka-Glaz A, Torbe B, Menkiszak J . Association of common WRAP 53 variant with ovarian cancer risk in the Polish population. Mol Biol Rep. 2012; 40(3):2145-7. PMC: 3563948. DOI: 10.1007/s11033-012-2273-9. View

Stirnimann C, Petsalaki E, Russell R, Muller C . WD40 proteins propel cellular networks. Trends Biochem Sci. 2010; 35(10):565-74. DOI: 10.1016/j.tibs.2010.04.003. View

Henriksson S, Rassoolzadeh H, Hedstrom E, Coucoravas C, Julner A, Goldstein M . The scaffold protein WRAP53β orchestrates the ubiquitin response critical for DNA double-strand break repair. Genes Dev. 2014; 28(24):2726-38. PMC: 4265676. DOI: 10.1101/gad.246546.114. View

Tycowski K, Shu M, Kukoyi A, Steitz J . A conserved WD40 protein binds the Cajal body localization signal of scaRNP particles. Mol Cell. 2009; 34(1):47-57. PMC: 2700737. DOI: 10.1016/j.molcel.2009.02.020. View

10.

Kolas N, Chapman J, Nakada S, Ylanko J, Chahwan R, Sweeney F . Orchestration of the DNA-damage response by the RNF8 ubiquitin ligase. Science. 2007; 318(5856):1637-40. PMC: 2430610. DOI: 10.1126/science.1150034. View

11.

Polo S, Jackson S . Dynamics of DNA damage response proteins at DNA breaks: a focus on protein modifications. Genes Dev. 2011; 25(5):409-33. PMC: 3049283. DOI: 10.1101/gad.2021311. View

12.

Falck J, Coates J, Jackson S . Conserved modes of recruitment of ATM, ATR and DNA-PKcs to sites of DNA damage. Nature. 2005; 434(7033):605-11. DOI: 10.1038/nature03442. View

13.

Zhong F, Savage S, Shkreli M, Giri N, Jessop L, Myers T . Disruption of telomerase trafficking by TCAB1 mutation causes dyskeratosis congenita. Genes Dev. 2011; 25(1):11-6. PMC: 3012932. DOI: 10.1101/gad.2006411. View

14.

Mahmoudi S, Henriksson S, Weibrecht I, Smith S, Soderberg O, Stromblad S . WRAP53 is essential for Cajal body formation and for targeting the survival of motor neuron complex to Cajal bodies. PLoS Biol. 2010; 8(11):e1000521. PMC: 2970535. DOI: 10.1371/journal.pbio.1000521. View

15.

Mailand N, Bekker-Jensen S, Faustrup H, Melander F, Bartek J, Lukas C . RNF8 ubiquitylates histones at DNA double-strand breaks and promotes assembly of repair proteins. Cell. 2007; 131(5):887-900. DOI: 10.1016/j.cell.2007.09.040. View

16.

Schildkraut J, Goode E, Clyde M, Iversen E, Moorman P, Berchuck A . Single nucleotide polymorphisms in the TP53 region and susceptibility to invasive epithelial ovarian cancer. Cancer Res. 2009; 69(6):2349-57. PMC: 2666150. DOI: 10.1158/0008-5472.CAN-08-2902. View

17.

Huen M, Grant R, Manke I, Minn K, Yu X, Yaffe M . RNF8 transduces the DNA-damage signal via histone ubiquitylation and checkpoint protein assembly. Cell. 2007; 131(5):901-14. PMC: 2149842. DOI: 10.1016/j.cell.2007.09.041. View

18.

Garvin S, Tiefenbock K, Farnebo L, Thunell L, Farnebo M, Roberg K . Nuclear expression of WRAP53β is associated with a positive response to radiotherapy and improved overall survival in patients with head and neck squamous cell carcinoma. Oral Oncol. 2014; 51(1):24-30. DOI: 10.1016/j.oraloncology.2014.10.003. View

19.

Venteicher A, Abreu E, Meng Z, McCann K, Terns R, Veenstra T . A human telomerase holoenzyme protein required for Cajal body localization and telomere synthesis. Science. 2009; 323(5914):644-8. PMC: 2728071. DOI: 10.1126/science.1165357. View

20.

Xu C, Min J . Structure and function of WD40 domain proteins. Protein Cell. 2011; 2(3):202-14. PMC: 4875305. DOI: 10.1007/s13238-011-1018-1. View