Error-prone DNA repair activity during somatic hypermutation in shark B lymphocytes

Abstract

Sharks are representatives of the earliest vertebrates that possess an immune system utilizing V(D)J recombination to generate Ag receptors. Their Ab repertoire diversity is based in part on a somatic hypermutation process that introduces adjacent nucleotide substitutions of 2-5 bp. We have isolated mutant nonfunctional Ig rearrangements and intronic flank sequences to characterize the nonselected, intrinsic properties of this phenomenon; changes unique to shark were observed. Duplications and deletions were associated with N additions, suggesting participation of a DNA polymerase with some degree of template independence during the repair of DNA breaks initiated by activation-induced cytidine deaminase. Other mutations were consistent with some in vitro activities of mammalian translesion DNA polymerase η: tandem base substitutions, strand slippage, and small insertions/deletions. The nature of substitution patterns shows that DNA lesions at shark Ig genes recruit DNA repair factors with a species-specific repertoire of activities. We speculate that the tandem mutations are introduced by direct sequential misinsertions and that, in shark B cells, the mispairs tend to be extended rather than proofread. Despite extensive changes undergone by some mutants, the physical range of mutational activity remained restricted to VDJ and within the first 2-kb portion of the 6.8-kb J-C intron, perhaps a self-regulating aspect of activation-induced cytidine deaminase action that is conserved in evolution.

Figure 1

Organization of the Ig L and H chain genes used in this study. Elasmobranch IgL and IgH genes are organized as miniloci that rearrange independently. The NS5-2 L chains and the G2A and G2B H chains are encoded by single copy genes. Top, IgL NS5-2 gene. There are four L chain isotypes in the nurse shark: kappa, lambda, sigma, NS5. Of the two functional, rearranging genes of the NS5 L chain isotype, one is preferentially amplified in these studies [22]. Leader, V and J gene segments, and C exon of L chain gene represented by filled blocks. Recombination signal sequences (RSS) with 12 bp spacer shown as black triangle, 23 bp spacer as white triangle. The distance from J to C is approximate, as the entire sequence could not be determined. Bottom, IgH G2A gene. There are 9-12 functional IgH genes, grouped into subfamilies: G1, G2 (A, B), G3, G4 (A-G, variable number of members), and G5 [21]. Every animal so far examined carries G2A and G2B as single-copy genes [21, 23, 24]. Leader, V, D1, D2 and JH gene segments shown as gray blocks, the first Cmu exon as black block. The J-C intron varies 6-10 kb among IgH genes. Relative distances between V gene segments according to the scale. Forward and reverse PCR primers (arrows) are described in Methods.

Figure 2

Frequency of mutation distributed over rearranged VDJ and 3′ flanking sequence. Fifty-six sequences were analyzed, of which 18 were mutants. Mutations in the 16 G2A sequences were tallied, excluding CDR3, and their distribution over the V region and downstream flank calculated as the number of substitutions in 50 bp segments divided by 50, divided by the number of sequences [28]. A diagram of the H chain gene is shown beneath the graph and the asterisk indicates the position of CDR3, which has been omitted. A second graph underneath depicts the number of insertions and deletions per 50 bp segment in increments of 1. The changes are mostly represented by nonfunctional sequences (13/16) which also contained more indels.

Figure 3

The boundaries of mutation in the shark H chain gene. The fully rearranged VDDJ is shown, together with a scale marking distance from the JH gene segment to the first C exon (6.8 kb). Genomic G2A rearrangements cloned with 5357 bp (C9, A2, A3) or 6848 bp (s series) of 3′ flanking sequence were scored for total substitutions (left column). The extent of the 5′-most and 3′-most tandem changes is depicted as a bracket for each clone. The lighter bracket in C9 includes an isolated group of seven mutations distributed in the 3′-most 400 bp. Some of the mutants contained extensive deletion, and the true distance from the JH border is indicated in parentheses if there is a discrepancy of >100 bp. For example, the 3′-most doublet in A3 is at position 3496 bp according to the J-C intron reference sequence, but it is located 1995 bp from its JH border. Asterisks mark the related clones A2 and s122; the hypothetical ancestral mutant “A2/122” is discussed in the text. The error for the Expand Long Template enzyme mix (Roche) is 0.11% (4 substitutions/3696 bp) as scored in nonrearranged clones.

Figure 4

Comparison of related IgH mutants. The reference germline (GL) gene G2A is shown without intergenic sequence in VH, D1, D2 and JH gene segments (labeled in boldface, gaps indicated by slashes). The start of the VH gene segment is indicated over the splice acceptor site and is the divided into framework (FR) and complementarity-determining (CDR) regions. The beginning of the J-C intron is indicated over the splice donor site. Clones A2 and s122 are aligned with respect to the GL and the PCR primers are italicized. Differences from the GL are highlighted in blue and those shared between the mutants are highlighted in purple. Changes in CDR3 are not marked. Although the rearrangement is in-frame, the less mutated A2 CDR3 contains two stops. The VDJ was probably never functional, and its first mutations (in CDR1, AT to GA generated TGA) were deleterious. Dashes indicate gaps. Duplicated sequences are highlighted and their template underlined. Lower case n is placed where point deletion was introduced during PCR slippage. Asterisks show sites of inserted nt, crosshatches deletions. The numbering of the reference sequence includes leader intron and coding VH gene segments (1-474) and 3′ flank (475-2289).

Figure 5

Insertions and deletions in Ig sequence. A. Portion of the J-C intron from clone G20-88 containing 3 different insertion events. B-K. Ten L chain sequences with indels from experiments shown in Table I. B. JNS5-38, nonproductive, insertion in CDR1. C. JNS5-39, nonproductive, insertion in JL. The letter “i” over T indicates insertion in inversely duplicated sequence D. JNS5-46, nonproductive, deletion in FR3. E. JNS5-48, nonproductive, deletion in CDR1. F. JNS5-61, nonproductive, deletion in CDR1; deletion and incomplete duplication in FR3. G. JNS5-80, nonproductive, deletion in CDR3. H. JNS5-83, in-frame, insertion in CDR3. I. SPH-156, in-frame but lacking structural TTC in JL, duplication in FR3; insertion in CDR3. The distance between insert and upstream template is 46 bp. J. SPH2-2, nonproductive, insertion in CDR2. K. SPH4, nonproductive, duplication in CDR2; insertion in FR3. The sequences are compared with the J-C intron or NS5 reference sequence, identities indicated by vertical bars. Mutations are shown in lower case. Duplicated nt are shaded and the template sequence is underlined. In those instances that could involve T nucleotides, both insert and flank template are marked with arrows indicating their relative orientation.

Figure 6

Changes in A/T-rich areas in the J-C intron. Top. Clones with substitutions in two regions of the J-C intron of the G2A gene are shown, with numbering according to the reference sequence in Fig. 4. G/C nucleotides are highlighted in yellow and mutations in blue. An inserted duplication (AAACA) is shown for clone A2 at position 640. Dashes indicate deleted sequence. Lower case n is placed where point deletion was introduced during PCR slippage. Bottom. Hypothetical pathway by which CT at position 887-888 in clone G20-88 are mutated to TC through transient misalignment during gap repair. Step 1: Dissociation of DNA polymerase causes fraying, followed by Step 2: strand misalignment. Step 3: Distributive DNA synthesis. Polymerase dissociates, with attendant fraying. Step 4: Primer and template realign. Step 5: Extension of primer stabilizes mismatches. Tandem mutations CT to TC established.