Skip to Content
Merck
  • The proximity ligation assay reveals that at DNA double-strand breaks WRAP53β associates with γH2AX and controls interactions between RNF8 and MDC1.

The proximity ligation assay reveals that at DNA double-strand breaks WRAP53β associates with γH2AX and controls interactions between RNF8 and MDC1.

Nucleus (Austin, Tex.) (2016-01-07)
Hanif Rassoolzadeh, Christos Coucoravas, Marianne Farnebo
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.

MATERIALS
Product Number
Brand
Product Description

Supelco
1,2-Dichloropropane, analytical standard
Pricing and availability is not currently available.
Sigma-Aldrich
Monoclonal Anti-β-Actin antibody produced in mouse, clone AC-15, ascites fluid
Pricing and availability is not currently available.
Sigma-Aldrich
Anti-phospho-Histone H2A.X (Ser139) Antibody, clone JBW301, clone JBW301, Upstate®, from mouse
Pricing and availability is not currently available.
Sigma-Aldrich
Anti-RNF168 Antibody, from rabbit, purified by affinity chromatography
Pricing and availability is not currently available.