Ku70/Ku80 and DNA-dependent Protein Kinase Catalytic Subunit Modulate RAG-mediated Cleavage: IMPLICATIONS FOR THE ENFORCEMENT OF THE 12/23 RULE

Dennis J Sawchuk,Jorge Mansilla-Soto,Claudio Alarcon,Netai C Singha,Hanno Langen,Marco E Bianchi,Susan P Lees-Miller,Michel C Nussenzweig,Patricia Cortes

doi:10.1074/jbc.m403706200

Abstract

The 12/23 rule is a critical step for regulation of V(D)J recombination. To date, only the RAG proteins and high mobility group protein 1 or 2 have been implicated in 12/23 regulation. Through protein fractionation and biochemical experiments, we find that Ku70/Ku80 and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) modulate RAG-mediated cleavage. Modulation of cleavage by Ku70/80 and DNA-PKcs results in preferential inhibition of 12/12 and 23/23 DNA cleavage, thus increasing 12/23 rule specificity. This observation indicates that DNA repair factors, Ku70/80 and DNA-PKcs, might be present upstream of the DNA cleavage events and not recruited downstream as is currently thought, assigning new nonrepair functions to the DNA-dependent protein kinase.

Highlights

The 12/23 rule is a critical step for regulation of V(D)J recombination
Through protein fractionation and biochemical experiments, we find that Ku70/Ku80 and DNAdependent protein kinase catalytic subunit (DNA-PKcs) modulate RAG-mediated cleavage
Identification of a 12/23 Rule Enforcing Activity—Our previous work demonstrated that RAG1 and RAG2 complemented with 293T whole cell extract (WCE) show strict 12/23 regulation in vitro [19]

Summary

EXPERIMENTAL PROCEDURES

The mammalian expression vectors for GST-core RAG1 and GSTcore RAG2 have been described [34]. Plasmids pEF-FT-RAG1 and pEFHT-RAG2 encode thioredoxin (Trx) fusions to core RAG1 380 –1040) or core RAG2 (residues 1–383), tagged at the N terminus with a FLAG (RAG1) or an HA (RAG2) epitope. Subcloning details can be obtained upon request. Recombination substrates pJH290 12/23 [35], 12/12, and 23/23 [19] were PCR-amplified using RA2 and RA14 primers to yield a 560-bp amplicon containing the RSSs. The PCR fragment was cloned into PCR2.1 vector using TA cloning (Invitrogen). The subcloned deletion substrates are identified as pDS12/23, pDS12/12, and pDS23/23

Purification of Recombinant Proteins

Preparation of DNA Cleavage Substrate

Cleavage Assay

Western Blotting

RESULTS

DISCUSSION