Activation-Induced Cytidine Deaminase-Dependent DNA Breaks in Class Switch Recombination Occur during G1 Phase of the Cell Cycle and Depend upon Mismatch Repair

Carol E Schrader,Janet Stavnezer,Jeroen E J Guikema,Erin K Linehan,Erik Selsing

doi:10.4049/jimmunol.179.9.6064

Abstract

Ab class switching occurs by an intrachromosomal recombination and requires generation of double-strand breaks (DSBs) in Ig switch (S) regions. Activation-induced cytidine deaminase (AID) converts cytosines in S regions to uracils, which are excised by uracil DNA glycosylase (UNG). Repair of the resulting abasic sites would yield single-strand breaks (SSBs), but how these SSBs are converted to DSBs is unclear. In mouse splenic B cells, we find that AID-dependent DSBs occur in Smu mainly in the G(1) phase of the cell cycle, indicating they are not created by replication across SSBs. Also, G(1) phase cells express AID, UNG, and mismatch repair (MMR) proteins and possess UNG activity. We find fewer S region DSBs in MMR-deficient B cells than in wild-type B cells, and still fewer in MMR-deficient/SmuTR(-/-) B cells, where targets for AID are sparse. These DSBs occur predominantly at AID targets. We also show that nucleotide excision repair does not contribute to class switching. Our data support the hypothesis that MMR is required to convert SSBs into DSBs when SSBs on opposite strands are too distal to form DSBs spontaneously.

Highlights

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In mouse splenic B cells, we find that Activation-induced cytidine deaminase (AID)-dependent double-strand breaks (DSBs) occur in S␮ mainly in the G1 phase of the cell cycle, indicating they are not created by replication across single-strand breaks (SSBs)
The results from our studies support the hypothesis that distal SSBs are converted into DSBs by mismatch repair (MMR) recognition and repair of dU:dG mismatches introduced by AID

Summary

Abbreviations used in this paper

CSR, class switch recombination; DSB, doublestrand break; AID, activation-induced cytidine deaminase; TR, tandem repeat; UNG, uracil DNA glycosylase; APE, AP endonuclease; SSB, single-strand break; NER, nucleotide excision repair; MMR, mismatch repair; WT, wild type; LM-PCR, ligation-mediated PCR; XPF, Xeroderma pigmentosum F. Because MMR is required for recombination outside the S␮TR region where AID hot spots are relatively infrequent, we have proposed that MMR is needed to convert SSBs into DSBs when the single-strand nicks are too far apart to form spontaneous DSBs [29]. This hypothesis is consistent with the finding that CSR does not require MMR, but is reduced 2to 5-fold in MMR-deficient B cells, i.e., at least 50% of CSR events require MMR, depending on the isotype (27, 30 –34). The data support the hypothesis that distal SSBs are converted by MMR into DSBs, constituting an important step in CSR

Materials and Methods

Results

Discussion