MSH2-MSH6 stimulates DNA polymerase eta, suggesting a role for A:T mutations in antibody genes - PubMed (original) (raw)

MSH2-MSH6 stimulates DNA polymerase eta, suggesting a role for A:T mutations in antibody genes

Teresa M Wilson et al. J Exp Med. 2005.

Abstract

Activation-induced cytidine deaminase deaminates cytosine to uracil (dU) in DNA, which leads to mutations at C:G basepairs in immunoglobulin genes during somatic hypermutation. The mechanism that generates mutations at A:T basepairs, however, remains unclear. It appears to require the MSH2-MSH6 mismatch repair heterodimer and DNA polymerase (pol) eta, as mutations of A:T are decreased in mice and humans lacking these proteins. Here, we demonstrate that these proteins interact physically and functionally. First, we show that MSH2-MSH6 binds to a U:G mismatch but not to other DNA intermediates produced during base excision repair of dUs, including an abasic site and a deoxyribose phosphate group. Second, MSH2 binds to pol eta in solution, and endogenous MSH2 associates with the pol in cell extracts. Third, MSH2-MSH6 stimulates the catalytic activity of pol eta in vitro. These observations suggest that the interaction between MSH2-MSH6 and DNA pol eta stimulates synthesis of mutations at bases located downstream of the initial dU lesion, including A:T pairs.

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Figures

Figure 1.

Figure 1.

The MSH2–MSH6 heterodimer binds to a U:G mismatch. Increasing amounts of the MSH2–MSH6 complex (0–60 nM) were incubated with 1 fmol of the various labeled 50-mer DNA substrates, which are depicted at the top of the figure. Bound complexes were detected by a shift in the substrate after electrophoresis.

Figure 2.

Figure 2.

Pol η binds to MSH2–MSH3 and MSH2–MSH6 heteroduplexes by ELISA analysis. Wells were coated with MSH2–MSH3 (diamonds), MSH2–MSH6 (squares), or BSA (triangles) and incubated with increasing amounts of pol η. Bound protein was detected with anti–pol η antibody. Error bars were calculated as the standard deviation of three experiments.

Figure 3.

Figure 3.

Pol η interacts with MSH2 in vitro and in vivo. (A) Coomassie-stained gel. An SDS-PAGE gel shows the purity of the GST-tagged COOH-terminal pols before they were coupled to glutathione-Sepharose beads. (B) GST pull-down assay. 35S-labeled MSH2, MSH3, and MSH6 proteins were incubated with the pol-coupled beads and resolved on an SDS-PAGE gel. Input lanes represent 100% of the MSH protein added to the assay tube. (C) Immunoprecipitation. FLAG-tagged pol η was overexpressed in HeLa cells and immunoprecipitated (IP) with or without antibody to FLAG protein. Precipitates were solubilized and separated by electrophoresis. The gel was immunoblotted (IB) for Western analyses using an antibody to FLAG to detect pol η and an antibody to MSH2. Input lanes represent 30% of the cell extracts used in the assay.

Figure 4.

Figure 4.

Effect of mismatch repair proteins on DNA replication by pols η and ι. All reactions were performed for 10 min. (A) Dose–response experiments depicting pol η–catalyzed primer extension in the presence of MSH2–MSH3 or MSH2–MSH6 with AT as the first two template bases to be copied. (B) Dose–response experiments depicting pol ι–catalyzed primer extension in the presence of MSH2–MSH6 using the same template as in A. (C) Influence of template sequence context on the MSH2–MSH6-dependent stimulation of primer extension by pol η. TA, GT, and CT represent the first two bases to be copied.

Figure 5.

Figure 5.

MSH2–MSH6 dependent stimulation of primer extension by pol η using matched (C:G) and mismatched (T:G or U:G) termini. All reactions were performed for 10 min. The graphs indicate quantitation of the gels and plot primer elongation catalyzed by pol η alone (closed circles) or in the presence of MSH2–MSH6 (open circles) as a percentage of total primer termini.

Figure 6.

Figure 6.

Effect of MSH2–MSH6 on processivity and fidelity of pol η. The local template sequence is shown to the left of each set of gels. (A) Processivity of pol η was measured in the absence and presence of MSH2–MSH6 in two-min reactions containing all four nucleotides and herring sperm DNA as a trap for pol molecules that dissociate from the radiolabeled primer template. (B) Nucleotide incorporation specificity of pol η in the absence and presence of MSH2–MSH6 was measured in 10-min reactions containing 100 μM of each individual nucleotide.

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