Involvement of Rad18 in somatic hypermutation

Abstract

Somatic hypermutation of Ig genes is initiated by transcriptioncoupled cytidine deamination in Ig loci. Error-prone processing of the resultant DNA lesions is thought to cause extensive mutagenesis, but it is presently an enigma how and why error-prone rather than error-free repair pathways are recruited. During DNA replication, recruitment of error-prone translesion polymerases may be mediated by Rad6͞Rad18-mediated ubiquitination of proliferating cell nuclear antigen, a major switchboard controlling the fidelity of DNA lesion bypass in eukaryotes. By inactivation of Rad18 in the DT40 B cell line, we show that the Rad6 pathway is involved in somatic hypermutation in these cells. Our findings imply that targeted recruitment of mutagenic polymerases by the Rad6 pathway contributes to the complex process of somatic hypermutation and provide a framework for more detailed mechanistic studies of the mutagenesis phase of secondary Ig diversification.proliferating cell nuclear antigen ͉ ubiquitination ͉ Rad6 pathway G iven the many challenges that damage cellular DNA daily, the faithful maintenance of genetic information requires the interplay of multiple DNA repair pathways. In most cases, these mechanisms ensure restoration of the original DNA sequence, preventing mutations or aberrations that may lead to impairment of cellular function. In some instances, though, a certain imprecision may be tolerated or even favored by the cell to allow for survival in critical situations. DNA repair mechanisms that are inherently imprecise are the basis of the spontaneous mutability of all genomes and may be recruited and adapted for situations where high genetic variability is mandatory for survival.The adaptive immune system of vertebrates has been shaped in coevolution with pathogenic organisms of high genetic diversity and variability. The high diversity of the primary immune repertoire established during V(D)J recombination in B and T cells generates sufficient potential for recognition of any pathogen, but the affinity of binding is often not sufficient for effective neutralization. Therefore, a second wave of diversification during acute infections ensures that the binding affinity of the antibodies produced by B lymphocytes is fine-tuned to the task at hand. The underlying mechanism in humans is somatic hypermutation, which modifies the antigen binding region by targeted mutagenesis in the variable region of the Ig genes (1). An alternative diversification mechanism prevalent in chicken and some mammals, Ig gene conversion alters the same region by targeted homologous recombination with upstream Ig pseudogenes (2).Somatic hypermutation and Ig gene conversion are related processes (3) originating from the same initial DNA lesion. The transcription-coupled deamination of cytosines by activationinduced cytidine deaminase (AID) leads to uracil that may be processed to abasic sites or strand breaks by the excision repair pathway, i.e., uracil-N-glycosylase (4, 5). The resultant DNA lesions may be repaired by mutagenesis or reco...

DNA polymerase η is a limiting factor for A:T mutations in Ig genes and contributes to antibody affinity maturation

“…It remains to be determined whether human carriers of POLH [31]. RAD 18 is an E3 ubiquitin ligase which, together with RAD6, monoubiquitinates PCNA on stalled replication forks and has been implicated in SHM of Ig genes in chicken DT40 cells [32,33]. REV1 has been shown to interact with multiple Y-family DNA polymerases [34] and contribute to the generation of C to G and G to C transversions in Ig genes in mice [10,11].…”

Ubiquitylated PCNA plays a role in somatic hypermutation and class-switch recombination and is required for meiotic progression

“…On the basis of previous work with yeast (12), human cells (11), and DT40 chicken cells (16,17,19), we examined whether the K164R mutation would interfere with the second phase of SHM that is responsible for the mutations in A:T residues, which have *Theoretical maximum number of unique mutations: number of sites ϫ the number of mice in the category ϫ 3 (because 3 possible substitutions can occur at each site). † Observed number of unique mutations: within each mouse, identical mutations were counted once.…”

“…The monoubiquitylation of PCNA is regulated by a complex mechanism that involves the E2 and E3 ubiquitin ligases, RAD6 and RAD18, and deubiquitylation primarily by USP1 (12,15,16). Recent studies in the DT40 chicken B cell line (16,17) suggest that the recruitment and activation of Pol to the Ig V regions is mediated by PCNA that is monoubiquitylated at residue K164.…”

“…PCNA is a DNA-encircling homotrimer that is central to all forms of DNA replication and serves as a sliding platform that recruits MMR and activates polymerases and other factors in a reversible and competitive fashion (13,14). The monoubiquitylation of PCNA is regulated by a complex mechanism that involves the E2 and E3 ubiquitin ligases, RAD6 and RAD18, and deubiquitylation primarily by USP1 (12,15,16). Recent studies in the DT40 chicken B cell line (16,17) suggest that the recruitment and activation of Pol to the Ig V regions is mediated by PCNA that is monoubiquitylated at residue K164.…”

“…The monoubiquitylation of PCNA is regulated by a complex mechanism that involves the E2 and E3 ubiquitin ligases, RAD6 and RAD18, and deubiquitylation primarily by USP1 (12,15,16). Recent studies in the DT40 chicken B cell line (16,17) suggest that the recruitment and activation of Pol to the Ig V regions is mediated by PCNA that is monoubiquitylated at residue K164.As the work described below was being completed it was reported that a mouse expressing PCNA with a lysine-to-arginine mutation at residue 164 that prevented the mono-and polyubiquitylation had a decrease in V region somatic mutations at A:T bases but no significant defect in CSR (18). We have also generated a mouse that expresses PCNA with a lysine-to-arginine mutation at residue 164.…”