10-10 Mismatch and base-excision repair pathways contribute to somatic hypermutation following initiation by AID.
The uridine produced by AID represents a dual lesion in DNA; not only is uridine foreign to normal DNA, but it is now a mismatch with the guanosine nucleoside on the opposite DNA strand. The presence of uridine in DNA can trigger several types of DNA repair—including the mismatch repair and the base-excision repair pathways—which further alter the DNA sequence. The various repair processes lead to different mutational outcomes (Fig. 10.19). In the mismatch repair pathway, the presence of uridine is detected by the mismatch repair proteins MSH2 and MSH6 (MSH2/6). They recruit nucleases that remove the complete uridine nucleotide along with several adjacent nucleotides from the damaged DNA strand. This is followed by a fill-in ‘patch repair’ by a DNA polymerase; unlike the process in all other cells, in B cells this DNA synthesis is error-prone and tends to introduce mutations at nearby A:T base pairs.
The initial steps in the base-excision repair pathway are shown in Fig. 10.20. In this pathway, the enzyme uracil-DNA glycosylase (UNG) removes the uracil base from the uridine to create an abasic site in the DNA. If no further modification is made, this will result at the next round of DNA replication in the random insertion of a nucleotide opposite the abasic site by DNA polymerase, leading to mutation. The action of UNG may, however, be followed by the action of another enzyme, apurinic/apyrimidinic endonuclease 1 (APE1), which excises the abasic residue to create a single-strand discontinuity (known as a single-strand nick) in the DNA at the site of the original cytidine. Repair of the single-strand nick proceeding through double-strand breaks may result in gene conversion. Gene conversion is not used in the diversification of immunoglobulin genes in humans and mice, but is of importance in some other mammals and in birds.
Fig. 10.20 The base-excision repair pathway produces single-strand nicks in DNA by the sequential actions of AID, uracil-DNA glycosylase (UNG), and apurinic/ apyrimidinic endonuclease 1 (APE1). Double-stranded DNA (first panel) can be made accessible to AID by transcription that unwinds the DNA helix locally (second panel). AID, which is specifically expressed in activated B cells, converts cytidine residues to uridines (third panel). The ubiquitous base-excision repair enzyme UNG can then remove the uracil ring from uridine, creating an abasic site (fourth panel). The repair endonuclease APE1 then cuts the sugar–phosphate DNA backbone next to the abasic residue (fifth panel), thereby forming a single-strand nick in the DNA (sixth panel). APE1 does not excise ribose to form a single-strand nick in DNA, but rather cuts the DNA backbone to yield a 5ʹ-deoxyribosephosphate terminus that is then removed by, for example, DNA polymerase b.
Somatic hypermutation involves both mutation at the original cytidines targeted by AID and mutation at nearby non-cytidine nucleotides. If the original
U:G mismatch is recognized by UNG, then an abasic site will be generated in the DNA (see Fig. 10.19). If no further modification is made to this site, it can be replicated without instructive base pairing from the template strand by a class of error-prone ‘translesion’ DNA polymerases that normally repair gross damage to DNA, such as that caused by ultraviolet (UV) radiation. These polymerases can incorporate any nucleotide into the new DNA strand opposite the abasic site, and after a further round of DNA replication this can result in a stable mutation at the site of the original C:G base pair.