Excision Repair and DNA Damage Tolerance in the Context of Nucleosomes

Chromatin is occlusive to DNA damage response factors that process DNA lesions to reduce mutations and forego programmed cell death; paradoxically, chromatin is essential for cell viability because it is required for the compaction of genomic DNA in the nucleus and modulation of metabolic processes. The primary structural component of chromatin is the nucleosome, which consists of ~147 base pairs of DNA wrapped around a histone octamer. To understand how histones contribute to DNA damage response, we analyzed amino-terminal tail (N-tail) deletions of all four canonical histones, both individually and in combination, in the budding yeast Saccharomyces cerevisiae. We found that combinatorial N-tail deletions of histones H2A and H3 (tH2A:tH3) sensitize yeast cells to the DNA alkylating agent methyl methanesulfonate (MMS), and epitasis analyses indicate that multiple DNA damage response pathways are involved in this MMS sensitivity phenotype. Interestingly, the tH2A:tH3 mutant is deficient in base excision repair (BER), a repair pathway with two subpathways that differ in repair patch sizes. We developed an in vitro assay to differentiate between these subpathways and showed that DNA lesions located in the nucleosome core are preferentially repaired by DNA polymerase β that insert short repair patches. Additionally, the tH2A:tH3 mutant was shown to be deficient in nucleotide excision repair (NER), a repair pathway for DNA helix-distorting lesions, such as those induced by UV light. NER also has two subpathways differentiated by the presence or absence of the transcription machinery. We found that transcription-coupled NER requires the removal of the posttranslational ubiquitin moiety from H2B at lysine residue 123 to destabilize chromatin structure and allow for accessibility by repair enzymes. Altogether, our data show that even though chromatin is inhibitory to DNA damage response factors, chromatin provides signals to promote different DNA damage response pathways and subpathways, including those of BER and NER

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

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

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

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