Postcleavage sequence specificity in V(D)J recombination

Abstract

Unintended DNA rearrangements in a differentiating lymphocyte can have severe, oncogenic consequences, but the mechanisms for avoiding pathogenic outcomes in V(D)J recombination are not well understood. The first level at which fidelity is instituted is in discrimination by the recombination proteins between authentic and inauthentic recombination signal sequences. Nevertheless, this discrimination is not absolute and cannot fully eliminate targeting errors. To learn more about the basis of specificity during V(D)J recombination, we have investigated whether it is possible for the recombination machinery to detect an inaccurately targeted sequence subsequent to cleavage. These studies indicate that even postcleavage steps in V(D)J recombination are sequence specific and that noncanonical sequences will not efficiently support the resolution of recombination intermediates in vivo. Accordingly, interventions after a mistargeting event conceivably occur at a late stage in the joining process and the likelihood may well be crucial to enforcing fidelity during antigen receptor gene rearrangement.

FIG. 1

Configuration-specific differences in recombination outcome. RSS orientation dictates the type of junction that will be recovered after recombination. Three different outcomes, a signal joint via deletion, a signal and coding joint via inversion, and a coding joint via deletion via represent the recovered products resulting from the configurations shown in sections A, B, and C, respectively. Regardless of configuration, stage 1 operations involve cleavage at the heptamers of each RSS (shown as an open triangle for the 12-RSS and as a solid triangle for the 23-RSS). At the end of stage 1, as shown in the middle column (‘Cleaved intermediate’), two blunt signal ends and two hairpin-terminated coding ends are formed in every case. Subsequently, during stage 2, there are different operations required for resolution of the coding and/or signal ends. The identity of stage 1 operations and the divergence of stage 2 operations with different configurations forms the basis for distinguishing the postcleavage effects of RSS sequence in vivo.

FIG. 2

Recombination substrates. In all substrates, 12-RSSs occur at sites 1 or 2, and the canonical 23-RSS is inserted at site 3. The frequency with which a given test RSS positioned at site 1 recombines is tested relative to the frequency with which the invariant sequence at site 2 is rearranged. In each section of the figure, labeled bars represent the oligonucleotide probes that are used in the typing of recombinants (see Materials and Methods). (A) The SJΔ substrate and two possible recombinant deletion products, involving either the site 1 or site 2 RSS. (B) Inv substrate with the inversional recombinant resulting from site-1-to-site-3 rearrangement and site 2 deletion product. (C) The CJΔ substrate is shown with the deleted coding joint product. This plasmid is cotransfected with the site 2 control plasmid, which gives rise to the site 2 deletion product.

FIG. 3

Variant RSSs and configuration-specific RJP. The sequence of each tested RSS is given, indicating matches to the canonical RSS in uppercase letters. Spacer sequences were changed according to the dictates of a given experiment. The 12a spacer is an arbitrary sequence as given previously (25). The 12b spacer is identical to the spacer of the RSS appearing in site 2, and the 12c spacer is a continuation of the CA repeat. The total number of recombinants typed as either site 1 or site 2 rearrangements is given for each of the SJΔ and Inv constructs. For experiments where the site 2 control was introduced on a separate plasmid, the values shown are calculated as described in Materials and Methods (a sample calculation is given in Fig. 4). The RJP is defined as the ratio of site 1 to site 2 recombinants. The ratio of inversion to SJΔ provides a means by which to compare the stage 2 impact of a different RSS variants. Superscript numbers: 1, cotransfection of p6131-SJΔno2 and the site 2 control plasmid (see Fig. 2C); 2, cotransfection of p6131-Invno2 and the site 2 control plasmid; 3, cotransfection of p6131-Inv(506) (in which the distance between sites 1 and 3 was increased from 279 to 506 bp) and the site 2 control plasmid; and 4, cotransfection of p6131-CJΔ(546) (in which the distance between sites 1 and 3 was increased from 319 to 546 bp) and the site 2 control plasmid.

FIG. 4

Reproducibility of the measured RJP. (A) Nine transfections of p6131-SJΔ were conducted and screened. The top four transfections were performed 4 years earlier than the bottom five. These numbers correspond to the pooled data reported in Fig. 3. (B) Ten transfections of p6131-CJΔ cointroduced with the site 2 control plasmid were analyzed. Colonies selected on chloramphenicol were typed to determine the number of site 1 and site 2 recombinants as usual (columns A and B). Colonies isolated on ampicillin after DpnI digestion of the harvested DNA were typed to determine the numbers of each parental plasmid successfully transfected as described in Materials and Methods (columns C and D). Colony counts were normalized as shown (two subsequent columns) to give the RJP (rightmost column).

FIG. 5

Coding joints. (A) Sequences of coding joints from randomly selected inversions (Inv) and deletions (CJΔ). Underlined nucleotides can be assigned to either end. (B) Variant target recognition of a CA repeat. As seen in analysis of coding joints in panel A, as well as of signal joints (not shown), a CA repeat is recognized in alternative frames displaced by 2 bp. Heptamer and nonamer elements are in boldface, and capital letters indicate matches to the canonical sequence.