DNA-Dependent Protein Kinase Inhibits AID-Induced Antibody Gene Conversion

Abstract

Affinity maturation and class switching of antibodies requires activation-induced cytidine deaminase (AID)-dependent hypermutation of Ig V(D)J rearrangements and Ig S regions, respectively, in activated B cells. AID deaminates deoxycytidine bases in Ig genes, converting them into deoxyuridines. In V(D)J regions, subsequent excision of the deaminated bases by uracil-DNA glycosylase, or by mismatch repair, leads to further point mutation or gene conversion, depending on the species. In Ig S regions, nicking at the abasic sites produced by AID and uracil-DNA glycosylases results in staggered double-strand breaks, whose repair by nonhomologous end joining mediates Ig class switching. We have tested whether nonhomologous end joining also plays a role in V(D)J hypermutation using chicken DT40 cells deficient for Ku70 or the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Inactivation of the Ku70 or DNA-PKcs genes in DT40 cells elevated the rate of AID-induced gene conversion as much as 5-fold. Furthermore, DNA-PKcs-deficiency appeared to reduce point mutation. The data provide strong evidence that double-strand DNA ends capable of recruiting the DNA-dependent protein kinase complex are important intermediates in Ig V gene conversion.

GANP Regulates the Choice of DNA Repair Pathway by DNA-PKcs Interaction in AID-Dependent IgV Region Diversification

“…This is further confirmed by the fact that AID attacks C on both strands simultaneously (67). AID-initiated DSBs are the intermediates in IgV L SHM and GCV (12,19,26,53), and HR and NHEJ are the two conserved main pathways evolved to deal with DNA breaks, from yeast to vertebrates. HR plays a dominant role in DSB repair in yeast, whereas NHEJ is the predominant repair mechanism in vertebrates, including birds.…”

“…DNA-PKcs suppresses both spontaneous and DSB-induced HR at the initial step of DNA repair (56). This was clearly demonstrated by its suppression of GCV in DT40 cells (19,53). The promotion of GCV/HR by GANP (Fig.…”

“…Although DT40 cells are commonly used for their very high rate of gene targeting through HR, NHEJ remains a predominant repair mechanism in DT40 cells. NHEJ repair poses significant competition or hindrance for GCV/HR, both at the level of GCV and IgV region diversification (19,21,53), as well as at the level of general HR in DSB repair (52). From our data, abrogating NHEJ in Ku70 2/2 , a core NHEJ molecule, greatly augments GCV/HR compared with DT40 CL18 GFP (Fig.…”

“…Many factors, including DNA-PKcs, can regulate the mechanistic overlap or competition between NHEJ and HR (16,(53)(54)(55)68). DNA-PKcs suppresses both spontaneous and DSB-induced HR at the initial step of DNA repair (56).…”

Checkpoint kinase 2 is required for efficient immunoglobulin diversification

“…However, this may not provide a satisfactory explanation for the decreased somatic hypermutation activity, as NHEJ defects should not affect most of the mutagenic processes involved here, which do not involve double strand breaks, 48 even though a potential role for DNA-PKcs in promoting mutagenesis has been suggested. 49 As a potential alternative and complementary explanation for increased homologous recombination activity in Chk2-deficient cells, we considered upregulation of the Chk1 axis via crossregulation of the 2 checkpoint signaling pathways. Indeed, Chk2 inhibition caused an increased activatory Chk1 phosphorylation in the human cell lines used for hypermutation analyses ( Fig.…”