DNA repair in antibody somatic hypermutation

Abstract

Somatic hypermutation (SHM) underlies the generation of a diverse repertoire of high-affinity antibodies. It is effected by a two-step process: (i) DNA lesions initiated by activation-induced cytidine deaminase (AID), and (ii) lesion repair by the combined intervention of DNA replication and repair factors that include mismatch repair (MMR) proteins and translesion DNA synthesis (TLS) polymerases. AID and TLS polymerases that are crucial to SHM, namely polymerase (pol) theta, pol zeta and pol eta, are induced in B cells by the stimuli that are required to trigger this process: B-cell receptor crosslinking and CD40 engagement by CD154. These polymerases, together with MMR proteins and other DNA replication and repair factors, could assemble to form a multimolecular complex ("mutasome") at the site of DNA lesions. Molecular interactions in the mutasome would result in a "polymerase switch", that is, the substitution of the high-fidelity replicative pol delta and pol epsilon with the TLS pol theta, pol eta, Rev1, pol zeta and, perhaps, pol iota, which are error-prone and crucially insert mismatches or mutations while repairing DNA lesions. Here, we place these concepts in the context of the existing in vivo and in vitro findings, and discuss an integrated mechanistic model of SHM.

Figure 1

BCR cross-linking and T-cell contact through CD40:CD154 engagement and CD86/CD80:CD28 co-engagement are required for the induction of SHM. CD40:CD154 upregulates AID expression (large red circles). BCR crosslinking upregulates the error-prone TLS pol θ (large pink ovals) and pol ζ catalytic subunit Rev3 (brown ovals), which, with other TLS polymerases, namely, pol η (dark blue ovals), Rev1 (light green circles) and, perhaps, pol ι (orange ovals), are recruited into the DNA repair process that results in the insertion of mutations. The small inset depicts mutation frequency in V(D)J and C regions. The large inset depicts the polymerase switch. Somatic mutations (red crosses) are introduced by TLS polymerase(s) during DNA synthesis, while bypassing an abasic site or while copying undamaged DNA in patch DNA re-synthesis of MMR or, perhaps, a mutagenic long-patch BER. Abasic site bypass requires the sequential action of two DNA polymerases: one, such as pol θ, pol η, Rev1 or, perhaps, pol ι, that inserts a nucleotide opposite the damaged template nucleotide (inserter), and the other, such as pol θ or pol ζ, that extends from the inserted nucleotide (extender). Pol η is highly inefficient at inserting a nucleotide opposite to an abasic site, but its recruitment by PCNA greatly stimulates the ability of this TLS polymerase to insert a nucleotide opposite this lesion. Pol θ is the first DNA polymerase known to bypass abasic sites efficiently by functioning as a mispair inserter and a mispair extender.

Figure 2

An integrated model of SHM. This assumes that AID deaminates dC in both DNA strands. dU is not relevant to DNA and the dU:dG mismatch is ‘replicated over’ or dealt with by the DNA repair machinery. Replicating over dU results in a dC→dT transition mutation (Phase 1a), whereas dU deglycosylation by Ung gives rise to an abasic site. In the presence of PCNA (orange ring), DNA synthesis opposite the abasic site by TLS pol θ, which has both nucleotide inserter and extender activity, or by the nucleotide inserter pol η, Rev1 or, perhaps, pol ι, followed by the nucleotide extender pol θ or pol ζ, yields dC→dT transitions and dC→dA or dC→dG transversions (Phase 1b). Alternatively, the abasic site can be recognized and excised by APE or the Mre11–Rad50 lyase to create a DNA nick. This nick can be repaired by DNA pol β (light pink circles) in an error-free fashion (short-patch BER) or repaired in an error-prone fashion by a TLS polymerase through a long-patch BER also involving PCNA and Fen1. dU:dG mispairs can also be recognized by the MMR machinery, resulting in a DNA-gap formation through the intervention of an unidentified endonuclease or MRN and Exo I. Subsequently, TLS pol θ, pol η, Rev1, pol ζ and, perhaps, pol ι can effect DNA re-synthesis as part of a patch repair, thereby inserting mismatches (Phase 2). In the long-patch BER or MMR, RPA (large brown ovals) and PCNA would recruit other repair proteins to the lesion and co-ordinate their actions. MMR proteins are indicated as large green ovals. Mutated nucleotides are shown in red.