DNA polymerase β prevents AID-instigated mutagenic non-canonical mismatch DNA repair

ABSTRACTIn B cells, the error-prone repair of activation-induced cytidine deaminase (AID)-induced lesions in immunoglobulin variable genes cause somatic hypermutation (SHM) of antibody genes. Due to clonal selection in the germinal centers (GC) this active mutation process provides the molecular basis for antibody affinity maturation. AID deaminates cytosine (C) to create uracil (U) in DNA. Typically, the short patch base excision repair (spBER) effectively restores genomic U lesions. We here demonstrate that GC B cells actively degrade DNA polymerase β (Polβ), resulting in the inactivation of the gap-filling step of spBER. Consequently, lesions instigated by AID, and likely other base damages, are channeled towards mutagenic non-canonical mismatch repair (mncMMR), responsible for the vast majority of mutations at adenine and thymine (A:T) bases. Apparently, GC B cells prohibit faithful spBER, thereby favoring A:T mutagenesis during SHM. Lastly, our data suggest that the loss of Polβ relates to hypoxia that characterizes the GC microenvironment.

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DNA polymerase β is able to repair breaks in switch regions and plays an inhibitory role during immunoglobulin class switch recombination

Journal of Experimental Medicine ◽

10.1084/jem.20070756 ◽

2007 ◽

Vol 204 (7) ◽

pp. 1677-1689 ◽

Cited By ~ 43

Author(s):

Xiaoming Wu ◽

Janet Stavnezer

Keyword(s):

B Cells ◽

Dna Polymerase ◽

Excision Repair ◽

Cytidine Deaminase ◽

Class Switch Recombination ◽

Strand Break ◽

Dna Polymerase Β ◽

Single Strand Break ◽

Class Switch ◽

Base Excision

Immunoglobulin (Ig) class switch recombination (CSR) is initiated by activation-induced cytidine deaminase (AID), which converts cytosines to uracils in switch (S) regions. Subsequent excision of dU by uracil DNA glycosylase (UNG) of the base excision repair (BER) pathway is required to obtain double-strand break (DSB) intermediates for CSR. Since UNG normally initiates faithful repair, it is unclear how the AID-instigated S region lesions are converted into DSBs rather than correctly repaired by BER. Normally, DNA polymerase β (Polβ) would replace the dC deaminated by AID, leading to correct repair of the single-strand break, thereby preventing CSR. We address the question of whether Polβ might be specifically down-regulated during CSR or inhibited from accessing the AID-instigated lesions, or whether the numerous AID-initiated S region lesions might simply overwhelm the BER capacity. We find that nuclear Polβ levels are induced upon activation of splenic B cells to undergo CSR. When Polβ−/− B cells are activated to switch in culture, they switch slightly better to IgG2a, IgG2b, and IgG3 and have more S region DSBs and mutations than wild-type controls. We conclude that Polβ attempts to faithfully repair S region lesions but fails to repair them all.

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MSH2–MSH6 stimulates DNA polymerase η, suggesting a role for A:T mutations in antibody genes

Journal of Experimental Medicine ◽

10.1084/jem.20042066 ◽

2005 ◽

Vol 201 (4) ◽

pp. 637-645 ◽

Cited By ~ 136

Author(s):

Teresa M. Wilson ◽

Alexandra Vaisman ◽

Stella A. Martomo ◽

Patsa Sullivan ◽

Li Lan ◽

...

Keyword(s):

Dna Polymerase ◽

Base Excision Repair ◽

Somatic Hypermutation ◽

Excision Repair ◽

Cytidine Deaminase ◽

Abasic Site ◽

Cell Extracts ◽

Activation Induced Cytidine Deaminase ◽

Base Excision

Activation-induced cytidine deaminase deaminates cytosine to uracil (dU) in DNA, which leads to mutations at C:G basepairs in immunoglobulin genes during somatic hypermutation. The mechanism that generates mutations at A:T basepairs, however, remains unclear. It appears to require the MSH2–MSH6 mismatch repair heterodimer and DNA polymerase (pol) η, as mutations of A:T are decreased in mice and humans lacking these proteins. Here, we demonstrate that these proteins interact physically and functionally. First, we show that MSH2–MSH6 binds to a U:G mismatch but not to other DNA intermediates produced during base excision repair of dUs, including an abasic site and a deoxyribose phosphate group. Second, MSH2 binds to pol η in solution, and endogenous MSH2 associates with the pol in cell extracts. Third, MSH2–MSH6 stimulates the catalytic activity of pol η in vitro. These observations suggest that the interaction between MSH2–MSH6 and DNA pol η stimulates synthesis of mutations at bases located downstream of the initial dU lesion, including A:T pairs.

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FAM72A antagonizes UNG2 to promote mutagenic uracil repair during antibody maturation

10.1101/2020.12.23.423975 ◽

2020 ◽

Author(s):

Yuqing Feng ◽

Conglei Li ◽

Jessica Stewart ◽

Philip Barbulescu ◽

Noe Seija Desivo ◽

...

Keyword(s):

B Cells ◽

Mismatch Repair ◽

Somatic Hypermutation ◽

Excision Repair ◽

Cytidine Deaminase ◽

Antibody Maturation ◽

Genome Wide ◽

A Genome ◽

Repair Pathways ◽

Base Excision

Activation-induced cytidine deaminase (AID) catalyzes the deamination of deoxycytidines within Immunoglobulin (Ig) genes to induce somatic hypermutation (SHM) and class switch recombination (CSR). AID-induced deoxyuracils within Ig loci are recognized and processed by subverted base excision and mismatch repair pathways that ensure a mutagenic outcome in B lymphocytes. However, it is unclear why DNA repair pathways that remove deoxyuracil from DNA are not efficient at faithfully repairing AID-induced lesions. Here, we identified through a genome-wide CRISPR screen that FAM72A, a protein with no ascribed function, is a major determinant for the error-prone processing of deoxyuracil. Fam72a-deficient CH12F3-2 B cells and primary B cells from Fam72a-/- mice have reduced CSR and SHM frequencies. The SHM spectrum in B cells from Fam72a-/- mice is opposite to that observed in Ung2-/- mice, suggesting that UNG2 is hyperactive in FAM72A-deficient cells. Indeed, FAM72A binds to UNG2 resulting in reduced UNG2 activity, and significantly reduced protein levels in the G1 phase, coinciding with peak AID activity. This effect leads to increased genome-wide deoxyuracils in B cells. By antagonizing UNG2, FAM72A therefore increases U:G mispairs that engage mutagenic mismatch repair promoting error-prone processing of AID-induced deoxyuracils. This work shows that FAM72A bridges base-excision repair and mismatch repair to modulate antibody maturation.

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The human checkpoint sensor and alternative DNA clamp Rad9–Rad1–Hus1 modulates the activity of DNA ligase I, a component of the long-patch base excision repair machinery

Biochemical Journal ◽

10.1042/bj20050211 ◽

2005 ◽

Vol 389 (1) ◽

pp. 13-17 ◽

Cited By ~ 41

Author(s):

Ekaterina SMIRNOVA ◽

Magali TOUEILLE ◽

Enni MARKKANEN ◽

Ulrich HÜBSCHER

Keyword(s):

Quality Control ◽

Dna Polymerase ◽

Base Excision Repair ◽

Excision Repair ◽

Dna Ligase ◽

Dna Polymerase Β ◽

Flap Endonuclease 1 ◽

Important Target ◽

Dna Ligase I ◽

Base Excision

The human checkpoint sensor and alternative clamp Rad9–Rad1–Hus1 can interact with and specifically stimulate DNA ligase I. The very recently described interactions of Rad9–Rad1–Hus1 with MutY DNA glycosylase, DNA polymerase β and Flap endonuclease 1 now complete our view that the long-patch base excision machinery is an important target of the Rad9–Rad1–Hus1 complex, thus enhancing the quality control of DNA.

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