The translesion DNA polymerase θ plays a dominant role in immunoglobulin gene somatic hypermutation

Abstract

Immunoglobulin (Ig) somatic hypermutation (SHM) critically underlies the generation of high-affinity antibodies. Mutations can be introduced by error-prone polymerases such as polymerase f (Rev3), a mispair extender, and polymerase g, a mispair inserter with a preference for dA/dT, while repairing DNA lesions initiated by AIDmediated deamination of dC to yield dU:dG mismatches. The partial impairment of SHM observed in the absence of these polymerases led us to hypothesize a main role for another translesion DNA polymerase. Here, we show that deletion in C57BL/6J mice of the translesion polymerase h, which possesses a dual nucleotide mispair inserter-extender function, results in greater than 60% decrease of mutations in antigen-selected V186.2DJ H transcripts and greater than 80% decrease in mutations in the Ig H chain intronic J H 4-iEl sequence, together with significant alterations in the spectrum of the residual mutations. Thus, polymerase h plays a dominant role in SHM, possibly by introducing mismatches while bypassing abasic sites generated by UDG-mediated deglycosylation of AID-effected dU, by extending DNA past such abasic sites and by synthesizing DNA during dU:dG mismatch repair.

DNA polymerases in adaptive immunity

“…Two groups have reported contrasting results on the impact of a complete inactivation of Polθ on SHM in mice. In one report, deletion of Polq (the gene encoding Polθ) leads to a large decrease in the frequency of mutations, with a pattern biased towards transition mutations 89 . In another report, deletion of this gene only induces a moderate reduction in mutation frequency, without alteration of the pattern of mutations 90 .…”

DNA Replication to Aid Somatic Hypermutation

“…The virtual absence of dC → dG transversions in the nontranscribed strand of the Vλ1 segment DNA in Rev1 deficient mice is consistent with the role of Rev1 in inserting dC across abasic sites during SHM (Jansen et al, 2006). Pol ζ and pol θ are the only TLS polymerases known to efficiently extend the nascent DNA strand past an abasic site by copying undamaged portion of DNA template, consistent with their major role in SHM Zan et al, 2001;Masuda et al, 2005;Zan et al, 2005). Pol ζ is relatively error-free when copying undamaged DNA template.…”

“…Both BCR crosslinking and CD154:CD40 engagement upregulate error-prone pol θ and the Rev3 catalytic subunit of pol ζ in both human and mice. Pol η is upregulated in germinal center B cells in mice, but not in humans (Poltoratsky et al, 2001;Zan et al, 2001;Zeng et al, 2001;Zan et al, 2005). An integrated model of SHM during DNA replication.…”

“…In mice immunized with carrier-conjugated hapten or sheep red blood cells (SRBC), germinal centers appear within four days (Jacob et al, 1993;Wang and Carter, 2005) and expression of AID, which is induced by CD154 and/or cytokines such as IL-4 and TGF-β, begins within hours and peaks at day five (Muramatsu et al, 1999). In addition, expression of TLS polymerases critical to SHM, such as DNA polymerase (pol) θ, pol η and pol ζ, is significantly upregulated in B cells by the same stimuli that induce AID expression and SHM (Poltoratsky et al, 2001;Zan et al, 2001;Zeng et al, 2001;Zan et al, 2005), suggesting that SHM is a tightly regulated active process (Figure 1). Mutations in V(D)J DNA are detected thereafter (Jacob et al, 1993), supporting the contention that after generation of initial dU lesions by AID, B cell division and accompanying DNA replication are necessary for mutations to be fixed in the genome.…”

Mouse models of DNA polymerases

“…DSBs can be repaired by nonhomologous end-joining (NHEJ) or homologous recombination (HR). The DNA polymerases Pol e, Pol b, Pol f, Pol h, Pol y, Pol k, Pol l function in DSB repair by HR or NHEJ [Holbeck et al, 1993;Jessberger et al, 1993;Wang et al, 2004;Xu et al, 2005;Zan et al, 2005;Rattray and Strathern, 2005;Masuda et al, 2006;Nick McElhinny, 2008;Kidane et al, 2010;Moldovan et al, 2010]. The pathways of homologous recombination and nonhomologous end-joining have been reviewed in Chaudhuri and Alt [2004] and San Filippo et al [2008].…”