Ku DNA End-Binding Activity Promotes Repair Fidelity and Influences End-Processing During Nonhomologous End-Joining in Saccharomyces cerevisiae.

The Ku heterodimer acts centrally in nonhomologous end-joining (NHEJ) of DNA double-strand breaks (DSB). Saccharomyces cerevisiae Ku, like mammalian Ku, binds and recruits NHEJ factors to DSB ends. Consequently, NHEJ is virtually absent in yeast Ku null (yku70∆ or yku80∆) strains. Previously, we unexpectedly observed imprecise NHEJ proficiency in a yeast Ku mutant with impaired DNA end-binding (DEB). However, how DEB impairment supported imprecise NHEJ was unknown. Here, we found imprecise NHEJ proficiency to be a feature of a panel of DEB-impaired Ku mutants and that DEB impairment resulted in a deficiency in precise NHEJ. These results suggest that DEB-impaired Ku specifically promotes error-prone NHEJ. Epistasis analysis showed that classical NHEJ factors, as well as novel and previously characterized NHEJ-specific residues of Ku, are required for the distinct error-prone repair in a Ku DEB mutant. However, sequencing of repair junctions revealed that imprecise repair in Ku DEB mutants was almost exclusively characterized by small deletions, in contrast to the majority of insertions that define imprecise repair in wild-type strains. Notably, while sequencing indicated a lack of Pol4-dependent insertions at the site of repair, Pol2 exonuclease activity, which mediates small deletions in NHEJ, contributed to imprecise NHEJ in a Ku DEB mutant. The deletions were smaller than in Ku-independent microhomology-mediated end-joining (MMEJ) and were neither promoted by Mre11 nuclease activity nor Sae2 Thus, the quality of Ku's engagement at the DNA end influences end-processing during NHEJ and DEB impairment unmasks a Ku-dependent error-prone pathway of end-joining distinct from MMEJ.

The ATM- and ATR-related SCD domain is over-represented in proteins involved in nervous system development.

ATM and ATR are cellular kinases with a well-characterized role in the DNA-damage response. Although the complete set of ATM/ATR targets is unknown, they often contain clusters of S/TQ motifs that constitute an SCD domain. In this study, we identified putative ATM/ATR targets that have a conserved SCD domain across vertebrates. Using this approach, we have identified novel putative ATM/ATR targets in pathways known to be under direct control of these kinases. Our analysis has also unveiled significant enrichment of SCD-containing proteins in cellular pathways, such as vesicle trafficking and actin cytoskeleton, where a regulating role for ATM/ATR is either unknown or poorly understood, hinting at a much broader and overarching role for these kinases in the cell. Of particular note is the overrepresentation of conserved SCD-containing proteins involved in pathways related to neural development. This finding suggests that ATM/ATR could be directly involved in controlling this process, which may be linked to the adverse neurological effects observed in patients with mutations in ATM.

Signaling networks in MS: a systems-based approach to developing new pharmacological therapies.

The pathogenesis of multiple sclerosis (MS) involves alterations to multiple pathways and processes, which represent a significant challenge for developing more-effective therapies. Systems biology approaches that study pathway dysregulation should offer benefits by integrating molecular networks and dynamic models with current biological knowledge for understanding disease heterogeneity and response to therapy. In MS, abnormalities have been identified in several cytokine-signaling pathways, as well as those of other immune receptors. Among the downstream molecules implicated are Jak/Stat, NF-Kb, ERK1/3, p38 or Jun/Fos. Together, these data suggest that MS is likely to be associated with abnormalities in apoptosis/cell death, microglia activation, blood-brain barrier functioning, immune responses, cytokine production, and/or oxidative stress, although which pathways contribute to the cascade of damage and can be modulated remains an open question. While current MS drugs target some of these pathways, others remain untouched. Here, we propose a pragmatic systems analysis approach that involves the large-scale extraction of processes and pathways relevant to MS. These data serve as a scaffold on which computational modeling can be performed to identify disease subgroups based on the contribution of different processes. Such an analysis, targeting these relevant MS-signaling pathways, offers the opportunity to accelerate the development of novel individual or combination therapies.

Using a PyMOL activity to reinforce the connection between genotype and phenotype in an undergraduate genetics laboratory.

With the purpose of developing an activity that would help clarify genetic concepts related to the connection between genotype and phenotype and the nature of mutations, we designed a three hour teaching module using the PyMol software. The activity starts with two pre-laboratory assignments, one to learn how to use PyMol and the other to read about a specific protein or protein family. During the laboratory students are given instructions where and how to find additional information on a specific disease and its causal mutations in order to prepare a 10-minute, in-class presentation. Using a post activity, anonymous quiz, we found a statistically significant different grade distribution in students that participated in the PyMol activity relative to a control group. We also found a significant improvement in the student's comprehension when answering questions regarding the nature of mutations and protein structure. This demonstrates the utility of this simulation activity as a vehicle to improve student's understanding of specific key genetic concepts.

SCDFinder, a web-based tool for the identification of putative novel ATM and ATR targets.

MOTIVATION: The S/TQ cluster domain (SCD) constitutes a new type of protein domain that is not defined by sequence similarity but by the presence of multiple S/TQ motifs within a variable stretch of amino acids. SCDs are recognized targets for DNA damage response (DDR) kinases like ATM and ATR. Characterizing DDR targets is of significant interest. The aim of this work was to develop a web-based tool to allow for easy identification and visualization of SCDs within specific proteins or in whole proteome sets, a feature not supported by current domain and motif search tools. RESULTS: We have developed an algorithm that (i) generates a list of all proteins in an organism containing at least one user-defined SCD within their sequence, or (ii) identifies and renders a visual representation of all user-defined SCDs present in a single sequence or batch of sequences. AVAILABILITY AND IMPLEMENTATION: The application was developed using Pearl and Python, and is available at the following URL: http://ustbioinfo.webfactional.com/scd/.

TRF2 interaction with Ku heterotetramerization interface gives insight into c-NHEJ prevention at human telomeres.

Telomeres are protected from nonhomologous end-joining (NHEJ) to avoid deleterious chromosome fusions, yet they associate with the Ku heterodimer that is principal in the classical NHEJ (c-NHEJ) pathway. T-loops have been proposed to inhibit Ku's association with telomeric ends, thus inhibiting c-NHEJ; however, deficiencies in the t-loop model suggest additional mechanisms are in effect. We demonstrate that TRF2 interacts with Ku at telomeres and via residues in Ku70 helix 5 (α5), which are vital for NHEJ. We show that Ku's interaction with a TRF2 mutant that induces telomeric fusions is significantly impaired. Additionally, we demonstrate that Ku70 α5 is required for Ku self-association in live cells, which can bridge DNA ends. Together, these findings lead us to propose a model in which telomeres are directly protected from c-NHEJ via TRF2 impeding Ku's ability to synapse telomere ends.

An S/T-Q cluster domain census unveils new putative targets under Tel1/Mec1 control.

BACKGROUND: The cellular response to DNA damage is immediate and highly coordinated in order to maintain genome integrity and proper cell division. During the DNA damage response (DDR), the sensor kinases Tel1 and Mec1 in Saccharomyces cerevisiae and ATM and ATR in human, phosphorylate multiple mediators which activate effector proteins to initiate cell cycle checkpoints and DNA repair. A subset of kinase substrates are recognized by the S/T-Q cluster domain (SCD), which contains motifs of serine (S) or threonine (T) followed by a glutamine (Q). However, the full repertoire of proteins and pathways controlled by Tel1 and Mec1 is unknown. RESULTS: To identify all putative SCD-containing proteins, we analyzed the distribution of S/T-Q motifs within verified Tel1/Mec1 targets and arrived at a unifying SCD definition of at least 3 S/T-Q within a stretch of 50 residues. This new SCD definition was used in a custom bioinformatics pipeline to generate a census of SCD-containing proteins in both yeast and human. In yeast, 436 proteins were identified, a significantly larger number of hits than were expected by chance. These SCD-containing proteins did not distribute equally across GO-ontology terms, but were significantly enriched for those involved in processes related to the DDR. We also found a significant enrichment of proteins involved in telophase and cytokinesis, protein transport and endocytosis suggesting possible novel Tel1/Mec1 targets in these pathways. In the human proteome, a wide range of similar proteins were identified, including homologs of some SCD-containing proteins found in yeast. This list also included high concentrations of proteins in the Mediator, spindle pole body/centrosome and actin cytoskeleton complexes. CONCLUSIONS: Using a bioinformatic approach, we have generated a census of SCD-containing proteins that are involved not only in known DDR pathways but several other pathways under Tel1/Mec1 control suggesting new putative targets for these kinases.

Ku must load directly onto the chromosome end in order to mediate its telomeric functions.

The Ku heterodimer associates with the Saccharomyces cerevisiae telomere, where it impacts several aspects of telomere structure and function. Although Ku avidly binds DNA ends via a preformed channel, its ability to associate with telomeres via this mechanism could be challenged by factors known to bind directly to the chromosome terminus. This has led to uncertainty as to whether Ku itself binds directly to telomeric ends and whether end association is crucial for Ku's telomeric functions. To address these questions, we constructed DNA end binding-defective Ku heterodimers by altering amino acid residues in Ku70 and Ku80 that were predicted to contact DNA. These mutants continued to associate with their known telomere-related partners, such as Sir4, a factor required for telomeric silencing, and TLC1, the RNA component of telomerase. Despite these interactions, we found that the Ku mutants had markedly reduced association with telomeric chromatin and null-like deficiencies for telomere end protection, length regulation, and silencing functions. In contrast to Ku null strains, the DNA end binding defective Ku mutants resulted in increased, rather than markedly decreased, imprecise end-joining proficiency at an induced double-strand break. This result further supports that it was the specific loss of Ku's telomere end binding that resulted in telomeric defects rather than global loss of Ku's functions. The extensive telomere defects observed in these mutants lead us to propose that Ku is an integral component of the terminal telomeric cap, where it promotes a specific architecture that is central to telomere function and maintenance.

Poly(ADP-ribose) polymerase inhibitor ABT-888 potentiates the cytotoxic activity of temozolomide in leukemia cells: influence of mismatch repair status and O6-methylguanine-DNA methyltransferase activity.

The poly(ADP-ribose) polymerase (PARP) inhibitor ABT-888 potentiates the antitumor activity of temozolomide (TMZ). TMZ resistance results from increased O(6)-methylguanine-DNA methyltransferase (MGMT) activity and from mismatch repair (MMR) system mutations. We evaluated the relative importance of MGMT activity, MMR deficiency, nonhomologous end joining (NHEJ), and PARP activity in ABT-888 potentiation of TMZ. MMR-proficient and MMR-deficient leukemia cells with varying MGMT activity, as well as primary leukemia samples, were used to determine TMZ IC(50) alone and with ABT-888. ABT-888 effectively inhibited PARP activity and enhanced TMZ growth inhibition in most leukemia cells. ABT-888 potentiation was most effective in MMR-deficient cells with low MGMT activity [potentiation factor (PF) = 21]. ABT-888 also potentiated TMZ activity in MMR-deficient cells with elevated MGMT activity. Unexpectedly, ABT-888 also enhanced TMZ activity in MMR-proficient cells (PF = 3-7). ABT-888 potentiation was unrelated to NHEJ activity. ABT-888 potentiated TMZ (PF = 2-5) in two of four acute myeloid leukemia patient samples but showed little potentiation in primary acute lymphoblastic leukemia. In conclusion, although ABT-888 potentiation of TMZ was most pronounced in MMR-deficient cells with low MGMT activity, neither MMR proficiency nor MGMT overexpression completely abrogated ABT-888 potentiation of TMZ.

Distinct faces of the Ku heterodimer mediate DNA repair and telomeric functions.

The Ku heterodimer, comprised of Ku70 and Ku80 subunits, is a conserved complex involved in nonhomologous end-joining (NHEJ). However, it also functions in maintenance of telomeres, chromosome termini normally resistant to end-joining events. To elucidate the spatial organization of these functions, we rationally guided Ku mutagenesis in yeast with real-valued evolutionary trace (rvET). This revealed two ancestrally related alpha-helices: one on the Ku70 surface that is required in yeast for NHEJ, and a second on the Ku80 surface that is required in yeast for telomeric heterochromatin formation. When bound to a DNA end, the surface containing the NHEJ-specific Ku70 helix is oriented toward the DNA terminus, whereas the surface containing the telomeric function-specific Ku80 helix faces inward, toward telomeric chromatin, when bound to a telomere. We propose a 'two-face' model for Ku and that divergent evolution of these faces allowed Ku's dual role in NHEJ and telomere maintenance.