•Nonhomologous end-joining protein Ku70 is similar at interstitial and subtelomeric DSBs.•Repair proteins RAD51, BRCA1 and CtIP are greatly increased at subtelomeric DSBs.•ATM is reduced at ...subtelomeric DSBs compared to interstitial DSBs.•ATR is increased at subtelomeric DSBs compared to interstitial DSBs.•DSBs near telomeres are more likely to be inappropriately processed.
Telomeres are nucleoprotein structures that are required to protect chromosome ends. Dysfunctional telomeres are recognized as DNA double-strand breaks (DSBs), and elicit the activation of a DNA damage response (DDR). We have previously reported that DSBs near telomeres are poorly repaired, resulting in a high frequency of large deletions and gross chromosome rearrangements (GCRs). Our previous genetic studies have demonstrated that this sensitivity of telomeric regions to DSBs is a result of excessive processing. In the current study, we have further investigated the sensitivity of telomeric regions to DSBs through the analysis of repair proteins associated with DSBs at interstitial and telomeric sites. Following the inducible expression of I-SceI endonuclease, chromatin immunoprecipitation (ChIP) and real-time quantitative PCR were used to compare the recruitment of repair proteins at I-SceI-induced DSBs at interstitial and subtelomeric sites. We observed that proteins that are specifically associated with processing of DSBs during homologous recombination repair, RAD51, BRCA1, and CtIP, are present at a much greater abundance at subtelomeric DSBs. In contrast, Ku70, which is specifically involved in classical nonhomologous end joining, showed no difference at interstitial and subtelomeric DSBs. Importantly, ATM was lower in abundance at subtelomeric DSBs, while ATR was in greater abundance at subtelomeric DSBs, consistent with the accumulation of processed DSBs near telomeres, since processing is accompanied by a transition from ATM to ATR binding. Combined, our results suggest that excessive processing is responsible for the increased frequency of large deletions and GCRs at DSBs near telomeres.
Natural organic matter poses an increasing challenge to water managers because of its potential adverse impacts on water treatment and distribution, and subsequently human health. Projections were ...made of impacts of climate change on dissolved organic carbon (DOC) in the primarily agricultural Boyne catchment which is used as a potable water supply in Ireland. The results indicated that excluding a potential rise in extreme precipitation, future projected loads are not dissimilar to those observed under current conditions. This is because projected increases in DOC concentrations are offset by corresponding decreases in precipitation and hence river flow. However, the results presented assume no changes in land use and highlight the predicted increase in DOC loads from abstracted waters at water treatment plants.
Display omitted
•Potential impacts of climate change on NOM were projected.•Results show future behaviours are similar to current conditions.•Projected increases are offset by parallel decreases in precipitation and flow.
DNA polymerase theta (POLQ)-mediated end joining (TMEJ) is a distinct pathway for mediating DNA double-strand break (DSB) repair. TMEJ is required for the viability of
-mutated cancer cells. It is ...crucial to identify tumors that rely on POLQ activity for DSB repair, because such tumors are defective in other DSB repair pathways and have predicted sensitivity to POLQ inhibition and to cancer therapies that produce DSBs. We define here the
-associated mutation signatures in human cancers, characterized by short insertions and deletions in a specific range of microhomologies. By analyzing 82 COSMIC (Catalogue of Somatic Mutations in Cancer) signatures, we found that
-mutated cancers with a higher level of
expression have a greatly enhanced representation of the small insertion and deletion signature 6, as well as single base substitution signature 3. Using human cancer cells with disruptions of
, we further show that TMEJ dominates end joining of two separated DSBs (distal EJ). Templated insertions with microhomology are enriched in POLQ-dependent distal EJ. The use of this signature analysis will aid in identifying tumors relying on POLQ activity.
Replication stress causes DNA damage at fragile sites in the genome. DNA damage at telomeres can initiate breakage-fusion-bridge cycles and chromosome instability, which can result in replicative ...senescence or tumor formation. Little is known about the extent of replication stress or telomere dysfunction in human embryonic stem cells (hESCs). hESCs are grown in culture with the expectation of being used therapeutically in humans, making it important to minimize the levels of replication stress and telomere dysfunction. Here, the hESC line UCSF4 was cultured in a defined medium with growth factor Activin A, exogenous nucleosides, or DNA polymerase inhibitor aphidicolin. We used quantitative fluorescence in situ hybridization to analyze individual telomeres for dysfunction and observed that it can be increased by aphidicolin or Activin A. In contrast, adding exogenous nucleosides relieved dysfunction, suggesting that telomere dysfunction results from replication stress. Whether these findings can be applied to other hESC lines remains to be determined. However, because the loss of telomeres can lead to chromosome instability and cancer, we conclude that hESCs grown in culture for future therapeutic purposes should be routinely checked for replication stress and telomere dysfunction.
We previously reported that a single DNA double-strand break (DSB) near a telomere in mouse embryonic stem cells can result in chromosome instability. We have observed this same type of instability ...as a result of spontaneous telomere loss in human tumor cell lines, suggesting that a deficiency in the repair of DSBs near telomeres has a role in chromosome instability in human cancer. We have now investigated the frequency of the chromosome instability resulting from DSBs near telomeres in the EJ-30 human bladder carcinoma cell line to determine whether subtelomeric regions are sensitive to DSBs, as previously reported in yeast. These studies involved determining the frequency of large deletions, chromosome rearrangements, and chromosome instability resulting from I-
SceI endonuclease-induced DSBs at interstitial and telomeric sites. As an internal control, we also analyzed the frequency of small deletions, which have been shown to be the most common type of mutation resulting from I-
SceI-induced DSBs at interstitial sites. The results demonstrate that although the frequency of small deletions is similar at interstitial and telomeric DSBs, the frequency of large deletions and chromosome rearrangements is much greater at telomeric DSBs. DSB-induced chromosome rearrangements at telomeric sites also resulted in prolonged periods of chromosome instability. Telomeric regions in mammalian cells are therefore highly sensitive to DSBs, suggesting that spontaneous or ionizing radiation-induced DSBs at these locations may be responsible for many of the chromosome rearrangements that are associated with human cancer.
Previous work has shown that high charge and energy particle irradiation of human cells evokes a mutagenic repair phenotype, defined by increased mutagenic repair of new double-strand breaks that are ...introduced enzymatically, days or weeks after the initial irradiation. The effect was seen originally with 600 MeV/u 56Fe particles, which have a linear energy transfer (LET) value of 174 keV/μm, but not with X rays or γ rays (LET ≤ 2 keV/μm). To better define the radiation quality dependence of the phenomenon, we tested two ions with intermediate LET values, 1,000 MeV/u 48Ti (LET = 108 keV/μm) and 300 MeV/u 28Si (LET = 69 keV/μm). These experiments used a previously validated assay, where a rare-cutting nuclease introduces double-strand breaks in two reporter transgene cassettes, which are located on different chromosomes. Deletions of a block of sequence in one of the cassettes, or translocations between cassettes, are measured independently using a multicolor fluorescence assay. The results showed that 48Ti was a potent, but transient, inducer of mutagenic repair, based on increased frequency of nuclease-induced translocations. The 48Ti ions did not affect the frequency of nuclease-induced deletions. The 28Si ions had no measurable effect on either endpoint. There was a close correlation between the induction of the mutagenic repair phenomenon and the frequency of micronuclei in the targeted population (R2 = 0.74), whereas there was no apparent correlation with radiation-induced cell inactivation. Together, these results better define the radiation quality dependence of the mutagenic repair phenomenon and establish its correlation, or lack of correlation, with other endpoints.
The carcinogenic risk of high-charge and energy (HZE) particle exposure arises from its ability to both induce complex DNA damage and from its ability to evoke deleterious, non-DNA targeted effects. ...We investigate here whether these nontargeted effects involve dysregulation of double-strand break repair, such that a history of HZE exposure heightens the risks from future injury. We used a new human cell reporter line, in which expression of the I-SceI meganuclease stimulates both translocations on different chromosomes, and deletions on the same chromosome. Exposure to 1.0 Gy of 600 MeV/u 56Fe ions led to a doubling in the frequency of I-SceI-mediated translocations and a smaller, but nevertheless significant, increase in the frequency of I-SceI-mediated deletions. This mutagenic repair phenotype persisted for up to two weeks and eight population doublings. The phenotype was not induced by low-linear energy transfer radiation or by a lower dose of HZE-particle radiation (0.3 Gy) indicating that the effect is radiation quality and dose dependent. The mutagenic repair phenotype was associated with the presence of micronuclei and persistent DSB repair foci, consistent with a hypothesis that genomic stress is a causative factor.
The mobilisation and impact of roadway runoff on dairy farms has been established as a sub-component of the nutrient transfer continuum. It is acknowledged that fresh nutrient sources deposited on ...roadways dominate runoff and recent work has shown that available phosphorus (P) in roadway substrates is also an important source component. The objectives of this study were to understand spatial and temporal available P concentrations in roadway substrate (aggregate and soil mix) across dairy and beef farms during the open (February–October, when cows use the roadway network and fields) and closed (November–January, when cows were housed) periods and identify locations which could be considered critical source areas (CSA). For the study, roadway substrate samples were taken at 18 locations on each farm, across eight sampling periods, and were analysed for available P. Results showed that mean available P concentrations (Morgan’s P) in the sampling locations ranged from 15.9 to 101.4 mg L -1 for the Dairy Farm, from 4.1 to 59.4 mg L -1 for Beef Farm 1 and from 6.3 to 23.2 mg L -1 for Beef Farm 2. In open period, the results showed that mean available P concentrations were 75.4 ± 30.9, 14.9 ± 3.2 and 13.4 ± 2.5 mg L -1 for the Dairy Farm, Beef Farm 1 and Beef Farm 2, respectively. In closed period, the mean available P were 40.3 ± 17.2, 10.4 ± 1.0 and 9.8 ± 0.2 mg L -1 for Dairy Farm, Beef Farm 1 and Beef Farm 2, respectively. Overall, P concentrations on the Dairy Farm roadways was up to 4 times greater than that in the Beef Farms’ roadways. Compared to soils in adjacent fields, P concentrations in the Dairy and Beef Farms roadways was up-to 7 and 2 times higher, respectively. Critical roadway sections that required mitigation were two in the Dairy Farm, one in Beef Farm 1 and two in Beef Farm 2. In addition to fresh faeces and urine (i.e., dominant source of nutrients in farm roadways), this study showed that nutrient enriched roadway materials are a labile P source. Thus, future mitigation of roadway runoff must consider all roadway nutrient sources, including livestock fresh excreta as well as used roadway surface materials.
Telomerase serves to maintain telomeric repeat sequences at the ends of chromosomes. However, telomerase can also add telomeric repeat sequences at DNA double-strand breaks (DSBs), a process called ...chromosome healing. Here, we employed a method of inducing DSBs near telomeres to query the role of two proteins, PIF1 and NBS1, in chromosome healing in mammalian cells. PIF1 was investigated because the PIF1 homolog in
Saccharomyces cerevisiae inhibits chromosome healing, as shown by a 1000-fold increase in chromosome in PIF1-deficient cells. NBS1 was investigated because the functional homolog of NBS1 in
S. cerevisiae, Xrs2, is part of the Mre11/Rad50/Xrs2 complex that is required for chromosome healing due to its role in the processing of DSBs and recruitment of telomerase. We found that disruption of
mPif1 had no detectable effect on the frequency of chromosome healing at DSBs near telomeres in murine embryonic stem cells. Moreover, the Nbs1
ΔB hypomorph, which is defective in the processing of DSBs, also had no detectable effect on the frequency of chromosome healing, DNA degradation, or gross chromosome rearrangements (GCRs) that result from telomeric DSBs. Although we cannot rule out small changes in chromosome healing using this system, it is clear from our results that knockout of PIF1 or the Nbs1
ΔB hypomorph does not result in large differences in chromosome healing in murine cells. These results represent the first genetic assessment of the role of these proteins in chromosome healing in mammals, and suggest that murine cells have evolved mechanisms to ensure the functional redundancy of Pif1 or Nbs1 in the regulation of chromosome healing.
PDF
