Automating data analysis for hydrogen/deuterium exchange mass spectrometry using data-independent acquisition methodology.

We present a hydrogen/deuterium exchange workflow coupled to tandem mass spectrometry (HX-MS2) that supports the acquisition of peptide fragment ions alongside their peptide precursors. The approach enables true auto-curation of HX data by mining a rich set of deuterated fragments, generated by collisional-induced dissociation (CID), to simultaneously confirm the peptide ID and authenticate MS1-based deuteration calculations. The high redundancy provided by the fragments supports a confidence assessment of deuterium calculations using a combinatorial strategy. The approach requires data-independent acquisition (DIA) methods that are available on most MS platforms, making the switch to HX-MS2 straightforward. Importantly, we find that HX-DIA enables a proteomics-grade approach and wide-spread applications. Considerable time is saved through auto-curation and complex samples can now be characterized and at higher throughput. We illustrate these advantages in a drug binding analysis of the ultra-large protein kinase DNA-PKcs, isolated directly from mammalian cells.

Prevalence and Prognostic Significance of PIK3CA Mutation and CNV Status and Phosphorylated AKT Expression in Patients With Cervical Cancer Treated With Primary Surgery.

Currently, there are limited and conflicting reports on the prognostic utility of PIK3CA and associated pathway markers for cervical cancers treated with primary surgical management. Moreover, current studies are lacking complete characterization of adjuvant treatment with RT and/or chemotherapy. We aimed to document the prevalence, clinicopathologic, adjuvant treatment details, and prognostic value of PI3K/AKT pathway mutations and copy number variation and phosphorylated AKT status in patients with cervical cancers treated with primary surgery. A clinicopathologic review was performed on a retrospective cohort of 185 patients with cervical cancer, treated with primary surgery at a single tertiary institution. Next-generation sequencing and digital PCR was used to determine PI3K/AKT pathway mutational status and PIK3CA copy number variation, respectively, and fluorescent immunohistochemistry measured phosphorylated AKT expression. In all, 179 of 185 (96.8%) of tumors were successfully sequenced; 48 (26.8%) were positive for PI3K/AKT pathway mutations-the majority (n=37, 77.1%) PIK3CA mutations. PIK3CA mutation was associated with pathologically positive lymph nodes [12 (32%) vs. 22 (16%); P =0.022] and indication for postoperative chemoradiotherapy [17 (45.9%) vs. 32 (22.5%); P =0.004]. On multivariable analysis, PIK3CA status was not associated with overall survival ( P =0.103) or progression-free survival ( P =0.240) at 5 yrs, nor was PIK3CA copy number variation status. phosphorylated AKT ≤ median significantly predicted for progression-free survival [multivariable hazard ratio 0.39 (0.17-0.89; P =0.025)] but not overall survival ( P =0.087). The correlation of PIK3CA with pathologic positive lymph node status yet lack of association with survival outcomes may be due to the use of adjuvant postoperative therapy. PIK3CA assessment before radical hysterectomy may help identify patients with a higher risk of node-positive disease.

High-Efficiency Enrichment by Saturating Nanoliters of Protein Affinity Media.

Affinity-purification mass spectrometry (AP-MS) is an established technique for identifying protein-protein interactions (PPIs). The basic technology involves immobilizing a high-specificity ligand to a solid-phase support (e.g., an agarose or magnetic bead) to pull down protein(s) of interest from cell lysates. Although these supports are engineered to minimize interactions with background protein, the conventional method recovers mostly nonspecific binders. The law of mass action for dilute solutions has taught us to use an excess of beads to capture all target proteins, especially weakly interacting ones. However, modern microbead technology presents a binding environment that is much different from a dilute solution. We describe a fluidic platform that captures and processes ultralow nanoliter quantities of magnetic particles, simultaneously increasing the efficiency of PPI detection and strongly suppressing nonspecific binding. We demonstrate the concept with synthetic mixtures of tagged protein and illustrate performance with a variety of AP-MS experiment types. These include a BioID experiment targeting lamin-A interactors from HeLa cells and pulldowns using GFP-tagged proteins associated with a double-strand DNA repair mechanism. We show that efficient extraction requires saturation of the solid-phase support and that <10 nL of beads is sufficient to generate comprehensive protein interaction maps.

How to fix DNA breaks: new insights into the mechanism of non-homologous end joining.

Non-homologous end joining (NHEJ) is the major pathway for the repair of ionizing radiation-induced DNA double-strand breaks (DSBs) in human cells and is essential for the generation of mature T and B cells in the adaptive immune system via the process of V(D)J recombination. Here, we review how recently determined structures shed light on how NHEJ complexes function at DNA DSBs, emphasizing how multiple structures containing the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) may function in NHEJ. Together, these studies provide an explanation for how NHEJ proteins assemble to detect and protect DSB ends, then proceed, through DNA-PKcs-dependent autophosphorylation, to a ligation-competent complex.

Cryo-EM visualization of DNA-PKcs structural intermediates in NHEJ.

DNA double-strand breaks (DSBs), one of the most cytotoxic forms of DNA damage, can be repaired by the tightly regulated nonhomologous end joining (NHEJ) machinery (Stinson and Loparo and Zhao et al.). Core NHEJ factors form an initial long-range (LR) synaptic complex that transitions into a DNA-PKcs (DNA-dependent protein kinase, catalytic subunit)-free, short-range state to align the DSB ends (Chen et al.). Using single-particle cryo-electron microscopy, we have visualized three additional key NHEJ complexes representing different transition states, with DNA-PKcs adopting distinct dimeric conformations within each of them. Upon DNA-PKcs autophosphorylation, the LR complex undergoes a substantial conformational change, with both Ku and DNA-PKcs rotating outward to promote DNA break exposure and DNA-PKcs dissociation. We also captured a dimeric state of catalytically inactive DNA-PKcs, which resembles structures of other PIKK (Phosphatidylinositol 3-kinase-related kinase) family kinases, revealing a model of the full regulatory cycle of DNA-PKcs during NHEJ.

A genetic roadmap to the response to genotoxic agents in human cells.

To maintain genome fidelity and prevent diseases such as cancer, our cells must constantly detect, and efficiently and precisely repair, DNA damage. Paradoxically, DNA-damaging agents in the form of radiation and chemotherapy are also used to treat cancer. Olivieri et al. used a CRISPR-based screen to identify genes that, when disrupted, lead to sensitivity or resistance to 27 different DNA-damaging agents used in the lab and/or in the clinic to treat cancer patients1. Their results reveal multiple new genes and connections that regulate these critical DNA damage repair pathways, with implications for basic and clinical research as well as cancer therapy.

KRCC1, a modulator of the DNA damage response.

The lysine-rich coiled-coil 1 (KRCC1) protein is overexpressed in multiple malignancies, including ovarian cancer, and overexpression correlates with poor overall survival. Despite a potential role in cancer progression, the biology of KRCC1 remains elusive. Here, we characterize the biology of KRCC1 and define its role in the DNA damage response and in cell cycle progression. We demonstrate that KRCC1 associates with the checkpoint kinase 1 (CHK1) upon DNA damage and regulates the CHK1-mediated checkpoint. KRCC1 facilitates RAD51 recombinase foci formation and augments homologous recombination repair. Furthermore, KRCC1 is required for proper S-phase progression and subsequent mitotic entry. Our findings uncover a novel component of the DNA damage response and a potential link between cell cycle, associated damage response and DNA repair.

Purification of DNA-Dependent Protein Kinase Catalytic Subunit (DNA-PKcs) from HeLa Cells.

With a predicted molecular mass of 469 kDa, expression of recombinant DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is challenging. However, DNA-PKcs is relatively abundant in human cells, making it possible to purify the endogenous protein. Here we describe a method to purify DNA-PKcs and its binding partner Ku70/80 from HeLa cells and describe conditions for transfer of DNA-PKcs and other large polypeptides for immunoblotting.

Function and Molecular Mechanism of the DNA Damage Response in Immunity and Cancer Immunotherapy.

The DNA damage response (DDR) is an organized network of multiple interwoven components evolved to repair damaged DNA and maintain genome fidelity. Conceptually the DDR includes damage sensors, transducer kinases, and effectors to maintain genomic stability and accurate transmission of genetic information. We have recently gained a substantially improved molecular and mechanistic understanding of how DDR components are interconnected to inflammatory and immune responses to stress. DDR shapes both innate and adaptive immune pathways: (i) in the context of innate immunity, DDR components mainly enhance cytosolic DNA sensing and its downstream STimulator of INterferon Genes (STING)-dependent signaling; (ii) in the context of adaptive immunity, the DDR is needed for the assembly and diversification of antigen receptor genes that is requisite for T and B lymphocyte development. Imbalances between DNA damage and repair impair tissue homeostasis and lead to replication and transcription stress, mutation accumulation, and even cell death. These impacts from DDR defects can then drive tumorigenesis, secretion of inflammatory cytokines, and aberrant immune responses. Yet, DDR deficiency or inhibition can also directly enhance innate immune responses. Furthermore, DDR defects plus the higher mutation load in tumor cells synergistically produce primarily tumor-specific neoantigens, which are powerfully targeted in cancer immunotherapy by employing immune checkpoint inhibitors to amplify immune responses. Thus, elucidating DDR-immune response interplay may provide critical connections for harnessing immunomodulatory effects plus targeted inhibition to improve efficacy of radiation and chemotherapies, of immune checkpoint blockade, and of combined therapeutic strategies.

Structural insights into the role of DNA-PK as a master regulator in NHEJ.

DNA-dependent protein kinase catalytic subunit DNA-PKcs/PRKDC is the largest serine/threonine protein kinase of the phosphatidyl inositol 3-kinase-like protein kinase (PIKK) family and is the most highly expressed PIKK in human cells. With its DNA-binding partner Ku70/80, DNA-PKcs is required for regulated and efficient repair of ionizing radiation-induced DNA double-strand breaks via the non-homologous end joining (NHEJ) pathway. Loss of DNA-PKcs or other NHEJ factors leads to radiation sensitivity and unrepaired DNA double-strand breaks (DSBs), as well as defects in V(D)J recombination and immune defects. In this review, we highlight the contributions of the late Dr. Carl W. Anderson to the discovery and early characterization of DNA-PK. We furthermore build upon his foundational work to provide recent insights into the structure of NHEJ synaptic complexes, an evolutionarily conserved and functionally important YRPD motif, and the role of DNA-PKcs and its phosphorylation in NHEJ. The combined results identify DNA-PKcs as a master regulator that is activated by its detection of two double-strand DNA ends for a cascade of phosphorylation events that provide specificity and efficiency in assembling the synaptic complex for NHEJ.

Decreased ATM Protein Expression Is Substantiated with PTEN Loss in Defining Aggressive Phenotype of Prostate Cancer Associated with Lethal Disease.

BACKGROUND: Ataxia Telangiectasia Mutated (ATM) serine/threonine protein kinase is a known tumor suppressor, involved in DNA damage repair. It has prognostic and predictive therapeutic implications and is associated with aggressive prostate cancer (PCa). OBJECTIVE: To investigate the prognostic value of ATM protein expression in PCa patients and assessed the combined value of ATM, ERG, and PTEN status. DESIGN SETTING AND PARTICIPANTS: This study consisted of 303 patients with incidental, locally advanced, and castrate-resistant PCa by transurethral resection of the prostate (TURP). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: TURP samples from 303 PCa patients were assessed by immunohistochemistry (IHC for ATM, ERG, and PTEN. Individual and combined marker status were correlated with International Society of Urological Pathology Gleason grade group, overall survival (OS), and PCa-specific mortality (PCSM). RESULTS AND LIMITATIONS: Decreased ATM expression (negative/weak intensity) occurred in 164/303 (54.1%) patients, and was associated with shorter OS and higher PCSM (p = 0.015 and p = 0.001, respectively). Negative/weak ATM expression was significantly associated with PCSM with a hazard ratio of 2.09 (95% confidence interval 1.34-3.27, p = 0.001). Assessment of Combined ATM/PTEN expression showed improved prognostic power to predict OS and PCSM, independent of Gleason grade groups. CONCLUSIONS: Decreased ATM protein expression is associated with poor outcomes in advanced PCa patients. Patients with combined low ATM/PTEN negative expression are at the highest risk for reduced OS and PCSM. Assessing the combined status of ATM/PTEN by IHC in PCa patients may aid in risk stratification relative to OS and PCSM. Moreover, since ATM plays an integral role in DNA damage response pathways, future studies will enhance our understanding of how outcomes of patients with altered ATM and PTEN expression can be improved further with poly-ADP ribose polymerase inhibitors (PARPi), combinations of PARPi and androgen receptor-targeted therapies, as well as platinum-based chemotherapies. PATIENT SUMMARY: Lower ATM intensity is associated with increased cancer-specific mortality in prostate cancer patients. Patients with lower ATM and PTEN negative expression showed decreased overall survival and increased cancer mortality compared with controls.

Long Noncoding RNA NIHCOLE Promotes Ligation Efficiency of DNA Double-Strand Breaks in Hepatocellular Carcinoma.

Long noncoding RNAs (lncRNA) are emerging as key players in cancer as parts of poorly understood molecular mechanisms. Here, we investigated lncRNAs that play a role in hepatocellular carcinoma (HCC) and identified NIHCOLE, a novel lncRNA induced in HCC with oncogenic potential and a role in the ligation efficiency of DNA double-stranded breaks (DSB). NIHCOLE expression was associated with poor prognosis and survival of HCC patients. Depletion of NIHCOLE from HCC cells led to impaired proliferation and increased apoptosis. NIHCOLE deficiency led to accumulation of DNA damage due to a specific decrease in the activity of the nonhomologous end-joining (NHEJ) pathway of DSB repair. DNA damage induction in NIHCOLE-depleted cells further decreased HCC cell growth. NIHCOLE was associated with DSB markers and recruited several molecules of the Ku70/Ku80 heterodimer. Further, NIHCOLE putative structural domains supported stable multimeric complexes formed by several NHEJ factors including Ku70/80, APLF, XRCC4, and DNA ligase IV. NHEJ reconstitution assays showed that NIHCOLE promoted the ligation efficiency of blunt-ended DSBs. Collectively, these data show that NIHCOLE serves as a scaffold and facilitator of NHEJ machinery and confers an advantage to HCC cells, which could be exploited as a targetable vulnerability. SIGNIFICANCE: This study characterizes the role of lncRNA NIHCOLE in DNA repair and cellular fitness in HCC, thus implicating it as a therapeutic target.See related commentary by Barcena-Varela and Lujambio, p. 4899.

Structural basis of long-range to short-range synaptic transition in NHEJ.

DNA double-strand breaks (DSBs) are a highly cytotoxic form of DNA damage and the incorrect repair of DSBs is linked to carcinogenesis1,2. The conserved error-prone non-homologous end joining (NHEJ) pathway has a key role in determining the effects of DSB-inducing agents that are used to treat cancer as well as the generation of the diversity in antibodies and T cell receptors2,3. Here we applied single-particle cryo-electron microscopy to visualize two key DNA-protein complexes that are formed by human NHEJ factors. The Ku70/80 heterodimer (Ku), the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), DNA ligase IV (LigIV), XRCC4 and XLF form a long-range synaptic complex, in which the DNA ends are held approximately 115 Å apart. Two DNA end-bound subcomplexes comprising Ku and DNA-PKcs are linked by interactions between the DNA-PKcs subunits and a scaffold comprising LigIV, XRCC4, XLF, XRCC4 and LigIV. The relative orientation of the DNA-PKcs molecules suggests a mechanism for autophosphorylation in trans, which leads to the dissociation of DNA-PKcs and the transition into the short-range synaptic complex. Within this complex, the Ku-bound DNA ends are aligned for processing and ligation by the XLF-anchored scaffold, and a single catalytic domain of LigIV is stably associated with a nick between the two Ku molecules, which suggests that the joining of both strands of a DSB involves both LigIV molecules.

The active DNA-PK holoenzyme occupies a tensed state in a staggered synaptic complex.

In the non-homologous end-joining (NHEJ) of a DNA double-strand break, DNA ends are bound and protected by DNA-PK, which synapses across the break to tether the broken ends and initiate repair. There is little clarity surrounding the nature of the synaptic complex and the mechanism governing the transition to repair. We report an integrative structure of the synaptic complex at a precision of 13.5 Å, revealing a symmetric head-to-head arrangement with a large offset in the DNA ends and an extensive end-protection mechanism involving a previously uncharacterized plug domain. Hydrogen/deuterium exchange mass spectrometry identifies an allosteric pathway connecting DNA end-binding with the kinase domain that places DNA-PK under tension in the kinase-active state. We present a model for the transition from end-protection to repair, where the synaptic complex supports hierarchical processing of the ends and scaffold assembly, requiring displacement of the catalytic subunit and tension release through kinase activity.

Uncovering DNA-PKcs ancient phylogeny, unique sequence motifs and insights for human disease.

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a key member of the phosphatidylinositol-3 kinase-like (PIKK) family of protein kinases with critical roles in DNA-double strand break repair, transcription, metastasis, mitosis, RNA processing, and innate and adaptive immunity. The absence of DNA-PKcs from many model organisms has led to the assumption that DNA-PKcs is a vertebrate-specific PIKK. Here, we find that DNA-PKcs is widely distributed in invertebrates, fungi, plants, and protists, and that threonines 2609, 2638, and 2647 of the ABCDE cluster of phosphorylation sites are highly conserved amongst most Eukaryotes. Furthermore, we identify highly conserved amino acid sequence motifs and domains that are characteristic of DNA-PKcs relative to other PIKKs. These include residues in the Forehead domain and a novel motif we have termed YRPD, located in an α helix C-terminal to the ABCDE phosphorylation site loop. Combining sequence with biochemistry plus structural data on human DNA-PKcs unveils conserved sequence and conformational features with functional insights and implications. The defined generally progressive DNA-PKcs sequence diversification uncovers conserved functionality supported by Evolutionary Trace analysis, suggesting that for many organisms both functional sites and evolutionary pressures remain identical due to fundamental cell biology. The mining of cancer genomic data and germline mutations causing human inherited disease reveal that robust DNA-PKcs activity in tumors is detrimental to patient survival, whereas germline mutations compromising function are linked to severe immunodeficiency and neuronal degeneration. We anticipate that these collective results will enable ongoing DNA-PKcs functional analyses with biological and medical implications.

Visualizing functional dynamicity in the DNA-dependent protein kinase holoenzyme DNA-PK complex by integrating SAXS with cryo-EM.

Assembly of KU and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) at DNA double strand breaks (DSBs) forms DNA-PK holoenzyme as a critical initiating step for non-homologous end joining (NHEJ) repair of DSBs produced by radiation and chemotherapies. Advanced cryo-electron microscopy (cryo-EM) imaging together with breakthrough macromolecular X-ray crystal (MX) structures of KU and DNA-PKcs recently enabled visualization of the ∼600 kDa DNA-PK assembly at near atomic resolution. These important static structures provide the foundation for definition and interpretation of functional movements crucial to mechanistic understanding that can be tested through solution state structure analysis. We herein therefore leverage Cryo-EM and MX structures for the interpretation of synchrotron small-angle X-ray scattering (SAXS) data on DNA-PK conformations in solution to inform the structural mechanism for NHEJ initiation. SAXS, which measures thermodynamic solution-state conformational states and assemblies outside of cryo- and solid-state conditions, unveils the inherent flexibility of KU, DNA-PKcs and DNA-PK. The combined structural measurements reveal mobility of KU80 C-terminal region (KU80CTR), motion/plasticity of HEAT (DNA-PKcs Huntingtin, Elongation Factor 3, PP2 A, and TOR1) regions, allosteric switching upon DNA-PKcs autophosphorylation, and dimeric arrangements of DNA-PK assembly. Importantly, the results uncover displacement of the N-terminal HEAT domain during autophosphorylation as suitable for a regulated release mechanism of DNA-PKcs from DNA-PK to control unproductive access to toxic and mutagenic DNA repair intermediates. These integrated analyses show that the marriage of SAXS with cryo-EM leverages the strengths of both techniques to enable assessment of functional conformations and flexibility defining atomic-resolution molecular mechanisms for DSB repair.

Mechanism of efficient double-strand break repair by a long non-coding RNA.

Mechanistic studies in DNA repair have focused on roles of multi-protein DNA complexes, so how long non-coding RNAs (lncRNAs) regulate DNA repair is less well understood. Yet, lncRNA LINP1 is over-expressed in multiple cancers and confers resistance to ionizing radiation and chemotherapeutic drugs. Here, we unveil structural and mechanistic insights into LINP1's ability to facilitate non-homologous end joining (NHEJ). We characterized LINP1 structure and flexibility and analyzed interactions with the NHEJ factor Ku70/Ku80 (Ku) and Ku complexes that direct NHEJ. LINP1 self-assembles into phase-separated condensates via RNA-RNA interactions that reorganize to form filamentous Ku-containing aggregates. Structured motifs in LINP1 bind Ku, promoting Ku multimerization and stabilization of the initial synaptic event for NHEJ. Significantly, LINP1 acts as an effective proxy for PAXX. Collective results reveal how lncRNA effectively replaces a DNA repair protein for efficient NHEJ with implications for development of resistance to cancer therapy.

PIK3CA mutation and CNV status and post-chemoradiotherapy survival in patients with cervical cancer.

PURPOSE: This study aimed to describe the prognostic value of PI3K/AKT pathway mutations in a large cohort of patients with cervical cancer. EXPERIMENTAL DESIGN: Patients with pre-treatment archival specimens, diagnosed with FIGO stages IB-IVA cervical cancer between 1998 and 2014 and treated with radical, curative intent chemoradiotherapy (CRT) at a single center were identified. Mutational status was determined by next generation sequencing and PIK3CA copy number (CNV) was assessed by digital PCR. RESULTS: 190 patients with available pre-treatment tumor specimens were identified. Median OS and PFS were 57.4 and 46.0 months, respectively. A total of 161 tumors were successfully sequenced; 60 (37.3%) had PI3K/AKT pathway mutations, with 50 (30.1%) having PIK3CA hotspot mutations. PIK3CA CNV gain was noted in 79 (59.2%) of the 154 successfully analyzed. On univariate analysis, PIK3CA mutation was associated with poor OS (HR 1.73; 95% CI: 1.03-2.92; p = .037) but not PFS (HR 1.38; 0.84-2.28; p = .204). Absence of any PI3K/AKT pathway mutation was associated with improved OS (HR 1.68; 1.01-2.81; p = .046) but not PFS (HR 1.50; 0.93-2.43; p = .202). Associations were not maintained when adjusting for clinical factors. On univariate analysis, PIK3CA mutation positive, CNV normal tumors were associated with poorer OS (HR 2.55; 1.18-5.50; p = .017) and trend to worse PFS (HR 1.87; 0.90-3.83; p = .094) when compared to those with CNV gain and wildtype PIK3CA. CONCLUSIONS: PI3K/AKT pathway mutations are common in cervical cancer. Consideration of PIK3CA mutational status with CNV status may be important in predicting outcome in cervical cancer patients.