DNA replication stress: Causes, resolution and disease

AbstractDNA replication is a fundamental process of the cell that ensures accurate duplication of the genetic information and subsequent transfer to daughter cells. Various pertubations, originating from endogenous or exogenous sources, can interfere with proper progression and completion of the replication process, thus threatening genome integrity. Coordinated regulation of replication and the DNA damage response is therefore fundamental to counteract these challenges and ensure accurate synthesis of the genetic material under conditions of replication stress. In this review, we summarize the main sources of replication stress and the DNA damage signaling pathways that are activated in order to preserve genome integrity during DNA replication. We also discuss the association of replication stress and DNA damage in human disease and future perspectives in the field

Parallel genome-wide screens identify synthetic viable interactions between the BLM helicase complex and Fanconi anemia.

Maintenance of genome integrity via repair of DNA damage is a key biological process required to suppress diseases, including Fanconi anemia (FA). We generated loss-of-function human haploid cells for FA complementation group C (FANCC), a gene encoding a component of the FA core complex, and used genome-wide CRISPR libraries as well as insertional mutagenesis to identify synthetic viable (genetic suppressor) interactions for FA. Here we show that loss of the BLM helicase complex suppresses FANCC phenotypes and we confirm this interaction in cells deficient for FA complementation group I and D2 (FANCI and FANCD2) that function as part of the FA I-D2 complex, indicating that this interaction is not limited to the FA core complex, hence demonstrating that systematic genome-wide screening approaches can be used to reveal genetic viable interactions for DNA repair defects

Map of synthetic rescue interactions for the Fanconi anemia DNA repair pathway identifies USP48

Defects in DNA repair can cause various genetic diseases with severe pathological phenotypes. Fanconi anemia (FA) is a rare disease characterized by bone marrow failure, developmental abnormalities and increased cancer risk that is caused by defective repair of DNA interstrand crosslinks (ICLs). By performing genome-wide loss-of-function screens across a panel of human haploid isogenic FA-defective cells (FANCA, FANCC, FANCG, FANCI, FANCD2), we identified the deubiquitylating enzyme USP48 as synthetic viable for FA gene deficiencies. Thus, as compared to FA-defective cells alone, FA-deficient cells additionally lacking USP48 are less sensitive to genotoxic stress induced by ICL agents and display enhanced, BRCA1-dependent, clearance of DNA damage. Consequently, USP48 inactivation reduces chromosomal instability of FA-defective cells. Our results highlight a role for USP48 in controlling DNA repair and suggest it as a potential target that could be therapeutically exploited for FA

Effect of partial and complete supression of the pimer ARF - double strand DNA break response pathway in cancer development: role in therapeutic approach

The DNA damage response (DDR) pathway and ARF function act as barriers of human cancer development. It has been considered that the DDR and ARF exert this function independently of each other. However, a few studies propose that ARF’s activity is positively regulated by the DDR pathway. Examining this hypothesis we performed a series of experiments using molecular techniques such as immunoblotting, immunohistochemistry, immunofluorescence, immunoprecipitation, Real Time Reverse Transcritpion polymerase chain reaction (RT-PCR) in biological material from cell culture or histological samples, as well as ectopic protein expression through plasmid transfections, proteomic analyses, ribosome RNA biogenesis assay and xenografts of human cancer cells. We surprisingly found that ATM suppressed, in a transcription-independent manner, ARF protein levels and activity. Specifically, ATM activated protein phosphatase 1 (PP1). PP1 antagonized Nek2-dependent phosphorylation of nucleophosmin (NPM), liberating ARF from NPM and rendering it susceptible to degradation by the ULF E3-ubiquitin ligase. In human clinical samples, loss of ATM expression correlated with increased ARF levels and in xenograft and tissue culture models, inhibition of ATM stimulated the tumour-suppressive effects of ARF. The importance of the proposed mechanism can be exploited through a therapeutic approach, especially in cases of tumours bearing loss of p53. In such tumours, DDR may act in favour of the tumour cells, since the major effector of the antiumour barriers of apoptosis and senescence is absent, but ATM inhbition could boost ARF’s tumour-suppressive function, contributing to an anti-tumour response

DNA Repair Cofactors ATMIN and NBS1 Are Required to Suppress T Cell Activation

<div><p>Proper development of the immune system is an intricate process dependent on many factors, including an intact DNA damage response. The DNA double-strand break signaling kinase ATM and its cofactor NBS1 are required during T cell development and for the maintenance of genomic stability. The role of a second ATM cofactor, ATMIN (also known as ASCIZ) in T cells is much less clear, and whether ATMIN and NBS1 function in synergy in T cells is unknown. Here, we investigate the roles of ATMIN and NBS1, either alone or in combination, using murine models. We show loss of NBS1 led to a developmental block at the double-positive stage of T cell development, as well as reduced TCRα recombination, that was unexpectedly neither exacerbated nor alleviated by concomitant loss of ATMIN. In contrast, loss of both ATMIN and NBS1 enhanced DNA damage that drove spontaneous peripheral T cell hyperactivation, proliferation as well as excessive production of proinflammatory cytokines and chemokines, leading to a highly inflammatory environment. Intriguingly, the disease causing T cells were largely proficient for both ATMIN and NBS1. <i>In vivo</i> this resulted in severe intestinal inflammation, colitis and premature death. Our findings reveal a novel model for an intestinal bowel disease phenotype that occurs upon combined loss of the DNA repair cofactors ATMIN and NBS1.</p></div

Functional interplay between the DNA-damage-response kinase ATM and ARF tumour suppressor protein in human cancer

The DNA damage response (DDR) pathway and ARF function as barriers to cancer development. Although commonly regarded as operating independently of each other, some studies proposed that ARF is positively regulated by the DDR. Contrary to either scenario, we found that in human oncogene-transformed and cancer cells, ATM suppressed ARF protein levels and activity in a transcription-independent manner. Mechanistically, ATM activated protein phosphatase 1, which antagonized Nek2-dependent phosphorylation of nucleophosmin (NPM), thereby liberating ARF from NPM and rendering it susceptible to degradation by the ULF E3-ubiquitin ligase. In human clinical samples, loss of ATM expression correlated with increased ARF levels and in xenograft and tissue culture models, inhibition of ATM stimulated the tumour-suppressive effects of ARF. These results provide insights into the functional interplay between the DDR and ARF anti-cancer barriers, with implications for tumorigenesis and treatment of advanced tumours.</p

Loss of ATMIN and NBS1 in T cells leads to the accumulation of DNA damage.

<p>(A) Representative images of FACS sorted (for TCRβ⁺ populations) splenic T cells from control, ATM^-/-, ATMIN^ΔL, NBS1^ΔL and ATMIN^ΔLNBS1^ΔL mice, analysed using the alkali comet assay. (B) Quantification of A. N = 3 mice per genotype. FACS sorted splenic T cells from NBS1^ΔL and ATMIN^ΔLNBS1^ΔL mice were additionally sorted and analysed based on YFP expression. (C) Splenic sections from control, ATM^-/-, ATMIN^ΔL, NBS1^ΔL and ATMIN^ΔLNBS1^ΔL mice were co-stained for TUNEL and γH2AX. Nuclei were counterstained with DAPI. (D) Western blot analysis of splenic cells from control, ATM^-/-, ATMIN^ΔL, NBS1^ΔL and ATMIN^ΔLNBS1^ΔL mice for pS15-p53, total p53 and actin. (E) Representative flow cytometry data of CD11b⁺Gr1⁺ neutrophils in the spleen of control, ATMIN^ΔL, NBS1^ΔL and ATMIN^ΔLNBS1^ΔL mice. (F) Quantification of E. N = 7–10 mice per genotype. Error bars represent SEM (*<i>P</i><0.05, **<i>P</i><0.01, **** <i>P</i><0.0001).</p

Loss of ATMIN in combination with NBS1, in T cells, leads to increased mortality due to T cell activation.

<p>(A) Kaplan-Meier survival curve of control, ATMIN^ΔL, NBS1^ΔL, ATMIN^ΔLNBS1^ΔL and ATM^-/- mice. Survival was monitored for 32 weeks. (B) Representative images of spleens of control, ATMIN^ΔL, NBS1^ΔL and ATMIN^ΔLNBS1^ΔL mice as well as a moribund ATMIN^ΔLNBS1^ΔL mouse. (C) Histological analysis of the spleen of a control and a moribund ATMIN^ΔLNBS1^ΔL mouse stained for T cells using an anti-CD3 antibody. (D) Representative flow cytometry data of CD4 and CD8 T cells (gated on the TCRβ⁺ population) in the spleen of control, ATMIN^ΔL, NBS1^ΔL and ATMIN^ΔLNBS1^ΔL mice. (E) Histological analysis by using an anti-CD3 antibody to visualize T cells in the liver and lung of control and ATMIN^ΔLNBS1^ΔL mice. (F) Representative flow cytometry data of activated CD62L^lowCD44⁺ T cells (gated on the TCRβ⁺ population) in the spleen of control, ATMIN^ΔL, NBS1^ΔL and ATMIN^ΔLNBS1^ΔL mice. (G) Quantification of F. N = 3–4 mice per genotype. (H) Flow cytometry data showing the percentage of antigen-experienced CD62L^lowCD4⁺ T cells (gated on the TCRβ⁺ population) in the spleen of control and ATMIN^ΔLNBS1^ΔL mice as well as moribund ATMIN^ΔLNBS1^ΔL mice. (I) Representative flow cytometry data of proliferating (BrdU⁺) T cells (gated on the TCRβ⁺ population) in the spleen of mice indicated in F, measured by <i>in vivo</i> BrdU incorporation over a period of 4 days. (J) Quantification of I. N = 3–4 mice per genotype. Error bars represent SEM (*<i>P</i><0.05, ***<i>P</i><0.001).</p

Mice deficient for ATMIN and NBS1 in T cells produce inflammatory cytokines in the intestine and are hypersensitive to colitis.

<p>(A-C) Quantitative RT-PCR analysis of expression of proinflammatory cytokines Il17a, Tnfα and Il1β in the IELs of control, ATMIN^ΔL, NBS1^ΔL and ATMIN^ΔLNBS1^ΔL with (‘anti-CD3/CD28’) or without (‘unstim’) 12 hour <i>in vitro</i> stimulation with anti-CD3 and anti-CD28 antibodies. Gene expression is normalized to mef1α control. N = 4–8 mice per genotype. (D) Percentage of weight change upon DSS treatment of ATMIN^ΔL, NBS1^ΔL, ATMIN^ΔLNBS1^ΔL and ATM^-/- mice for 8 days. Each genotype is normalized to its respective control. N = 4 mice per genotype. (E-G) Quantitative RT-PCR analysis of expression of proinflammatory cytokines Il17a, Tnfα and Ifnγ in small intestine of DSS treated (‘+DSS’) or untreated (‘-DSS’) control, ATMIN^ΔL, NBS1^ΔL and ATMIN^ΔLNBS1^ΔL mice. N = 4 mice per genotype. (H) Histological analysis by H&E staining of large intestine of DSS treated control and ATMIN^ΔLNBS1^ΔL mice. (I) Histological analysis of control and ATMIN^ΔLNBS1^ΔL mice without (water) or with DSS. Error bars represent SEM (*<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001).</p

Model for the role of ATMIN and NBS1 in suppressing T cell activation and inflammation.

<p>Proposed model depicting mechanisms of inflammation caused by concomitant deletion of ATMIN and NBS1 (details can be found in the text).</p