Absolute quantification of DNA damage response proteins

Timeless (Tim), a core circadian clock gene in Drosophila, is retained in mammals but has no apparent mammalian circadian clock function. Mammalian TIM is essential for ATR-dependent Chk1 activation and S-phase arrest. We report that TIM is likewise essential for ATM-dependent Chk2-mediated signaling of doxorubicin-induced DNA double strand breaks. TIM depletion attenuates doxorubicin-induced G(2)/M cell cycle arrest and sensitizes cancer cells to doxorubicin-induced cytotoxicity. TIM is, thereby, a potential novel anticancer drug target whose inhibition may enhance the therapeutic cytotoxicity of agents that activate DNA damage pathways as part of their mechanism.

This article refers to:A small ubiquitin binding domain inhibits ubiquitin-dependent protein recruitment to DNA repair foci

Primary cilia have been considered tumor-suppressing organelles in cholangiocarcinoma (CCA), though the mechanisms behind their protective role are not fully understood. This study investigates how the loss of primary cilia affects DNA damage response (DDR) and DNA repair processes. Human cholangiocyte cell lines were used to examine the colocalization of DNA repair proteins at the cilia and assess the impact of experimental deciliation on DNA repair pathways. Deciliation was induced using shRNA knockdown or CRISPR knockout of IFT20, IFT88, or KIF3A, followed by exposure to the genotoxic agents cisplatin, methyl methanesulfonate (MMS), or irradiation. Cell survival, cell cycle progression, and apoptosis rates were evaluated, and DNA damage was assessed using comet assays and phosphorylated H2AX (γH2AX) quantification. An in vivo liver-specific IFT88 knockout model, generated using Albumin-Cre/Lox recombination, was used to study the loss of primary cilia in the liver. Results showed that RAD51 localized predominantly at the base of the cilium, whereas Ataxia Telangiectasia and Rad3-related protein (ATR), PARP1, CHK1, and CHK2 were also detected within the ciliary shaft. Deciliated cells displayed dysregulation in critical DNA repair pathways. These cells also showed reduced survival and increased S-phase arrest after genotoxic challenges as compared with ciliated cells. Enhanced DNA damage was observed via increased γH2AX signals and comet assay results. An increase in γH2AX expression was also observed in our in vivo model, indicating elevated DNA damage. In addition, key DDR proteins such as Ataxia Telangiectasia Mutated protein (ATM), p53, and p21, were downregulated in deciliated cells after irradiation. This study underscores the crucial role of primary cilia in regulating DNA repair and suggests that targeting cilia-related mechanisms could present a novel therapeutic approach for CCA.NEW & NOTEWORTHY Our study reveals a novel link between primary cilia and DNA repair in cholangiocytes. We show that DNA damage response (DDR) and repair proteins localize to cilia, and deciliation impairs survival and induces S-phase arrest under genotoxic stress. Deciliated cells exhibit increased DNA damage after cisplatin, irradiation, or methyl methanesulfonate (MMS) challenge. Following irradiation, Ataxia Telangiectasia Mutated protein (ATM), p53, and p21 are downregulated in deciliated cells. Similarly, IFT88 knockout mice show heightened DNA damage, highlighting the role of primary cilia in genome stability.

Poly(ADP-ribose) polymerase 1 (PARP1) is a nuclear enzyme that is rapidly activated by DNA strand breaks and signals the presence of DNA lesions by attaching ADP-ribose units to chromatin-associated proteins. The therapeutic applications of PARP inhibitors in potentiating the killing action of ionizing radiation have been well documented and are attracting increasing interest as a cancer treatment. However, the initial kinetics underlying the recognition of multiple DNA lesions by PARP1 and how inhibition of PARP potentiates the activity of DNA-damaging agents are unknown. Here we report the spatiotemporal dynamics of PARP1 recruitment to DNA damage induced by laser microirradiation in single living cells. We provide direct evidence that PARP1 is able to accumulate at a locally induced DNA double strand break. Most importantly, we observed that the rapid accumulation of MRE11 and NBS1 at sites of DNA damage requires PARP1. By determining the kinetics of protein assembly following DNA damage, our study reveals the cooperation between PARP1 and the double strand break sensors MRE11 and NBS1 in the close vicinity of a DNA lesion. This may explain the sensitivity of cancer cells to PARP inhibitors.

Fanconi Anemia and the Response of Human Hematopoietic Cells to DNA Damage

Double-strand breaks (DSBs) of chromosomal DNA trigger the cellular response that activates the pathways for DNA repair and cell-cycle checkpoints, and sometimes the pathways leading to cell death if the damage is too severe to be tolerated. Evidence indicates that, upon generation of DNA DSBs, many nuclear proteins that are involved in DNA repair and checkpoints are recruited to chromatin around the DNA lesions. In the present study we used a proteomics approach to identify DNA-damage-induced chromatin-binding proteins in a systematic way. Two-dimensional gel analysis for protein extracts of chromatin from DNA-damage-induced and control HeLa cells identified four proteins as the candidates for DNA-damage-induced chromatin-binding proteins. MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS analysis identified these proteins to be NPM (nucleophosmin), hnRNP (heterogeneous nuclear ribonucleoprotein) C1, hnRNP C2 and 37-kDa laminin-receptor precursor, and the identity of these proteins was further confirmed by immunoblot analysis with specific antibodies. We then demonstrated with chromatin-binding assays that NPM and hnRNP C1/C2, the abundant nuclear proteins with pleiotropic functions, indeed bind to chromatin in a DNA-damage-dependent manner, implicating these proteins in DNA repair and/or damage response. Immunofluorescence experiments showed that NPM, normally present in the nucleoli, is mobilized into the nucleoplasm after DNA damage, and that neither NPM nor hnRNP C1/C2 is actively recruited to the sites of DNA breaks. These results suggest that NPM and hnRNP C1/C2 may function at the levels of the global context of chromatin, rather than by specifically targeting the broken DNA.

DNA double-strand breaks (DSBs) are the most severe type of DNA damage. Occurrence of DSBs in the cell activates the DNA damage response (DDR), which involves signaling cascades that sense and respond to the damage. Promptly after DSB induction, DDR proteins accumulate surrounding both DNA ends and form microscopically-visible foci. Recently, we demonstrated that the key DDR protein MDC1 directly binds RAP80, an additional DDR protein that recruits BRCA1 to DSBs. We provided evidences that the MDC1-RAP80 interaction depends on a ubiquitylation event on K-1977 of MDC1. However, it remained unknown whether K-1977 of MDC1 is required for the recruitment of RAP80 to DSBs. Here we show that K-1977 of MDC1 is necessary for focus formation by RAP80. Nevertheless, it has not effect on focus formation by γ-H2AX, MDC1 or 53BP1. The results imply a role for the MDC1-RAP80 interaction in focus formation by the RAP80-BRCA1 complex. In light of these recent results we discuss several aspects of the complexity of focus formation and present a model for the involvement of individual and complex recruitment mechanisms in focus formation.

The DNA damage, most notably DNA double-strand breaks, poses a serious threat to the stability of mammalian genome. Maintenance of genomic integrity is largely dependent on an efficient, accurate, and timely DNA damage response in the context of chromatin. Consequently, dysregulation of the DNA damage response machinery is fundamentally linked to the genomic instability and a likely predisposition to cancer. In turn, aberrant activation of DNA damage response pathways in human cancers enables tumor cells to survive DNA damages, thus, leading to the development of resistance of tumor cells to DNA damaging radio- and chemotherapies. A substantial body of experimental evidence has established that ATP-dependent chromatin remodeling and histone modifications play a central role in the DNA damage response. As a component of the nucleosome remodeling and histone deacetylase (NuRD) complex that couples both ATP-dependent chromatin remodeling and histone deacetylase activities, the metastasis-associated protein (MTA) family proteins have been recently shown to participate in the DNA damage response beyond its well-established roles in gene transcription. In this thematic review, we will focus on our current understandings of the role of the MTA family proteins in the DNA damage response and their potential implications in DNA damaging anticancer therapy.

2-Mercaptoethanol protects against DNA double-strand breaks after kidney ischemia and reperfusion injury through GPX4 upregulation.

Protein phosphorylation mediated by checkpoint kinases is crucial for the cellular
\nresponse to DNA damage. The sensor kinases Mec1 and Tel1 initiate the checkpoint
\nsignaling cascade by directly activating the checkpoint effector kinase Rad53. This
\ncheckpoint pathway, however, is responsive to normal endogenous replication as
\nwell. As a result an S-phase specific threshold for Rad53 activation exists, which
\nallows cells to tolerate endogenous damage-like structures.
\nHere we show that Rad53 itself is phosphorylated in a cell-cycle dependent manner
\nindependent of DNA damage signaling (Chapter 2). We propose that this is part of the
\ncell-cycle regulated sensitivity of Rad53 to activation. This phosphorylation occurs in
\nG2/M, persists until S phase onset and depends on both the polo-like kinase, Cdc5,
\nand the cyclin-dependent kinase, Cdc28. These cell-cycle dependent phosphorylation
\nevents are located in the C-terminal part of Rad53. Serines 774 and 789 were shown
\nto be phosphorylated by mass spectrometry. Mutation of these sites eliminated the
\ncell-cycle dependent phosphorylation of Rad53 and partially impaired the activation
\nof Rad53 in response to minor amounts of DNA damage in G2/M. This led to more
\nrapid checkpoint adaptation in response to irreparable DNA damage. Thus, cell-cycle
\ndependent phosphorylation in the C-terminal part of Rad53 enhances Rad53
\nactivation in response to DNA double strand breaks (DSBs).
\nMec1 and Tel1 initiate a response to DNA damage independently of Rad53. The
\nphosphorylation of histone H2A at serine 129 (γH2A) at DSBs by Mec1 and Tel1 has
\nan important role in mediating DNA repair. This study shows that the occurrence of
\nγH2A is not limited to DSBs, but also occurs at stalled replication forks (Chapter 3).
\nUsing chromatin immunoprecipitation high γH2A levels were monitored at
\nhydroxyurea-stalled replication forks and depended nearly exclusively upon Mec1
\nkinase activity. Furthermore our study showed that γH2A not only occurs at damaged
\nchromatin but in regions of normally replicating chromatin and near telomeres
\n(Chapter 4). High levels of γH2A could be monitored both in the rDNA of normally
\ngrowing yeast cells and at telomeres. Here γH2A depended mainly on Tel1 and
\nγH2A levels increased during S phase and during the elongation of critically short
\ntelomeres. We also provide evidence that γH2A contributes to telomeric anchoring in
\nS phase yeast cells in addition to the yKu and Sir4 anchoring pathways.

Differential expression of TP53 associated genes in Fanconi anemia cells after mitomycin C and hydroxyurea treatment

Lamin A/C, a core component of the nuclear lamina, forms a mesh-like structure beneath the inner nuclear membrane. While its structural role is well-studied, its involvement in DNA metabolism remains unclear. We conducted sequential protein fractionation to determine the subcellular localization of early DNA damage response (DDR) proteins. Our findings indicate that most DDR proteins, including ATM and the MRE11-RAD50-NBS1 (MRN) complex, are present in the nuclease – and high salt-resistant pellet fraction. Notably, ATM and MRN remain stably associated with these structures throughout the cell cycle, independent of ionizing radiation (IR)-induced DNA damage. Although Lamin A/C interacts with ATM and MRN, its depletion does not disrupt their association with nuclease-resistant structures. However, it impairs the IR-enhanced association of ATM with the nuclear matrix and ATM-mediated DDR signaling, as well as the interaction between ATM and MRN. This disruption impedes the recruitment of MRE11 to damaged DNA and the association of damaged DNA with the nuclear matrix. Additionally, Lamin A/C depletion results in reduced protein levels of CtIP and RAD51, which is mediated by transcriptional regulation. This, in turn, impairs the efficiency of homologous recombination (HR). Our findings indicate that Lamin A/C plays a pivotal role in DNA damage repair (DDR) by orchestrating ATM-mediated signaling, maintaining HR protein levels, and ensuring efficient DNA repair processes.

Colorectal cancer (CRC) is the second leading cause of cancer related deaths in the world, and 5-Fluorouracil (5-FU) based regimens are chiefly employed for its therapy. Modern regimens, based on 5-FU in combination with other drugs have improved the response rates in the terminal CRCs to ~50%. However, a lack of molecular understanding of the cellular response process upon 5-FU treatment has stalled development of more improved therapies. Here, it behooves to study the mechanistic aspects of the complex, but coordinated signaling pathway that gets activated upon DNA damage, the DNA damage response (DDR). Current focus is on understanding the action of chemotherapeutic drugs, especially nucleoside analogs like 5-FU, in the context of DDR to find molecular targets to enhance their efficacies. In the present study, we focus on understanding the molecular basis of 5-FU based neoadjuvant therapy and finding novel targets for improving the response rates in CRC patients. We find that 5-FU pretreatment radiosensitizes CRC cell lines to neocarzinostatin (NCS) and leads to persistent γ-H2AX, a marker for DNA double strand breaks. We further show that 5-FU reduces the ability of cells to perform homologous recombination repair (HRR) but not non-homologous end joining (NHEJ), which points towards HRR being the underlying molecular mechanism of radiosensitizaztion by 5-FU. The use of B02, an inhibitor of HRR also shows persistent γ-H2AX, further strengthening our hypothesis. Our subsequent investigations rule out impaired recruitment of repair proteins as the possible cause of 5-FU induced HRR reduction. On the other hand, ribonucleotide supplementation reduces 5-FU and NCS induced DNA damage suggesting that nucleotide pool disruption is a possible cause of 5-FU induced HRR reduction. Also the Thymidylate synthetase (TS) inhibitor raltitrexed does not synergize with NCS. Interestingly, we find complementary lethality between TS depletion and 5-FU; however, TS depletion using siRNA also does not synergize with NCS. We, therefore, postulate that 5-FU induced TS inhibition and DNA damage both are essential for reducing the HRR. We also find that Gemcitabine, another nucleoside analog, does not show any synergism with NCS, which render it unsuitable for studying DDR in SW480 cells. Interestingly, Ribonucleotide Reductase M2 (RRM2) depletion and hydroxyurea treatment also does not lead to synergism with NCS. Therefore, we conclude that Ribonucleotide reductase inhibition cannot synergize or affect the HRR in SW480 cells. Further, MK2 depletion, which rescues cells from gemcitabine-induced DNA damage does not exert such an effect in 5-FU treated cells, indicating that MK2 does not play any significant part in 5-FU induced DNA damage. In conclusion, our results strongly suggest that the molecular reason for 5-FU induced radiosensitization is a reduction in the cellular ability to carry out HRR. This leads us to postulate that HRR inhibitors like B02 can be used in future regimens to overcome 5-FU resistant colorectal cancers.

Melanoma and DNA damage from a distance (farstander effect).

No consensus has been reached on the proper time to add stable-isotope labeled (SIL) peptides in protein cleavage isotope dilution mass spectrometry workflows. While quantifying 24 monolignol pathway enzymes in the xylem tissue of Populus trichocarpa, we compared the protein concentrations obtained when adding the SIL standard peptides concurrently with the enzyme or after quenching of the digestion (i.e. postdigestion) and observed discrepancies for nearly all tryptic peptides investigated. In some cases, greater than 30-fold differences were observed. To explain these differences and potentially correct for them, we developed a mathematical model based on pseudo-first-order kinetics to account for the dynamic production and decay (e.g. degradation and precipitation) of the native peptide targets in conjunction with the decay of the SIL peptide standards. A time course study of the digests confirmed the results predicted by the proposed model and revealed that the discrepancy between concurrent and postdigestion introduction of the SIL standards was related to differential decay experienced by the SIL peptide and the native peptide in each method. Given these results, we propose concurrent introduction of the SIL peptide is most appropriate, though not free from bias. Mathematical modeling of this method reveals that overestimation of protein quantities would still result when rapid peptide decay occurs and that this bias would be further exaggerated by slow proteolysis. We derive a simple equation to estimate the bias for each peptide based on the relative rates of production and decay. According to this equation, nearly half of the peptides evaluated here were estimated to have quantitative errors greater than 10% and in a few cases over 100%. We conclude that the instability of peptides can often significantly bias the protein quantities measured in protein cleavage isotope dilution mass spectrometry-based assays and suggest peptide stability be made a priority when selecting peptides to use for quantification.