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.

Figure 1. Surface Ig Reversion of DT40 Clones

Accumulation (top) of sIg^+ve cells in sIg^−ve DT40 clones, or (bottom) of sIg^−ve cells in sIg^+ve DT40 clones after (A) 50 d of culture, or (B) 24 d of culture in two independent experiments (A or B) is depicted. The protein missing from each cell line is indicated. Control cells were (top) sIg^−ve DT40-CL18 cells, or (bottom) sIg^+ve DT40-CL18 cells. Each circle represents the frequency of sIg-reverted cells (on a log scale) detected in each clone by FACS after the culture period. The bar indicates the median reversion frequency. The numbers above each dataset give the number of clones analyzed. Asterisks indicate significant difference of the median reversion frequency from the control population: * p < 0.05, *** p < 0.001. Note: lack of surface Ig-expression in the XRCC3^−/− founder clones was due to Ig V(D)J point mutations and not due to the canonical CL18 frame shift carried by the control, Ku70^−/−, and DNA-PKcs^−/−/− clones (unpublished data). Thus, the reduced rate of Ig V gene conversion known to exist in these cells [43,45] did not significantly reduce their rate of surface Ig gain.

Figure 2. Gene Conversions Detected in CL18, DNA-PKcs^−/−/−, and Ku70^−/− Cell Lines

The consensus sequence for each clone is shown at the top and the mutated sequence carrying a gene conversion underneath. Dots indicate identity to the clone's starting (consensus) sequence and indicate the maximum extent of sequence that could have undergone gene conversion. The ΨV_λ gene with the longest stretch of identity to the mutated sequence is indicated as the gene conversion donor, with other possible donors listed in parentheses. (The identity in clone Cp14.1 with the “best” donor ΨV gene extends 5′ to that shown in the figure. Similarly, identity in clone Dp25.3 extends 3′ to that shown in the figure.) Colons indicate the insertion of a gap to maintain sequence alignment. Numbering indicates the sequence position of each gene conversion, with the translation start as base 1. Note: The same gene conversion with ΨV_λ occurred independently in two DNA-PKcs^−/−/−clones.

Figure 3. Models for Ig V Gene Conversion Based on Attack of Both DNA Strands by AID and Attack of One DNA Strand by AID

(A) Attack of both DNA strands by AID. In step 1, two complexes containing AID attack opposed strands at the same time. If both complexes directly recruited DNA-PKcs molecules, DNA-PKcs could dimerize and perhaps inhibit base excision by UNG. In step 2, excision by UNG and a lyase or exonuclease creates a staggered DSB. Step 3 shows that gene conversion requires the production of 3′-protruding ends, which may involve a 5′–3′ exonuclease, depending on the relative placement of the nicks. The 3′-protruding ends initiate gene conversion by invading a homologous ΨV gene. This step could be inhibited by the binding of dsDNA ends to the DNA-PK complex. In steps 4–6, nonhomologous (“mutated”) sequences are copied from the ΨV gene by mismatched end trimming, primer extension, template switch, further end trimming, and ligation. (B) Attack of one DNA strand by AID. In step 1, AID deamination in G1-phase may be ignored until S-phase, based on the model in [11]. In S-phase, shown in step 2, the dU base produced by AID is encountered by a replication fork, enabling access by UNG, which then creates an abasic site. Step 3 shows the abasic site is excised to create a dsDNA end, which can recruit the DNA-PK complex. In step 4, if DNA-PK is not recruited, the dsDNA end promotes gene conversion with an upstream ΨV gene. (C) Attack of one DNA strand by AID. As in (B), AID deamination is ignored until S-phase. In step 2, as in step 2 of (B), the dU base is encountered by a replication fork, but in this case lagging strand nicks (Okazaki fragments) are still present in the upstream ΨV genes. Step 3 shows the abasic site generated by UNG stalls the replication fork, without inducing nicking, and promotes strand invasion into an upstream ΨV gene. Step 4 shows a dsDNA end is created when primer extension encounters a lagging strand nick due to Okazaki fragments in the template ΨV gene. The replication fork stalls again. If DNA-PK binds the dsDNA end it inhibits the completion of HDR. In step 5, a second reconfiguration of the stalled replication fork occurs, and in step 6, resolution of the double cross-over completes gene conversion.