V(D)J Recombination Frequencies Can Be Profoundly Affected by Changes in the Spacer Sequence

Abstract

Each V, D, and J gene segment is flanked by a recombination signal sequence (RSS), composed of a conserved heptamer and nonamer separated by a 12- or 23-bp spacer. Variations from consensus in the heptamer or nonamer at specific positions can dramatically affect recombination frequency, but until recently, it had been generally held that only the length of the spacer, but not its sequence, affects the efficacy of V(D)J recombination. In this study, we show several examples in which the spacer sequence can significantly affect recombination frequencies. We show that the difference in spacer sequence alone of two VHS107 genes affects recombination frequency in recombination substrates to a similar extent as the bias observed in vivo. We show that individual positions in the spacer can affect recombination frequency, and those positions can often be predicted by their frequency in a database of RSS. Importantly, we further show that a spacer sequence that has an infrequently observed nucleotide at each position is essentially unable to support recombination in an extrachromosmal substrate assay, despite being flanked by a consensus heptamer and nonamer. This infrequent spacer sequence RSS shows only a 2-fold reduction of binding of RAG proteins, but the in vitro cleavage of this RSS is ∼9-fold reduced compared with a good RSS. These data demonstrate that the spacer sequence should be considered to play an important role in the recombination efficacy of an RSS, and that the effect of the spacer occurs primarily subsequent to RAG binding.

Single-Stranded DNA Structure and Positional Context of the Target Cytidine Determine the Enzymatic Efficiency of AID

“…In support of this rationale, we and others have shown that other properties of AID, such as WRC specificity, when measured in vitro, intimately match the pattern of hot-spot mutations in vivo (24,25,41). As another well-studied example of immunological relevance, the pattern of RAG1/2 cleavage and binding to different immunoglobulin recombination signal sequences in vitro correlates with the usage of gene segments in vivo (23,26,35,60).…”

The Extent of Histone Acetylation Correlates with the Differential Rearrangement Frequency of Individual VH Genes in Pro-B Cells

“…We had already shown that the three functional members of the V H S107 family have very different rearrangement frequencies in vivo, with V1 rearranging five times more than V11, and V13 rearranging 40 times less frequently than V1 (22). Although their RSS share the same heptamer and nonamer (28), we have previously determined that the difference in recombination frequency between V1 and V11 could partially be due to differences in the spacer sequence (29). However, other factors in addition to RSS efficiency must affect rearrangement frequency because the closely related V11 and V13 genes rearrange at very different frequencies, and their RSS are identical, including the spacer (22,29).…”

The B12/23 Restriction Is Critically Dependent on Recombination Signal Nonamer and Spacer Sequences

“…Since the acceptance of this manuscript, three papers (38)(39)(40) with findings relevant to spacer and nonamer sequence function have been published. a The V␤14 23RS and the D␤1 12RS were fixed at P1 and P2, respectively.…”