It is widely accepted that DNA replication fork stalling is a common occurrence during cell proliferation, but there are robust mechanisms to alleviate this and ensure DNA replication is completed prior to chromosome segregation. The SMC5/6 complex has consistently been implicated in the maintenance of replication fork integrity. However, the essential role of the SMC5/6 complex during DNA replication in mammalian cells has not been elucidated. In this study, we investigate the molecular consequences of SMC5/6 loss at the replication fork in mouse embryonic stem cells (mESCs), employing the auxin-inducible degron (AID) system to deplete SMC5 acutely and reversibly in the defined cellular contexts of replication fork stall and restart. In SMC5-depleted cells, we identify a defect in the restart of stalled replication forks, underpinned by excess MRE11-mediated fork resection and a perturbed localization of fork protection factors to the stalled fork. Previously, we demonstrated a physical and functional interaction of SMC5/6 with the COP9 signalosome (CSN), a cullin deneddylase that enzymatically regulates cullin ring ligase (CRL) activity. Employing a combination of DNA fiber techniques, the AID system, small-molecule inhibition assays, and immunofluorescence microscopy analyses, we show that SMC5/6 promotes the localization of fork protection factors to stalled replication forks by negatively modulating the COP9 signalosome (CSN). We propose that the SMC5/6-mediated modulation of the CSN ensures that CRL activity and their roles in DNA replication fork stabilization are maintained to allow for efficient replication fork restart when a replication fork stall is alleviated.

Tumors anomalously induce the expression of meiotic genes, which are otherwise restricted only to developing gametes. If and how these aberrantly expressed meiotic proteins influence DNA metabolism is not clear, but could have important implications for how tumors acquire and mitigate genomic instability. HORMAD1 is a highly conserved meiotic protein that is frequently expressed in lung adenocarincoma where its expression correlates with reduced patient survival and increased mutation burden. Here, we find that HORMAD1 associates with the replisome and is critical for protecting stalled DNA replication forks. Loss of HORMAD1 leads to nascent DNA strand degradation, an event which is mediated by the MRE11-DNA2-BLM pathway. We find that these phenotypes are due to limited RAD51 loading onto stalled replication forks in the absence of HORMAD1. Ultimately, loss of HORMAD1 leads to increased DNA breaks and chromosomal defects, which is exacerbated dramatically by induction of replication stress. Tumor cells proliferate despite encountering chronic replication stress, placing them on the precipice of catastrophic genomic damage. Our data support the hypothesis that the aberrant expression of HORMAD1 is engaged to attenuate the accumulation of excessive DNA damage due to chronic replication stress, which may otherwise lead to accumulation of toxic levels of genomic instability.
Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.

SUMMARY Tumors frequently activate the expression of genes that are only otherwise required for meiosis. HORMAD1, which is essential for meiotic recombination in multiple species, is expressed in over 50% of human lung adenocarcinoma cells (LUAD). We previously found that HORMAD1 promotes DNA double strand break (DSB) repair in LUAD. Here, we report that HORMAD1 takes on an additional role in protecting genomic integrity. Specifically, we find HORMAD1 is critical for protecting stalled DNA replication forks in LUAD. Loss of HORMAD1 leads to nascent DNA degradation, an event which is mediated by the MRE11-DNA2-BLM pathway. Moreover, following exogenous induction of DNA replication stress, HORMAD1 deleted cells accumulate single stranded DNA (ssDNA). We find that these phenotypes are the result of a lack of RAD51 and BRCA2 loading onto stalled replication forks. Ultimately, loss of HORMAD1 leads to increased DSBs and chromosomal aberrations in response to replication stress. Collectively, our data support a model where HORMAD1 expression is selected to mitigate DNA replication stress, which would otherwise induce deleterious genomic instability.

Double strand break (DSB) repair primarily occurs through 3 pathways: non-homologous end-joining (NHEJ), alternative end-joining (Alt-EJ), and homologous recombination (HR). Typical methods to measure pathway usage include integrated cassette reporter assays or visualization of DNA damage induced nuclear foci. It is now well understood that repair of Cas9-induced breaks also involves NHEJ, Alt-EJ, and HR pathways, providing a new format to measure pathway usage. Here, we have developed a simple Cas9-based system with validated repair outcomes that accurately represent each pathway and then converted it to a droplet digital PCR (ddPCR) readout, thus obviating the need for Next Generation Sequencing and bioinformatic analysis with the goal to make Cas9-based system accessible to more laboratories. The assay system has reproduced several important insights. First, absence of the key Alt-EJ factor Pol θ only abrogates ∼50% of total Alt-EJ. Second, single-strand templated repair (SSTR) requires BRCA1 and MRE11 activity, but not BRCA2, establishing that SSTR commonly used in genome editing is not conventional HR. Third, BRCA1 promotes Alt-EJ usage at two-ended DSBs in contrast to BRCA2. This assay can be used in any system, which permits Cas9 delivery and, importantly, allows rapid genotype-to-phenotype correlation in isogenic cell line pairs.
© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.

Investigations of CRISPR gene knockout editing profiles have contributed to enhanced precision of editing outcomes. However, for homology-directed repair (HDR) in particular, the editing dynamics and patterns in clinically relevant cells, such as human iPSCs and primary T cells, are poorly understood. Here, we explore the editing dynamics and DNA repair profiles after the delivery of Cas9-guide RNA ribonucleoprotein (RNP) with or without the adeno-associated virus serotype 6 (AAV6) as HDR donors in four cell types. We show that editing profiles have distinct differences among cell lines. We also reveal the kinetics of HDR mediated by the AAV6 donor template. Quantification of T50 (time to reach half of the maximum editing frequency) indicates that short indels (especially +A/T) occur faster than longer (>2 bp) deletions, while the kinetics of HDR falls between NHEJ (non-homologous end-joining) and MMEJ (microhomology-mediated end-joining). As such, AAV6-mediated HDR effectively outcompetes the longer MMEJ-mediated deletions but not NHEJ-mediated indels. Notably, a combination of small molecular compounds M3814 and Trichostatin A (TSA), which potently inhibits predominant NHEJ repairs, leads to a 3-fold increase in HDR efficiency.
© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.