The Transcriptional Response to DNA-Double-Strand Breaks in Physcomitrella patens

The model bryophyte Physcomitrella patens is unique among plants in supporting the generation of mutant alleles by facile homologous recombination-mediated gene targeting (GT). Reasoning that targeted transgene integration occurs through the capture of transforming DNA by the homology-dependent pathway for DNA double-strand break (DNA-DSB) repair, we analysed the genome-wide transcriptomic response to bleomycin-induced DNA damage and generated mutants in candidate DNA repair genes. Massively parallel (Illumina) cDNA sequencing identified potential participants in gene targeting. Transcripts encoding DNA repair proteins active in multiple repair pathways were significantly up-regulated. These included Rad51, CtIP, DNA ligase 1, Replication protein A and ATR in homology-dependent repair, Xrcc4, DNA ligase 4, Ku70 and Ku80 in non-homologous end-joining and Rad1, Tebichi/polymerase theta, PARP in microhomology-mediated end-joining. Differentially regulated cell-cycle components included up-regulated Rad9 and Hus1 DNA-damage-related checkpoint proteins and down-regulated D-type cyclins and B-type CDKs, commensurate with the imposition of a checkpoint at G2 of the cell cycle characteristic of homology-dependent DNA-DSB repair. Candidate genes, including ATP-dependent chromatin remodelling helicases associated with repair and recombination, were knocked out and analysed for growth defects, hypersensitivity to DNA damage and reduced GT efficiency. Targeted knockout of PpCtIP, a cell-cycle activated mediator of homology-dependent DSB resection, resulted in bleomycin-hypersensitivity and greatly reduced GT efficiency

Modeling double strand break susceptibility to interrogate structural variation in cancer

Abstract Background Structural variants (SVs) are known to play important roles in a variety of cancers, but their origins and functional consequences are still poorly understood. Many SVs are thought to emerge from errors in the repair processes following DNA double strand breaks (DSBs). Results We used experimentally quantified DSB frequencies in cell lines with matched chromatin and sequence features to derive the first quantitative genome-wide models of DSB susceptibility. These models are accurate and provide novel insights into the mutational mechanisms generating DSBs. Models trained in one cell type can be successfully applied to others, but a substantial proportion of DSBs appear to reflect cell type-specific processes. Using model predictions as a proxy for susceptibility to DSBs in tumors, many SV-enriched regions appear to be poorly explained by selectively neutral mutational bias alone. A substantial number of these regions show unexpectedly high SV breakpoint frequencies given their predicted susceptibility to mutation and are therefore credible targets of positive selection in tumors. These putatively positively selected SV hotspots are enriched for genes previously shown to be oncogenic. In contrast, several hundred regions across the genome show unexpectedly low levels of SVs, given their relatively high susceptibility to mutation. These novel coldspot regions appear to be subject to purifying selection in tumors and are enriched for active promoters and enhancers. Conclusions We conclude that models of DSB susceptibility offer a rigorous approach to the inference of SVs putatively subject to selection in tumors

Drosophila DNA polymerase theta utilizes both helicase-like and polymerase domains during microhomology-mediated end joining and interstrand crosslink repair

Double strand breaks (DSBs) and interstrand crosslinks (ICLs) are toxic DNA lesions that can be repaired through multiple pathways, some of which involve shared proteins. One of these proteins, DNA Polymerase theta (Pol theta), coordinates a mutagenic DSB repair pathway named microhomology-mediated end joining (MMEJ) and is also a critical component for bypass or repair of ICLs in several organisms. Pol theta contains both polymerase and helicase-like domains that are tethered by an unstructured central region. While the role of the polymerase domain in promoting MMEJ has been studied extensively both in vitro and in vivo, a function for the helicase-like domain, which possesses DNA-dependent ATPase activity, remains unclear. Here, we utilize genetic and biochemical analyses to examine the roles of the helicase-like and polymerase domains of Drosophila Pol theta. We demonstrate an absolute requirement for both polymerase and ATPase activities during ICL repair in vivo. However, similar to mammalian systems, polymerase activity, but not ATPase activity, is required for ionizing radiation-induced DSB repair. Using a site-specific break repair assay, we show that overall end-joining efficiency is not affected in ATPase-dead mutants, but there is a significant decrease in templated insertion events. In vitro, Pol theta can efficiently bypass a model unhooked nitrogen mustard crosslink and promote DNA synthesis following microhomology annealing, although ATPase activity is not required for these functions. Together, our data illustrate the functional importance of the helicase-like domain of Pol theta and suggest that its tethering to the polymerase domain is important for its multiple functions in DNA repair and damage tolerance

Ortholog of the polymerase theta helicase domain modulates DNA replication in Trypanosoma cruzi

DNA polymerase theta (Polθ), a member of the DNA polymerase family A, exhibits a polymerase C-terminal domain, a central domain, and an N-terminal helicase domain. Polθ plays important roles in DNA repair via its polymerase domain, regulating genome integrity. In addition, in mammals, Polθ modulates origin firing timing and MCM helicase recruitment to chromatin. In contrast, as a model eukaryote, Trypanosoma cruzi exhibits two individual putative orthologs of Polθ in different genomic loci; one ortholog is homologous to the Polθ C-terminal polymerase domain, and the other is homologous to the Polθ helicase domain, called Polθ-polymerase and Polθ-helicase, respectively. A pull-down assay using the T. cruzi component of the prereplication complex Orc1/Cdc6 as bait captured Polθ-helicase from the nuclear extract. Orc1/Cdc6 and Polθ-helicase directly interacted, and Polθ-helicase presented DNA unwinding and ATPase activities. A T. cruzi strain overexpressing the Polθ-helicase domain exhibited a significantly decreased amount of DNA-bound MCM7 and impaired replication origin firing. Taken together, these data suggest that Polθ-helicase modulates DNA replication by directly interacting with Orc1/Cdc6, which reduces the binding of MCM7 to DNA and thereby impairs the firing of replication origins

Using State Space Exploration to Determine How Gene Regulatory Networks Constrain Mutation Order in Cancer Evolution

Cancer develops via the progressive accumulation of somatic mutations, which subvert the normal operation of the gene regulatory network of the cell. However, little is known about the order in which mutations are acquired in successful clones. A particular sequence of mutations may confer an early selective advantage to a clone by increasing survival or proliferation, or lead to negative selection by triggering cell death. The space of allowed sequences of mutations is therefore constrained by the gene regulatory network. Here, we introduce a methodology for the systematic exploration of the effect of every possible sequence of oncogenic mutations in a cancer cell modelled as a qualitative network. Our method uses attractor identification using binary decision diagrams and can be applied to both synchronous and asynchronous systems. We demonstrate our method using a recently developed model of ER-negative breast cancer. We show that there are differing levels of constraint in the order of mutations for different combinations of oncogenes, and that the effects of ErbB2/HER2 over-expression depend on the preceding mutations

Measuring single cell divisions in human tissues from multi-region sequencing data.

Both normal tissue development and cancer growth are driven by a branching process of cell division and mutation accumulation that leads to intra-tissue genetic heterogeneity. However, quantifying somatic evolution in humans remains challenging. Here, we show that multi-sample genomic data from a single time point of normal and cancer tissues contains information on single-cell divisions. We present a new theoretical framework that, applied to whole-genome sequencing data of healthy tissue and cancer, allows inferring the mutation rate and the cell survival/death rate per division. On average, we found that cells accumulate 1.14 mutations per cell division in healthy haematopoiesis and 1.37 mutations per division in brain development. In both tissues, cell survival was maximal during early development. Analysis of 131 biopsies from 16 tumours showed 4 to 100 times increased mutation rates compared to healthy development and substantial inter-patient variation of cell survival/death rates

Evolution of a Higher Intracellular Oxidizing Environment in Caenorhabditis elegans under Relaxed Selection

We explored the relationship between relaxed selection, oxidative stress, and spontaneous mutation in a set of mutationaccumulation (MA) lines of the nematode Caenorhabditis elegans and in their common ancestor. We measured steady-state levels of free radicals and oxidatively damaged guanosine nucleosides in the somatic tissues of five MA lines for which nuclear genome base substitution and GC-TA transversion frequencies are known. The two markers of oxidative stress are highly correlated and are elevated in the MA lines relative to the ancestor; point estimates of the per-generation rate of mutational decay (DM) of these measures of oxidative stress are similar to those reported for fitness-related traits. Conversely, there is no significant relationship between either marker of oxidative stress and the per-generation frequencies of base substitution or GC-TA transversion. Although these results provide no direct evidence for a causative relationship between oxidative damage and base substitution mutations, to the extent that oxidative damage may be weakly mutagenic in the germline, the case for condition-dependent mutation is advanced