Genome-destabilizing and mutagenic effects of break-induced replication in Saccharomyces cerevisiae

DNA suffers constant damage, leading to a variety of lesions that require repair. One of the most devastating lesions is a double-strand break (DSB), which results in physical dissociation of two pieces of a chromosome. Necessarily, cells have evolved a number of DSB repair mechanisms. One mechanism of DSB repair is break-induced replication (BIR), which involves invasion of one side of the broken chromosome into a homologous template, followed by copying of the donor molecule through telomeric sequences. BIR is an important cellular process implicated in the restart of collapsed replication forks, as well as in various chromosomal instabilities. Furthermore, BIR uniquely combines processive replication involving a replication fork with DSB repair. This work employs a system in Saccharomyces cerevisiae to investigate genetic control, physical outcomes, and frameshift mutagenesis associated with BIR initiated by a controlled HO-endonuclease break in a chromosome. Mutations in POL32, which encodes a third, non-essential subunit of polymerase δ (Pol), as well as RAD9 and RAD24, which participate in the DNA damage checkpoint response, resulted in a BIR defect characterized by decreased BIR repair and increased loss of the broken chromosome. Also, increased incidence of chromosomal fusions determined to be half-crossover (HCO) molecules was confirmed in pol32Δ and rad24Δ, as well as a rad9Δrad50S double mutant. HCO formation was also stimulated by addition of a replication-inhibiting drug, methyl-methane sulfonate (MMS), to cells undergoing BIR repair. Based on these data, it is proposed that interruption of BIR after it has initiated is one mechanism of HCO formation. Addition of a frameshift mutation reporter to this system allowed mutagenesis associated with BIR DNA synthesis to be measured. It is demonstrated that BIR DNA synthesis is intrinsically inaccurate over the entire path of the replication fork, as the rate of frameshift mutagenesis during BIR is up to 2800-fold higher than normal replication. Importantly, this high rate of mutagenesis was observed not only close to the DSB where BIR is less stable, but also far from the DSB where the BIR replication fork is fast and stabilized. Pol delta proofreading and mismatch repair (MMR) are confirmed to correct BIR errors. Based on these data, it is proposed that a high level of DNA polymerase errors that is not fully compensated by error-correction mechanisms is largely responsible for mutagenesis during BIR. Pif1p, a helicase that is non-essential for DNA replication, and elevated dNTP levels during BIR also contributed to BIR mutagenesis. Taken together, this work characterizes BIR as an essential repair process that also poses risks to a cell, including genome destabilization and hypermutagenesis

Pre-existing Functional Heterogeneity of Tumorigenic Compartment as the Origin of Chemoresistance in Pancreatic Tumors

Summary: Adaptive drug-resistance mechanisms allow human tumors to evade treatment through selection and expansion of treatment-resistant clones. Here, studying clonal evolution of tumor cells derived from human pancreatic tumors, we demonstrate that in vitro cultures and in vivo tumors are maintained by a common set of tumorigenic cells that can be used to establish clonal replica tumors (CRTs), large cohorts of animals bearing human tumors with identical clonal composition. Using CRTs to conduct quantitative assessments of adaptive responses to therapeutics, we uncovered a multitude of functionally heterogeneous subpopulations of cells with differential degrees of drug sensitivity. High-throughput isolation and deep characterization of unique clonal lineages showed genetic and transcriptomic diversity underlying functionally diverse subpopulations. Molecular annotation of gemcitabine-naive clonal lineages with distinct responses to treatment in the context of CRTs generated signatures that can predict the response to chemotherapy, representing a potential biomarker to stratify patients with pancreatic cancer. : High-complexity lineage tracing shows that tumors growing in different environments are maintained by a common set of tumorigenic cells that enables the generation of clonal replica tumors (CRTs). Applying CRTs, Seth et al. unmask functional heterogeneity in response to therapeutics and identify a signature that predicts chemoresistance in pancreatic cancer. Keywords: tumor heterogeneity, functional heterogeneity, lineage tracing, clonal dynamics, clonal isolation, pancreatic cancer, drug resistance, subclonal gene signature, prognostic stratificatio