A Human MSH6 Germline Variant Associated With Systemic Lupus Erythematosus Induces Lupus-like Disease in Mice.

OBJECTIVE: To determine if single-nucleotide polymorphisms (SNPs) in DNA repair genes are enriched in individuals with systemic lupus erythematosus (SLE) and if they are sufficient to confer a disease phenotype in a mouse model. METHODS: Human exome chip data of 2499 patients with SLE and 1230 healthy controls were analyzed to determine if variants in 10 different mismatch repair genes (MSH4, EXO1, MSH2, MSH6, MLH1, MSH3, POLH, PMS2, ML3, and APEX2) were enriched in individuals with SLE. A mouse model of the MSH6 SNP, which was found to be enriched in individuals with SLE, was created using CRISPR/Cas9 gene targeting. Wildtype mice and mice heterozygous and homozygous for the MSH6 variant were then monitored for 2 years for the development of autoimmune phenotypes, including the presence of high levels of antinuclear antibodies (ANA). Additionally, somatic hypermutation frequencies and spectra of the intronic region downstream of the VH J558-rearranged JH4 immunoglobulin gene was characterized from Peyer's patches. RESULTS: Based on the human exome chip data, the MSH6 variant (rs63750897, p.Ser503Cys) is enriched among patients with SLE versus controls after we corrected for ancestry (odds ratio = 8.39, P = 0.0398). Mice homozygous for the MSH6 variant (Msh6S502C/S502C ) harbor significantly increased levels of ANA. Additionally, the Msh6S502C/S502C mice display a significant increase in the infiltration of CD68+ cells (a marker for monocytes and macrophages) into the lung alveolar space as well as apoptotic cells. Furthermore, characterization of somatic hypermutation in these mice reveals an increase in the DNA polymerase η mutational signature. CONCLUSION: An MSH6 mutation that is enriched in humans diagnosed with lupus was identified. Mice harboring this Msh6 mutation develop increased autoantibodies and an inflammatory lung disease. These results suggest that the human MSH6 variant is linked to the development of SLE.

The hematopoietic compartment is sufficient for lupus development resulting from the POLB-Y265C mutation.

Systemic lupus erythematosus is a chronic disease characterized by autoantibodies, renal and cutaneous disease, and immune complex formation. Emerging evidence suggests that aberrant DNA repair is an underlying mechanism of lupus development. We previously showed that the POLBY265C/C mutation, which results in development of an aberrant immune repertoire, leads to lupus-like disease in mice. To address whether the hematopoietic compartment is sufficient for lupus development, we transplanted bone marrow cells from POLBY265C/C and POLB+/+ into wild-type congenic mice. Only mice transplanted with the POLBY265C/C bone marrow develop high levels of antinuclear antibodies and renal disease. In conclusion, we show that the hematopoietic compartment harvested from the POLBY265C/C mice is sufficient for development of autoimmune disease.

DNA glycosylase deficiency leads to decreased severity of lupus in the Polb-Y265C mouse model.

The Polb gene encodes DNA polymerase beta (Pol ß), a DNA polymerase that functions in base excision repair (BER) and microhomology-mediated end-joining. The Pol ß-Y265C protein exhibits low catalytic activity and fidelity, and is also deficient in microhomology-mediated end-joining. We have previously shown that the PolbY265C/+ and PolbY265C/C mice develop lupus. These mice exhibit high levels of antinuclear antibodies and severe glomerulonephritis. We also demonstrated that the low catalytic activity of the Pol ß-Y265C protein resulted in accumulation of BER intermediates that lead to cell death. Debris released from dying cells in our mice could drive development of lupus. We hypothesized that deletion of the Neil1 and Ogg1 DNA glycosylases that act upstream of Pol ß during BER would result in accumulation of fewer BER intermediates, resulting in less severe lupus. We found that high levels of antinuclear antibodies are present in the sera of PolbY265C/+ mice deleted of Ogg1 and Neil1 DNA glycosylases. However, these mice develop significantly less severe renal disease, most likely due to high levels of IgM in their sera.

Differential immunomodulatory effect of PARP inhibition in BRCA1 deficient and competent tumor cells.

Poly-ADP-ribose polymerase (PARP) inhibitors are active against cells and tumors with defects in homology-directed repair as a result of synthetic lethality. PARP inhibitors (PARPi) have been suggested to act by either catalytic inhibition or by PARP localization in chromatin. In this study, we treat BRCA1 mutant cells derived from a patient with triple negative breast cancer and control cells for three weeks with veliparib, a PARPi, to determine if treatment with this drug induces increased levels of mutations and/or an inflammatory response. We show that long-term treatment with PARPi induces an inflammatory response in HCC1937 BRCA1 mutant cells. The levels of chromatin-bound PARP1 in the BRCA1 mutant cells correlate with significant upregulation of inflammatory genes and activation of the cyclic GMP-AMP synthase (cGAS)/signaling effector stimulator of interferon genes (STING pathway). In contrast, an increased mutational load is induced in BRCA1-complemented cells treated with a PARPi. Our results suggest that long-term PARP inhibitor treatment may prime both BRCA1 mutant and wild-type tumors for positive responses to immune checkpoint blockade, but by different underlying mechanisms.

The double-edged sword of cancer mutations: exploiting neoepitopes for the fight against cancer.

Defects in DNA repair have been linked to the accumulation of somatic mutations in tumours. These mutations can promote oncogenesis; however, recent developments have indicated that they may also lead to a targeted immune response against the tumour. This response is initiated by the development of new antigenic epitopes (neoepitopes) arising from mutations in protein-coding genes that are processed and then presented on the surface of tumour cells. These neoepitopes are unique to the tumour, thus enabling lymphocytes to launch an immune response against the cancer cells. Immunotherapies, such as checkpoint inhibitors (CPIs) and tumour-derived vaccines, have been shown to enhance the immunogenic response to cancers and have led to complete remission in some cancer patients. There are tumours that are not responsive to immunotherapy or conventional tumour therapeutics; therefore, there is a push for new treatments to combat these unresponsive cancers. Recently, combinatorial treatments have been developed to further utilise the immune system in the fight against cancer. These treatments have the potential to exploit the defects in DNA repair by inducing more DNA damage and mutations. This can potentially lead to the expression of high levels of neoepitopes on the surface of tumour cells that will stimulate an immunological response. Overall, exploiting DNA repair defects in tumours may provide an edge in this long fight against cancer.

Nucleosomes Regulate Base Excision Repair in Chromatin.

Chromatin is a significant barrier to many DNA damage response (DDR) factors, such as DNA repair enzymes, that process DNA lesions to reduce mutations and prevent cell death; yet, paradoxically, chromatin also has a critical role in many signaling pathways that regulate the DDR. The primary level of DNA packaging in chromatin is the nucleosome core particle (NCP), consisting of DNA wrapped around an octamer of the core histones H2A, H2B, H3 and H4. Here, we review recent studies characterizing how the packaging of DNA into nucleosomes modulates the activity of the base excision repair (BER) pathway and dictates BER subpathway choice. We also review new evidence indicating that the histone amino-terminal tails coordinately regulate multiple DDR pathways during the repair of alkylation damage in the budding yeast Saccharomyces cerevisiae.

DNA repair and systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with no known cure that affects at least five million people worldwide. Monozygotic twin concordance and familial aggregation studies strongly suggest that lupus results from genetic predisposition along with environmental exposures including UV light. The majority of the common risk alleles associated with genetic predisposition to SLE map to genes associated with the immune system. However, evidence is emerging that implicates a role for aberrant DNA repair in the development of lupus. Here we summarize our current knowledge of the potential association of lupus with mutations in DNA repair genes. We also discuss how defective or aberrant DNA repair could lead to the development of lupus.

Nucleosomes determine their own patch size in base excision repair.

Base excision repair (BER) processes non-helix distorting lesions (e.g., uracils and gaps) and is composed of two subpathways that differ in the number of nucleotides (nts) incorporated during the DNA synthesis step: short patch (SP) repair incorporates 1 nt and long patch (LP) repair incorporates 2-12 nts. This choice for either LP or SP repair has not been analyzed in the context of nucleosomes. Initial studies with uracil located in nucleosome core DNA showed a distinct DNA polymerase extension profile in cell-free extracts that specifically limits extension to 1 nt, suggesting a preference for SP BER. Therefore, we developed an assay to differentiate long and short repair patches in 'designed' nucleosomes containing a single-nucleotide gap at specific locations relative to the dyad center. Using cell-free extracts or purified enzymes, we found that DNA lesions in the nucleosome core are preferentially repaired by DNA polymerase ß and there is a significant reduction in BER polymerase extension beyond 1 nt, creating a striking bias for incorporation of short patches into nucleosomal DNA. These results show that nucleosomes control the patch size used by BER.

Histone ubiquitylation and its roles in transcription and DNA damage response.

DNA in human cells is constantly assaulted by endogenous and exogenous DNA damaging agents. It is vital for the cell to respond rapidly and precisely to DNA damage to maintain genome integrity and reduce the risk of mutagenesis. Sophisticated reactions occur in chromatin surrounding the damaged site leading to the activation of DNA damage response (DDR), including transcription reprogramming, cell cycle checkpoint, and DNA repair. Histone proteins around the DNA damage play essential roles in DDR, through extensive post-translational modifications (PTMs) by a variety of modifying enzymes. One PTM on histones, mono-ubiquitylation, has emerged as a key player in cellular response to DNA damage. In this review, we will (1) briefly summarize the history of histone H2A and H2B ubiquitylation (H2Aub and H2Bub, respectively), (2) discuss their roles in transcription, and (3) their functions in DDR.

Histone Sprocket Arginine Residues Are Important for Gene Expression, DNA Repair, and Cell Viability in Saccharomyces cerevisiae.

A critical feature of the intermolecular contacts that bind DNA to the histone octamer is the series of histone arginine residues that insert into the DNA minor groove at each superhelical location where the minor groove faces the histone octamer. One of these "sprocket" arginine residues, histone H4 R45, significantly affects chromatin structure in vivo and is lethal when mutated to alanine or cysteine in Saccharomyces cerevisiae (budding yeast). However, the roles of the remaining sprocket arginine residues (H3 R63, H3 R83, H2A R43, H2B R36, H2A R78, H3 R49) in chromatin structure and other cellular processes have not been well characterized. We have genetically characterized mutations in each of these histone residues when introduced either singly or in combination to yeast cells. We find that pairs of arginine residues that bind DNA adjacent to the DNA exit/entry sites in the nucleosome are lethal in yeast when mutated in combination and cause a defect in histone occupancy. Furthermore, mutations in individual residues compromise repair of UV-induced DNA lesions and affect gene expression and cryptic transcription. This study reveals simple rules for how the location and structural mode of DNA binding influence the biological function of each histone sprocket arginine residue.

The amino-terminal tails of histones H2A and H3 coordinate efficient base excision repair, DNA damage signaling and postreplication repair in Saccharomyces cerevisiae.

Histone amino-terminal tails (N-tails) are required for cellular resistance to DNA damaging agents; therefore, we examined the role of histone N-tails in regulating DNA damage response pathways in Saccharomyces cerevisiae. Combinatorial deletions reveal that the H2A and H3 N-tails are important for the removal of MMS-induced DNA lesions due to their role in regulating the basal and MMS-induced expression of DNA glycosylase Mag1. Furthermore, overexpression of Mag1 in a mutant lacking the H2A and H3 N-tails rescues base excision repair (BER) activity but not MMS sensitivity. We further show that the H3 N-tail functions in the Rad9/Rad53 DNA damage signaling pathway, but this function does not appear to be the primary cause of MMS sensitivity of the double tailless mutants. Instead, epistasis analyses demonstrate that the tailless H2A/H3 phenotypes are in the RAD18 epistasis group, which regulates postreplication repair. We observed increased levels of ubiquitylated PCNA and significantly lower mutation frequency in the tailless H2A/H3 mutant, indicating a defect in postreplication repair. In summary, our data identify novel roles of the histone H2A and H3 N-tails in (i) regulating the expression of a critical BER enzyme (Mag1), (ii) supporting efficient DNA damage signaling and (iii) facilitating postreplication repair.

UV damage-induced RNA polymerase II stalling stimulates H2B deubiquitylation.

Histone H2B monoubiquitylation plays an important role in RNA polymerase II (RNAPII) elongation. Whether this modification responds to RNAPII stalling is not yet known. We report that both yeast and human cells undergo a rapid and significant H2B deubiquitylation after exposure to UV irradiation. This deubiquitylation occurs concurrently with UV-induced transcription arrest and is significantly reduced in a DNA damage-bypassing RNAPII yeast mutant. Consistent with these results, yeast deubiquitylases Ubp8 and Ubp10 are associated with the RNAPII complex. Moreover, simultaneous deletion of Ubp8 and Ubp10 leads to a lack of H2B deubiquitylation after UV exposure. Consequently, nucleotide excision repair at an actively transcribed gene locus is decreased, whereas UV-induced RNAPII degradation is increased in ubp8Δubp10Δ mutant cells. These results indicate that eukaryotic cells respond to RNAPII arrest by deubiquitylating H2B to coordinate DNA repair and RNAPII degradation.

Analysis of the FliM/FliG motor protein interaction by two-hybrid mutation suppression analysis.

The Escherichia coli motor proteins FliM and FliG physically interact, presumably to control one or more of the functions of the bacterial flagellum clockwise/counterclockwise (CW/CCW) switch. We have previously demonstrated this interaction using the yeast two-hybrid system and have identified mutations in fliG that disrupt the interaction. Starting with the most interaction-defective of these fliG mutants, we mutagenized fliM to identify suppressor mutations that restore the FliM/FliG two-hybrid interaction. Certain fliM suppressor mutations exhibit allele specificity. These mutations help define a FliG-interaction surface on FliM. Moreover, the pattern of suppression suggests that two distinct sites on FliG interact with FliM, perhaps with two FliM molecules in a dimer per molecule of FliG.