Defining the nucleic acid substrate for somatic hypermutation

Abstract

Recent reports have more precisely defined the distribution of somatic mutations around rearranged mouse V-D-J genes. The 5' boundary of mutation is most likely in the region of the transcription start site (cap) and/or the promoter (P), implying that transcription may be a prerequisite for mutations to be generated. As more than 95% of somatic mutations lie downstream of the cap site, the transcription unit itself is implicated as the target of the mutational machinery. For heavy chain genes, the 3' boundary can extend into the enhancer region (E). For kappa light chain genes, the 3' boundary extends to approximately 700 bp beyond J kappa-5 (approximately 700 bp upstream of E). In a single study on mutated derivatives of the rearranged mouse lambda 1 light chain V-J gene, it was claimed that the 3' boundary fell within the constant region (C) exon. Although more data are required, the frequency of mutations around V-D-J genes appears asymmetrical, being positively skewed with a single mode centred near the V-D-J coding region and a long tail extending into the 3' non-translated region of the J-C intron. Such a distribution may place constraints on possible molecular mechanisms. It is suggested that the apparent asymmetrical pattern of mutation is best explained by models that assume localized error-prone DNA synthesis generating variable fragment lengths of mutated DNA or cDNA retrotranscripts. The frequency distribution of these lengths of mutated DNA is positively skewed into the 3' J-C intron, with a common terminus at or near the cap site. It is then assumed that they can be integrated into the target V-D-J region via a gene conversion or homologous recombination process. The model invoking reverse transcription may be preferred as it best explains the data without too many ad hoc assumptions.

The reverse transcriptase model of somatic hypermutation

“…Therefore V(D)J transgenes with a reporter tRNA in the J-C intron upstream of E i /MAR should not mutate in the V(D)J target region because cDNA synthesis will be prevented from proceeding through the tRNA sequence (¢gure 2a). The data published by Umar et al (1991) and Umar & Gearhart (1995) support this prediction (also see Steele et al (1992Steele et al ( , 1997 for further explanation and analyses of these data).…”

“…Alternatives suggested at the time were (i) premature termination of RNA synthesis; (ii) cleavage of pre-mRNA, coupled to hairpin loop RT priming; or (iii) speci¢c primer binding sites by analogy with tRNA primers in retroviral reverse transcription (Steele & Pollard 1987). Later, multiple RT-priming sites in the J-C intron were invoked (Steele et al 1992) to explain the asymmetrical distribution of somatic point mutations (Both et al 1990;Lebecque & Gearhart 1990;Weber et al 1991;Steele et al 1992;Rothen£uh et al 1993) (¢gure 1b). More recently, multiple priming sites and a number of alternative RT-priming mechanisms have also been advanced to attempt to explain both the asymmetry of the distribution and focusing to the particular V(D)J rearrangement undergoing hypermutation (discussed in Steele et al 1997; cf.…”

Somatic hypermutation in 5′ flanking regions of heavy chain antibody variable regions

“…A composite graph of a limited data set showing the distribution in mutation frequencies across a VDJH.~ gene suggested that the distribution of mutations around VDJ genes may be asymmetrical and that the mutation frequency 5' of the cap site was at least an order of magnitude lower than that 3' of the cap site (reviewed in [17]). An important outcome of this data review was that more sequences of 5' flanking regions was obviously required.…”

“…An earlier review of available data suggested that the distribution of somatic mutations around rearranged V regions may be positively skewed, with a single mode centred on the V(D)J gene and a long tail extending into the J-C intron [17], however it was pointed out that a larger data set was required for a more conclusive analysis. Therefore, to determine accurately the nature of the underlying frequen-cy distribution and 5' boundary for somatic hypermutation we have now sequenced an additional 12 somatically mutated, rearranged VH regions and their 5' flanking regions to a point approximately 520 bp upstream of the transcription start (cap) site.…”

The boundaries of the distribution of somatic hypermutation of rearranged immunoglobulin variable genes

“…The RT model still provides the best explanation for the gradual decline as explicitly stated by Steele, Rothenfluh and Both over a decade ago. 12 In this model, a family of errorfilled cDNA (produced by the RT activity of pol η ), initiated at various evenly spaced priming sites over the V(D)J and J-C intron of the pre-mRNA template, replace the original TS DNA in the populations of B cells assayed for SHM. Synthesis of cDNA on the pre-mRNA template should continue to the end of the template unless blocked by RNA structure orat the 5 ′ boundary.…”