Nucleolytic processing of abasic sites underlies PARP inhibitor hypersensitivity in ALC1-deficient BRCA mutant cancer cells.

Clinical success with poly (ADP-ribose) polymerase inhibitors (PARPi) is impeded by inevitable resistance and associated cytotoxicity. Depletion of Amplified in Liver Cancer 1 (ALC1), a chromatin-remodeling enzyme, can overcome these limitations by hypersensitizing BReast CAncer genes 1/2 (BRCA1/2) mutant cells to PARPi. Here, we demonstrate that PARPi hypersensitivity upon ALC1 loss is reliant on its role in promoting the repair of chromatin buried abasic sites. We show that ALC1 enhances the ability of the abasic site processing enzyme, Apurinic/Apyrimidinic endonuclease 1 (APE1) to cleave nucleosome-occluded abasic sites. However, unrepaired abasic sites in ALC1-deficient cells are readily accessed by APE1 at the nucleosome-free replication forks. APE1 cleavage leads to fork breakage and trapping of PARP1/2 upon PARPi treatment, resulting in hypersensitivity. Collectively, our studies reveal how cells overcome the chromatin barrier to repair abasic lesions and uncover cleavage of abasic sites as a mechanism to overcome limitations of PARPi.

A RAD18-UBC13-PALB2-RNF168 axis mediates replication fork recovery in BRCA1-deficient cancer cells.

BRCA1/2 proteins function in genome stability by promoting repair of double-stranded DNA breaks through homologous recombination and by protecting stalled replication forks from nucleolytic degradation. In BRCA1/2-deficient cancer cells, extensively degraded replication forks can be rescued through distinct fork recovery mechanisms that also promote cell survival. Here, we identified a novel pathway mediated by the E3 ubiquitin ligase RAD18, the E2-conjugating enzyme UBC13, the recombination factor PALB2, the E3 ubiquitin ligase RNF168 and PCNA ubiquitination that promotes fork recovery in BRCA1- but not BRCA2-deficient cells. We show that this pathway does not promote fork recovery by preventing replication fork reversal and degradation in BRCA1-deficient cells. We propose a mechanism whereby the RAD18-UBC13-PALB2-RNF168 axis facilitates resumption of DNA synthesis by promoting re-annealing of the complementary single-stranded template strands of the extensively degraded forks, thereby allowing re-establishment of a functional replication fork. We also provide preliminary evidence for the potential clinical relevance of this novel fork recovery pathway in BRCA1-mutated cancers, as RAD18 is over-expressed in BRCA1-deficient cancers, and RAD18 loss compromises cell viability in BRCA1-deficient cancer cells.

The SMC5/6 complex prevents genotoxicity upon APOBEC3A-mediated replication stress.

Mutational patterns caused by APOBEC3 cytidine deaminase activity are evident throughout human cancer genomes. In particular, the APOBEC3A family member is a potent genotoxin that causes substantial DNA damage in experimental systems and human tumors. However, the mechanisms that ensure genome stability in cells with active APOBEC3A are unknown. Through an unbiased genome-wide screen, we define the Structural Maintenance of Chromosomes 5/6 (SMC5/6) complex as essential for cell viability when APOBEC3A is active. We observe an absence of APOBEC3A mutagenesis in human tumors with SMC5/6 dysfunction, consistent with synthetic lethality. Cancer cells depleted of SMC5/6 incur substantial genome damage from APOBEC3A activity during DNA replication. Further, APOBEC3A activity results in replication tract lengthening which is dependent on PrimPol, consistent with re-initiation of DNA synthesis downstream of APOBEC3A-induced lesions. Loss of SMC5/6 abrogates elongated replication tracts and increases DNA breaks upon APOBEC3A activity. Our findings indicate that replication fork lengthening reflects a DNA damage response to APOBEC3A activity that promotes genome stability in an SMC5/6-dependent manner. Therefore, SMC5/6 presents a potential therapeutic vulnerability in tumors with active APOBEC3A.

DNA combing versus DNA spreading and the separation of sister chromatids.

DNA combing and DNA spreading are two central approaches for studying DNA replication fork dynamics genome-wide at single-molecule resolution by distributing labeled genomic DNA on coverslips or slides for immunodetection. Perturbations in DNA replication fork dynamics can differentially affect either leading or lagging strand synthesis, for example, in instances where replication is blocked by a lesion or obstacle on only one of the two strands. Thus, we sought to investigate whether the DNA combing and/or spreading approaches are suitable for resolving adjacent sister chromatids during DNA replication, thereby enabling the detection of DNA replication dynamics within individual nascent strands. To this end, we developed a thymidine labeling scheme that discriminates between these two possibilities. Our data suggests that DNA combing resolves sister chromatids, allowing the detection of strand-specific alterations, whereas DNA spreading typically does not. These findings have important implications when interpreting DNA replication dynamics from data obtained by these two commonly used techniques.