DNA Breaks in Ig V Regions Are Predominantly Single Stranded and Are Generated by UNG and MSH6 DNA Repair Pathways

Abstract

Antibody diversity is initiated by activation-induced deaminase (AID), which deaminates cytosine to uracil in DNA. Uracils in the Ig gene loci can be recognized by uracil DNA glycosylase (UNG) or mutS homologs 2 and 6 (MSH2-MSH6) proteins, and then processed into DNA breaks. Breaks in switch regions of the H chain locus cause isotype switching and have been extensively characterized as staggered and blunt double-strand breaks. However, breaks in V regions that arise during somatic hypermutation are poorly understood. In this study, we characterize AID-dependent break formation in J H introns from mouse germinal center B cells. We used a ligation-mediated PCR assay to detect single-strand breaks and double-strand breaks that were either staggered or blunt. In contrast to switch regions, V regions contained predominantly single-strand breaks, which peaked 10 d after immunization. We then examined the pathways used to generate these breaks in UNG-and MSH6-deficient mice. Surprisingly, both DNA repair pathways contributed substantially to break formation, and in the absence of both UNG and MSH6, the frequency of breaks was severely reduced. When the breaks were sequenced and mapped, they were widely distributed over a 1000-bp intron region downstream of J H 3 and J H 4 exons and were unexpectedly located at all 4 nt. These data suggest that during DNA repair, nicks are generated at distal sites from the original deaminated cytosine, and these repair intermediates could generate both faithful and mutagenic repair. During mutagenesis, single-strand breaks would allow entry for low-fidelity DNA polymerases to generate somatic hypermutation.

Deaminases and Why Mice Sometimes Lie in Immuno-Oncology Pre-Clinical Trials?

“…In inbred mice, the number of sites potentially targeted by deaminases as a part of an innate immune response is further restricted by homozygosity so that there is a limited opportunity of a further polymorphic heterozygous contribution to form diverse DBD heterodimers (or higher multimers) during deaminase-mediated somatic mutagenesis in cancer progression [17,20,21]. C-site deamination is thus important as it leads to mutagenic G•U (uracils via C-to-U) and mutagenic G •T mispairs (thymine via Cto-T at 5MeCpG sites) which can mature to Abasic sites (AP), and then to single stranded DNA nicks by the action of AP endonucleases at Transcription Bubbles and in post transcription DNA repair (with 3'OH ends on the transcribed DNA strand) as discussed already , Lindley and Steele 2013, Steele and Lindley 2017, and which is largely confirmed by the molecular data of the Gearhart group [15,[22][23][24].…”

“…Thus novel AID-associated heterodimer DBDs could initiate dysregulated Ig-SHM-like responses resulting in AID/APOBEC lesions opening the DNA to further mutagenic processes such as C-to-U at AID/APOBEC motifs (e.g. WRCW/RGYW), Abasic sites, AP endonuclease-mediated ssDNA nicks and error-prone DNA polymerases recruited via base excision repair (UNG) and mismatch repair (MSH2-MSH6) pathways [23][24][25].…”

A proposed reverse transcription mechanism for (CAG)n and similar expandable repeats that cause neurological and other diseases

“…This step is well documented in the immunoglobulin (Ig) SHM literature, see references in Franklin et al, (2004) and other reviews (Di Noia and Neuberger, 2007;Teng and Papavasiliou, 2007;Maul and Gearhart, 2010;Steele, 2016). A recent paper by the Gearhart group demonstrates further the generation of such single stranded nicks with 3 0 -OH ends (Zanotti et al, 2019). The focus here, and in Figure 1, is on how the TS is deaminated as it is this TS lesion which sets up the sequelae of downstream steps (Figure 1) that lead to the key diagnostic strand biases in Ig SHM data sets discussed in the text here, namely mutations at A sites exceed mutations at T sites (A >> T) and, in the same data sets, mutations at G sites exceed mutations at C sites (G >> C).…”

Function and Molecular Mechanism of the DNA Damage Response in Immunity and Cancer Immunotherapy

“…During SHM, AID deaminates a particular trinucleotide sequence in ssDNA of transcriptionally active genes, leaving behind numerous uracil residues and producing predominantly nucleotide substitutions in rearranged V genes on the heavy- and light-chain loci, and switch (S) regions, which precede most C genes on the heavy chain locus ( 217 , 218 ). The mutagenic outcome of uracil lesions can then be determined by one of the following DDR responses: (i) Uracil can act as a template for replication, resulting in a fixed C-T transition mutation; (ii) U-G mismatches can be recognized by the error‐prone MMR machinery, in which the MutSα complex (MSH2-MSH6) detects the mismatch and recruits MutLα complex (MLH1-PMS2) to nick the DNA, followed by the recruitment of Polη (DNA polymerase η) to generate mutations ( 219 , 220 ); (iii) Non‐canonical BER initiated by uracil DNA glycosylase (UNG) can be used to recruit proliferating cell nuclear antigen (PCNA) at the lesions, and low‐fidelity polymerases such as Polη, which can increase mutations during replication of common DNA fragile sites ( 221 ), then can be recruited by PCNA ubiquitination and utilized by both MMR and BER resulting in mutagenic repair ( 149 , 222 – 224 ). The nick generated by the UNG-dependent BER pathway is particularly important for CSR, as UNG1 knock out largely abolishes CSR ( 225 ).…”