Fork restart: unloading FANCD2 to travel ahead.

In this issue of Molecular Cell, Brunner et al.1 reveal that eliminating FANCD2 from stalled forks via FBXL12-mediated degradation enables cells to tolerate oncogene-induced replication stress, making FBXL12 a promising target for cancer treatment.

Single-stranded DNA Gap Accumulation is a Functional Biomarker for USP1 Inhibitor Sensitivity.

Recent studies suggest that PARP inhibitors and POLQ inhibitors confer synthetic lethality in BRCA1-deficient tumors by accumulation of single-stranded DNA (ssDNA) gaps at replication forks. Loss of USP1, a deubiquitinating enzyme, is also synthetic lethal with BRCA1 deficiency, and USP1 inhibitors are now undergoing clinical development for these cancers. Here, we show that USP1 inhibitors also promote the accumulation of ssDNA gaps during replication in BRCA1-deficient cells, and this phenotype correlates with the drug sensitivity. USP1 inhibition increased monoubiquitinated PCNA at replication forks, mediated by the ubiquitin ligase RAD18, and knockdown of RAD18 caused USP1 inhibitor resistance and suppression of ssDNA gaps. USP1 inhibition overcame PARP inhibitor resistance in a BRCA1-mutated xenograft model and induced ssDNA gaps. Furthermore, USP1 inhibition was synergistic with PARP and POLQ inhibition in BRCA1-mutant cells, with enhanced ssDNA gap accumulation. Finally, in patient-derived ovarian tumor organoids, sensitivity to USP1 inhibition alone or in combination correlated with the accumulation of ssDNA gaps. Assessment of ssDNA gaps in ovarian tumor organoids therefore represents a rapid approach for predicting response to USP1 inhibition in ongoing clinical trials.

Analysis of DNA Replication in Fission Yeast by Combing.

DNA replication studies based on population experiments give an average estimate of replication kinetics from many cells. This average replication profile masks the stochastic nature of origin firing in eukaryotes, which is revealed by using single-molecule techniques, such as DNA combing. The analysis of replication kinetics by DNA combing involves isolating DNA from cells that have been pulse-labeled with thymidine analogs and stretching it on a silanized coverslip. The analog-labeled patches on the stretched DNA fibers can then be detected using fluorescent antibodies against the analog. Each fiber represents a part of the genome from a single cell; therefore, it is possible to study the variation in behavior of individual origins from one cell to another. Furthermore, each DNA fiber is uniformly stretched, making it possible to measure distances accurately at kilobase resolution. It is also possible to stretch a high density of fibers on coverslips enabling quantitative data collection.