Genome instability is a characteristic enabling factor for carcinogenesis. HelQ helicase is a component of human DNA maintenance systems that prevent or reverse genome instability arising during DNA replication. Here, we provide details of the molecular mechanisms that underpin HelQ function—its recruitment onto ssDNA through interaction with replication protein A (RPA), and subsequent translocation of HelQ along ssDNA. We describe for the first time a functional role for the non-catalytic N-terminal region of HelQ, by identifying and characterizing its PWI-like domain. We present evidence that this domain of HelQ mediates interaction with RPA that orchestrates loading of the helicase domains onto ssDNA. Once HelQ is loaded onto the ssDNA, ATP-Mg2+ binding in the catalytic site activates the helicase core and triggers translocation along ssDNA as a dimer. Furthermore, we identify HelQ-ssDNA interactions that are critical for the translocation mechanism. Our data are novel and detailed insights into the mechanisms of HelQ function relevant for understanding how human cells avoid genome instability provoking cancers, and also how cells can gain resistance to treatments that rely on DNA crosslinking agents.

Co-opting of CRISPR-Cas ‘Interference’ reactions for editing the genomes of eukaryotic and prokaryotic cells has highlighted crucial support roles for DNA repair systems that strive to maintain genome stability. As front-runners in genome editing that targets DNA, the class 2 CRISPR-Cas enzymes Cas9 and Cas12a rely on repair of DNA double-strand breaks (DDSBs) by host DNA repair enzymes, using mechanisms that vary in how well they are understood. Data are emerging about the identities of DNA repair enzymes that support genome editing in human cells. At the same time, it is becoming apparent that CRISPR-Cas systems functioning in their native environment, bacteria or archaea, also need DNA repair enzymes. In this short review, we survey how DNA repair and CRISPR-Cas systems are intertwined. We consider how understanding DNA repair and CRISPR-Cas interference reactions in nature might help improve the efficacy of genome editing procedures that utilise homologous or analogous systems in human and other cells.

DNA strand breaks are repaired by DNA synthesis from an exposed DNA end paired with a homologous DNA template. DNA polymerase delta (Pol δ) catalyses DNA synthesis in multiple eukaryotic DNA break repair pathways but triggers genome instability unless its activity is restrained. We show that human HelQ halts DNA synthesis by isolated Pol δ and Pol δ-PCNA-RPA holoenzyme. Using novel HelQ mutant proteins we identify that inhibition of Pol δ is independent of DNA binding, and maps to a 70 amino acid intrinsically disordered region of HelQ. Pol δ and its POLD3 subunit robustly stimulated DNA single-strand annealing by HelQ, and POLD3 and HelQ interact physically via the intrinsically disordered HelQ region. This data, and inability of HelQ to inhibit DNA synthesis by the POLD1 catalytic subunit of Pol δ, reveal a mechanism for limiting DNA synthesis and promoting DNA strand annealing during human DNA break repair, which centres on POLD3.

DNA strand breaks can be repaired by base pairing with unbroken homologous DNA, forming a template for new DNA synthesis that patches over the break site. In eukaryotes multiple DNA break repair pathways utilize DNA polymerase δ to synthesise new DNA from the end of the broken DNA strand. Here we show that DNA synthesis by human Pol δ is halted by the HelQ DNA repair protein directly targeting isolated Pol δ or Pol δ in complex with PCNA and RPA. The mechanism of inhibition by HelQ is independent of DNA binding and maps to a region of HelQ that also modulates DNA binding by RPA. Interaction of HelQ with the POLD3 subunit of Pol δ stimulated DNA single-strand annealing activity of HelQ. The data suggest a mechanism for HelQ restraining the extent of DNA synthesis across multiple DNA break repair pathways.

Introduction: Acute Myeloid Leukemia (AML) is the most common acute leukemia in adults, with roughly 19,000 new diagnoses expected yearly in the United States. Mutations in fms-related tyrosine kinase 3 (FLT3) and nucleophosmin (NPM1) are observed in over one third of all AML patients. These mutations include internal tandem duplications within the juxtamembrane domain (FLT3-ITD; 15-30% of patients) and substitutions within the tyrosine kinase domain (FLT3-TKD; 5-10% of patients) of FLT3, along with 4bp insertions (15-30% of patients) within the C-terminal domain of NPM1. These mutations have significant impacts on prognosis; patients with FLT3-ITDs have poor prognosis while patients with NPM1 mutations without an associated FLT3-ITD mutation have better long-term outcomes. Since characterization of these mutations is critical for accurate therapeutic decisions, assays have been developed to accurately identify these mutations in AML patients. However, these assays lack greater context because they do not identify coexisting mutations in other AML associated genes. As such, they fail to characterize additional prognostic markers that may more fully predict and stratify AML patients’ disease progression. To investigate the limitations of AML individual mutations assays, we identified coexisting mutations in 22 AML patients with known FLT3 and NPM1 mutations using the MyAML™ targeted sequencing panel. Methods: Isolated DNA from 22 AML samples with known FLT3-ITD, FLT3-TKD and NPM1 mutation status was sheared then hybridized to MyAML oligonucleotide baits comprised of exons (coding and non-coding) and breakpoint hotspots from 194 genes known or predicted to be involved in AML pathogenesis. Targeted loci were sequenced on an Illumina MiSeq utilizing v3 chemistry with the 600-cycle kit. By indexing two samples per flowcell, we were able to sequence 12.6 to 32.9 M unique reads per sample, providing an average depth of 985x across the ~3.5Mb target. Using a custom bioinformatics pipeline, we performed mutation detection analyses to identify single nucleotide variants, indels, and structural variant breakpoints. We also calculated variant allelic frequencies to investigate potential aneuploidy, loss of heterozygosity and clonality. Results: Using individual PCR with capillary electrophoresis (PCR/CE) assays, 14 FLT3-ITD, 7 FLT-TKD and 10 NPM1 mutations were initially detected in the 22 AML patient samples. These mutations were 100% concordant with results from the MyAML sequencing data. However, while the individual PCR/CE assays were limited to detecting specific mutations in two genes, the MyAML panel detected 4,172 protein altering variants in 155 of the 192 additionally targeted genes. These include 35 potential mutations in five key AML genes: 13 in DNMT3A, 5 in IDH1, 8 in IDH2, 4 in KIT and 5 in CEBPA. Eight of the 22 patients contained at least two potentially pathogenic mutations in these five genes, with one patient containing mutations in four of the genes. Interestingly, while some co-existing mutations appear to have the same mutant to wild-type allelic ratio as the main FLT3 or NPM1 mutations, others have distinct ratios that may suggest the presence of subclonal cellular populations. For example, we identified an NPM1 Mutation A (c.859_860insTCTG; p.W288fs*>9; COSM158604) in 40.0% of a patient’s sequencing reads, while a co-existing CEPBA missense mutation (c.961A>G; p.N321D; COSM96570) was only present in 4.3% of the sequencing reads. Conclusions: While individual assays for mutations in FLT3, NPM1 and other common AML genes are useful for patient stratification and prognosis, it is crucial to understand these mutations in a greater genomic context. As more AML-related mutations are detected, such as resistance mutations to treatments with novel tyrosine kinase inhibitors, it becomes increasing important to fully characterize a patient’s tumor genome in order to successfully classify and treat their disease. In addition, as more AML patient samples are sequenced, relationships between mutations and their clonal populations can be elucidated, potentially leading to more effective combination therapies. MyAML targeted gene sequencing is the most comprehensive AML specific assay for the identification of somatic and germline driver mutations in their clonal context for the prediction of recurrence and response to various treatment regimens. Disclosures Patay: Genection, Inc.: Consultancy. Cubbon:LabPMM LLC: Employment. Stenzel:Invivoscribe, Inc.: Employment. Miller:Invivoscribe, Inc.: Employment, Equity Ownership.