Assembly of the nonhomologous end joining machinery.

The DNA double strand break (DSB), where the chromosome is physically fragmented, represents a serious threat to the integrity of a genome. Accordingly, two DSB repair mechanisms, homologous recombination (HR) and nonhomologous end joining (NHEJ), are conserved in all kingdoms of life. HR utilizes homologous sequences from sister chromatids to direct DNA synthesis spanning the DSB. In contrast, NHEJ involves direct processing and rejoining of the DSB ends. Because NHEJ plays essential roles in mutagenesis, immunological development and carcinogenesis, a detailed molecular understanding of this process is crucial. This dissertation focuses on determining the forces directing assembly of the NHEJ repair machinery using the robust genetic techniques available in Saccharomyces cerevisiae. In this yeast, NHEJ requires the Yku70-Yku80 (Ku), Mre11-Rad50-Xrs2 (MRX) and the Dnl4-Lif1-Nej1 (DNA ligase IV) protein complexes. We hypothesized that NHEJ requires contacts between these complexes to coordinate repair. A yeast two-hybrid screen of NHEJ proteins identified interactions between Yku80-Dn14, Xrs2-Lif1 and Mre11-Yku80. The Yku80-Dn14 interaction was refined to the Yku80 C-terminus and mutational analysis identified three conserved leucines, part of a putative amphipathic alpha helix, which determined interaction with Dn14. Mutation of the Yku80 C-terminus resulted in moderate NHEJ defects. The Xrs2-Lif1 interaction was refined to the Xrs2 fork-head associated (FHA) domain and the Lif1 C-terminus. Mutation of Xrs2 identified four residues, expected to contact phosphothreonines, which mediate interaction with Lif1. Correspondingly, two Lif1 threonines, T387 and T417, mediated interaction with Xrs2. Mutation of the Xrs2 FHA domain caused slight defects in NHEJ, but specifically and severely blocked NHEJ and recruitment of Dnl4 to a DSB when combined with the Yku80 C-terminal mutations. Overall, this work describes redundant interactions of DNA ligase IV with Ku and MRX that provide a framework for understanding assembly of the NHEJ machinery at a DSB. Finally, we demonstrated that the Mt-Ku and Mt-Lig proteins from the bacteria, Mycobacterium tuberculosis, are sufficient to reconstitute NHEJ in yeast, indicating that they represent a bona fide NHEJ system. Characterization of this simple system consisting of only two polypeptides provides a further model for exploration of the rules governing assembly of the NHEJ machinery.Ph.D.Biological SciencesGeneticsMolecular biologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/127027/2/3305054.pd

Mutations of the Yku80 C Terminus and Xrs2 FHA Domain Specifically Block Yeast Nonhomologous End Joining

The nonhomologous end-joining (NHEJ) pathway of DNA double-strand break repair requires three protein complexes in Saccharomyces cerevisiae: MRX (Mre11-Rad50-Xrs2), Ku (Ku70-Ku80), and DNA ligase IV (Dnl4-Lif1-Nej1). Much is known about the interactions that mediate the formation of each complex, but little is known about how they act together during repair. A comprehensive yeast two-hybrid screen of the NHEJ factors of S. cerevisiae revealed all known interactions within the MRX, Ku, and DNA ligase IV complexes, as well as three additional, weaker interactions between Yku80-Dnl4, Xrs2-Lif1, and Mre11-Yku80. Individual and combined deletions of the Yku80 C terminus and the Xrs2 forkhead-associated (FHA) domain were designed based on the latter two-hybrid results. These deletions synergistically blocked NHEJ but not the telomere and recombination functions of Ku and MRX, confirming that these protein regions are functionally important specifically for NHEJ. Further mutational analysis of Yku80 identified a putative C-terminal amphipathic α-helix that is both required for its NHEJ function and strikingly similar to a DNA-dependent protein kinase interaction motif in human Ku80. These results identify a novel role in yeast NHEJ for the poorly characterized Ku80 C-terminal and Xrs2 FHA domains, and they suggest that redundant binding of DNA ligase IV facilitates completion of this DNA repair event

Recruitment of Saccharomyces cerevisiae Dnl4–Lif1 Complex to a Double-Strand Break Requires Interactions With Yku80 and the Xrs2 FHA Domain

Nonhomologous end joining (NHEJ) in yeast depends on eight different proteins in at least three different functional complexes: Yku70–Yku80 (Ku), Dnl4–Lif1–Nej1 (DNA ligase IV), and Mre11–Rad50–Xrs2 (MRX). Interactions between these complexes at DNA double-strand breaks (DSBs) are poorly understood but critical for the completion of repair. We previously identified two such contacts that are redundantly required for NHEJ, one between Dnl4 and the C terminus of Yku80 and one between the forkhead-associated (FHA) domain of Xrs2 and the C terminus of Lif1. Here, we first show that mutation of the Yku80 C terminus did not impair Ku binding to DSBs, supporting specificity of the mutant defect to the ligase interaction. We next show that the Xrs2–Lif1 interaction depends on Xrs2 FHA residues (R32, S47, R48, and K75) analogous to those known in other proteins to contact phosphorylated threonines. Two potential target threonines in Lif1 (T417 and T387) were inferred by identifying regions similar to a site in the human Lif1 homolog, XRCC4, known to be bound by the FHA domain of polynucleotide kinase. Mutating these threonines, especially T417, abolished the Xrs2–Lif1 interaction and impaired NHEJ epistatically with Xrs2 FHA mutation. Combining mutations that selectively disable the Yku80–Dnl4 and Xrs2–Lif1 interactions abrogated both NHEJ and DNA ligase IV recruitment to a DSB. The collected results indicate that the Xrs–Lif1 and Yku80–Dnl4 interactions are important for formation of a productive ligase–DSB intermediate

Mycobacterial Ku and ligase proteins constitute a two-component NHEJ repair machine.

In mammalian cells, repair of DNA double-strand breaks (DSBs) by nonhomologous end-joining (NHEJ) is critical for genome stability. Although the end-bridging and ligation steps of NHEJ have been reconstituted in vitro, little is known about the end-processing reactions that occur before ligation. Recently, functionally homologous end-bridging and ligation activities have been identified in prokarya. Consistent with its homology to polymerases and nucleases, we demonstrate that DNA ligase D from Mycobacterium tuberculosis (Mt-Lig) possesses a unique variety of nucleotidyl transferase activities, including gap-filling polymerase, terminal transferase, and primase, and is also a 3' to 5' exonuclease. These enzyme activities allow the Mt-Ku and Mt-Lig proteins to join incompatible DSB ends in vitro, as well as to reconstitute NHEJ in vivo in yeast. These results demonstrate that prokaryotic Ku and ligase form a bona fide NHEJ system that encodes all the recognition, processing, and ligation activities required for DSB repair

Multigene Profiling of Circulating Tumor Cells (CTCs) for Prognostic Assessment in Treatment-Naïve Metastatic Hormone-Sensitive Prostate Cancer (mHSPC)

The substantial biological heterogeneity of metastatic prostate cancer has hindered the development of personalized therapeutic approaches. Therefore, it is difficult to predict the course of metastatic hormone-sensitive prostate cancer (mHSPC), with some men remaining on first-line androgen deprivation therapy (ADT) for several years while others progress more rapidly. Improving our ability to risk-stratify patients would allow for the optimization of systemic therapies and support the development of stratified prospective clinical trials focused on patients likely to have the greatest potential benefit. Here, we applied a liquid biopsy approach to identify clinically relevant, blood-based prognostic biomarkers in patients with mHSPC. Gene expression indicating the presence of CTCs was greater in CHAARTED high-volume (HV) patients (52% CTChigh) than in low-volume (LV) patients (23% CTChigh; * p = 0.03). HV disease (p = 0.005, q = 0.033) and CTC presence at baseline prior to treatment initiation (p = 0.008, q = 0.033) were found to be independently associated with the risk of nonresponse at 7 months. The pooled gene expression from CTCs of pre-ADT samples found AR, DSG2, KLK3, MDK, and PCA3 as genes predictive of nonresponse. These observations support the utility of liquid biomarker approaches to identify patients with poor initial response. This approach could facilitate more precise treatment intensification in the highest risk patients

Intraobserver Variability in Bladder Cancer Treatment Response Assessment With and Without Computerized Decision Support

We evaluated the intraobserver variability of physicians aided by a computerized decision-support system for treatment response assessment (CDSS-T) to identify patients who show complete response to neoadjuvant chemotherapy for bladder cancer, and the effects of the intraobserver variability on physicians\u27 assessment accuracy. A CDSS-T tool was developed that uses a combination of deep learning neural network and radiomic features from computed tomography (CT) scans to detect bladder cancers that have fully responded to neoadjuvant treatment. Pre- and postchemotherapy CT scans of 157 bladder cancers from 123 patients were collected. In a multireader, multicase observer study, physician-observers estimated the likelihood of pathologic T0 disease by viewing paired pre/posttreatment CT scans placed side by side on an in-house-developed graphical user interface. Five abdominal radiologists, 4 diagnostic radiology residents, 2 oncologists, and 1 urologist participated as observers. They first provided an estimate without CDSS-T and then with CDSS-T. A subset of cases was evaluated twice to study the intraobserver variability and its effects on observer consistency. The mean areas under the curves for assessment of pathologic T0 disease were 0.85 for CDSS-T alone, 0.76 for physicians without CDSS-T and improved to 0.80 for physicians with CDSS-T (P = .001) in the original evaluation, and 0.78 for physicians without CDSS-T and improved to 0.81 for physicians with CDSS-T (P = .010) in the repeated evaluation. The intraobserver variability was significantly reduced with CDSS-T (P < .0001). The CDSS-T can significantly reduce physicians\u27 variability and improve their accuracy for identifying complete response of muscle-invasive bladder cancer to neoadjuvant chemotherapy