RAG2’s Acidic Hinge Restricts Repair-Pathway Choice and Promotes Genomic Stability

Abstract

SUMMARY V(D)J recombination-associated DNA double-strand breaks (DSBs) are normally repaired by the high fidelity, classical non-homologous end joining (cNHEJ) machinery. Previous studies implicated the RAG/DNA post-cleavage complex (PCC) in regulating pathway choice by preventing access to inappropriate repair mechanisms such as homologous recombination (HR) and alternative NHEJ (aNHEJ). Here we report that RAG2’s “acidic hinge”, previously of unknown function, is critical for several key steps. Mutations that reduce the hinge’s negative charge destabilize the PCC, disrupt pathway choice, permit repair of RAG-mediated DSBs by the translocation-prone aNHEJ machinery, and reduce genomic stability in developing lymphocytes. Structural predictions and experimental results support our hypothesis that reduced flexibility of the hinge underlies these outcomes. Furthermore, sequence variants present in the human population reduce the hinge’s negative charge, permit aNHEJ, and diminish genomic integrity.

Restoration of ATM Expression in DNA-PKcs–Deficient Cells Inhibits Signal End Joining

“…The mechanism by which RAG-induced DSBs are restricted to the c-NHEJ pathway is poorly understood. Since certain RAG2 mutants defined by Roth and colleagues are deficient in this process, it has been suggested that the RAG endonuclease interacts with other factor(s) that facilitate restriction of RAG-induced DSBs to the c-NHEJ pathway (34, 65, 66). We considered that the difference between DNA-PKcs-deficient cells that are proficient versus deficient in signal end joining is not because the deficient cells lack a joining factor, but because the proficient cells lack a factor that restricts RAG-induced breaks to c-NHEJ.…”

An Autoregulatory Mechanism Imposes Allosteric Control on the V(D)J Recombinase by Histone H3 Methylation

“…Neutralization of charge in this interval is associated with aberrant repair of RAG-mediated DNA breaks, decreased stability of RAG-signal end complexes (SECs) and genomic instability (Coussens et al, 2013). While these effects appear to reflect events occurring after RSS recognition and DNA cleavage, they may be explained in part by our results.…”

Alternative end-joining pathway(s): Bricolage at DNA breaks

“…Furthermore, RAG mutants have been described that enabled A-EJ to access and rejoin RAG-dependent DSBs, even in C-NHEJ proficient cells albeit to a lesser extent [140][141][142], resulting in increased translocation frequency and high incidence of lymphomas in a p53-null murine genetic background [143]. A-EJ restriction is dependent on a particular RAG2 subdomain [144]. Consequently, under normal conditions, the role of A-EJ in the V(D)J recombination is reduced to the smallest share since it is highly unlikely that the A-EJ machinery has a chance to encounter and deal with RAG-induced DSBs.…”