Absence of DNA polymerase eta reveals targeting of C mutations on the nontranscribed strand in immunoglobulin switch regions - PubMed (original) (raw)
Absence of DNA polymerase eta reveals targeting of C mutations on the nontranscribed strand in immunoglobulin switch regions
Xianmin Zeng et al. J Exp Med. 2004.
Abstract
Activation-induced cytosine deaminase preferentially deaminates C in DNA on the nontranscribed strand in vitro, which theoretically should produce a large increase in mutations of C during hypermutation of immunoglobulin genes. However, a bias for C mutations has not been observed among the mutations in variable genes. Therefore, we examined mutations in the mu and gamma switch regions, which can form stable secondary structures, to look for C mutations. To further simplify the pattern, mutations were studied in the absence of DNA polymerase (pol) eta, which may produce substitutions of nucleotides downstream of C. DNA from lymphocytes of patients with xeroderma pigmentosum variant (XP-V) disease, whose polymerase eta is defective, had the same frequency of switching to all four gamma isotypes and hypermutation in mu-gamma switch sites (0.5% mutations per basepair) as control subjects. There were fewer mutations of A and T bases in the XP-V clones, similar to variable gene mutations from these patients, which confirms that polymerase eta produces substitutions opposite A and T. Most importantly, the absence of polymerase eta revealed an increase in C mutations on the nontranscribed strand. This data shows for the first time that C is preferentially mutated in vivo and pol eta generates hypermutation in the mu and gamma switch regions.
Figures
Figure 1.
Mutations are distributed throughout the μ-γ switch regions at a high frequency. The top schematic drawing shows the recombined μ (solid) and γ (striped) switch regions with an arrow indicating the breakpoint. On the abscissa, mutations are plotted on either side of the breakpoint for XP-V and control clones. On the ordinate, the frequency of mutation per 10 nucleotide increments was calculated as the number of mutations in the increment, divided by 10, divided by the number of times the increment was sequenced, and multiplied by 100.
Figure 2.
Mutations can occur sequentially in clonal progeny before and after switching. Clonal daughters were identified by identical breakpoints and unique mutations are depicted by arrows. (A) Before switching in the μ region, two clones (control 1, 2–21 and 93) with identical mutations in μ were joined to γ3 and γ1. (B) During switching, two unique clones (control 1: 2–24 and 4-B4) had nucleotide insertions at the site of joining. (C) After switching, three examples are shown. In the μ region, two clones (control 2: 11A9 and 11G9) with identical γ3 junctions had different mutations in μ. Also in the μ region, three clones (XP7BR: 12D12, 65, and 67) with identical γ1 regions had different mutations in μ. In the μ and γ regions, two clones (XP11BR: 5A1 and 5A2) with the same junction had different mutations in μ and γ. Sequences of these clones are shown in Figs. S1, S3, and S4.
Figure 3.
Pol η mutates A and T nucleotides in μ-γ switch regions. (A) Substitutions in clones from three XP-V and three control subjects show differences in spectra. Data have been corrected for nucleotide composition of the Sμ, Sγ1, Sγ2, Sγ3, and Sγ4 regions that were sequenced. (B) Substitutions from each individual are grouped as A:T and G:C pairs.
Figure 4.
Location of substitutions in the μ switch region. The 130-base region shown is from HSIGHMUS X54713 (nucleotides 142–271). Mutations from XP-V and control clones are shown above and below the germline sequence, respectively. Every 10th base is underlined.
Figure 5.
C bases in the μ switch region are preferentially mutated in pol η–deficient clones. (A) Substitutions were corrected for nucleotide composition of each clone. (B) Total mutations of each nucleotide are shown for XP-V and control clones, with standard errors for individual variation.
Figure 6.
Model of mutations introduced during repair and replication of deaminated cytosine in switch regions. AID deaminates C on the nontranscribed strand to uracil (U). U is removed by uracil glycosylase to produce an abasic site. (A) During gap-filling repair, pol η synthesizes a short distance (thick arrow). Mutations opposite T and A are introduced on the nontranscribed strand. (B) During replication, a translesion polymerase such as Rev1 inserts C opposite the abasic site as it synthesizes the transcribed strand (thick arrow). This would produce transversions of C to G as recorded from the nontranscribed strand.
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