Uracil excision by endogenous SMUG1 glycosylase promotes efficient Ig class switching and impacts on A:T substitutions during somatic mutation

Abstract

Excision of uracil introduced into the immunoglobulin loci by AID is central to antibody diversification. While predominantly carried out by the UNG uracil‐DNA glycosylase as reflected by deficiency in immunoglobulin class switching in Ung−/− mice, the deficiency is incomplete, as evidenced by the emergence of switched IgG in the serum of Ung−/− mice. Lack of switching in mice deficient in both UNG and MSH2 suggested that mismatch repair initiated a backup pathway. We now show that most of the residual class switching in Ung−/− mice depends upon the endogenous SMUG1 uracil‐DNA glycosylase, with in vitro switching to IgG1 as well as serum IgG3, IgG2b, and IgA greatly diminished in Ung−/−Smug1−/− mice, and that Smug1 partially compensates for Ung deficiency over time. Nonetheless, using a highly MSH2‐dependent mechanism, Ung−/−Smug1−/− mice can still produce detectable levels of switched isotypes, especially IgG1. While not affecting the pattern of base substitutions, SMUG1 deficiency in an Ung−/− background further reduces somatic hypermutation at A:T base pairs. Our data reveal an essential requirement for uracil excision in class switching and in facilitating noncanonical mismatch repair for the A:T phase of hypermutation presumably by creating nicks near the U:G lesion recognized by MSH2.

AID-associated DNA repair pathways regulate malignant transformation in a murine model of BCL6-driven diffuse large B-cell lymphoma

“…53 Absence of MSH2 or MSH6 causes ;50% to 80% reductions in CSR, whereas absence of UNG causes .95% reductions in CSR in mice and humans. [26][27][28][29]54,55 In addition, UNG deficiency in mice impairs formation of translocations between IgH and cMyc and reduces the incidence of post-GC plasmacytomas in Bcl-xL transgenic mice.…”

Differential expression of APE1 and APE2 in germinal centers promotes error-prone repair and A:T mutations during somatic hypermutation

“…UNG works very efficiently with APE1 (52), and the low level of APE1 might partially substitute for APE2 in its absence. Another uracil DNA glycosylase, such as SMUG1, could substitute for UNG (53), although SMUG1 is much less efficient than UNG. It binds AP sites very tightly and depends more on APE for its turnover (41,52).…”

Antibody diversification caused by disrupted mismatch repair and promiscuous DNA polymerases

“…Ung −/− mice have a normal mutation frequency but decreased C:G transversions, while A:T mutagenesis is unchanged [33]. Other uracil glycosylases such as single-strand-selective monofunctional uracil-DNA glycosylase (SMUG1) [34, 35] and methyl-CpG binding domain 4 [36, 37] are not involved in generating abasic sites for SHM under normal physiological conditions.…”

“…However, Pms2 −/− mice have a normal SHM frequency [42, 56, 67], indicating that either PMS2 is not the major nuclease or that compensatory nucleases can act efficiently in its absence. Neuberger and Rada proposed that the glycosylase component of SMUG1 could serve as the nick instigator by creating an abasic site that is then cleaved by APE [34, 68]. Although SHM is unaltered in SMUG1-deficient mice, a small but consistent decrease in A:T mutations occurs in an Ung −/− Smug1 −/− background.…”