Characterizing Ku’s Role as an AP lyase in Nonhomologous End Joining

Nonhomologous end joining (NHEJ) is important for the repair of ionizing radiation and radiomimetic drug-generated DSBs, which are often associated with ligation-obstructing nucleotide damage. To facilitate ligation at such breaks, NHEJ employs a host of processing factors (i.e. nucleases, polymerases, etc.) that prepare DNA ends for joining. While this mechanism is efficient at joining broken chromosomes, it can frequently be inaccurate (i.e. loss of sequence at the DSB), because repair is mediated without the assistance of a template. My dissertation demonstrates how NHEJ-mediated repair of DSBs with associated abasic sites is an exception to this phenomenon. I show that abasic sites at DSB termini severely block NHEJ's ligation step and must be excised for joining to proceed. Despite the many processing enzymes associated with NHEJ, none are capable of excising this damage. Instead we found that the NHEJ core factor, Ku, has intrinsic lyase activity that removes these abasic sites. Analysis of Ku's substrate specificity reveals that lyase activity is restricted to abasic sites near a 5' terminus that directly block ligation. Furthermore, sequence 5' of abasic sites embedded in double stranded DNA (+4 bps) is mostly preserved due to Ku's limited activity in this context. By characterizing Ku's active site I identified eight lysine residues that contribute to lyase activity and determined that the primary nucleophile is within the N-terminus of Ku70 (K31). These amino acids reside on the outer surface of the Ku heterodimer nearest the DNA end - an optimal position for interacting with abasic sites closest to the break terminus. My results provide mechanistic insight into how NHEJ deals with one type of damage induced by ionizing radiation and may explain why loss of Ku leads to severe radiation sensitivity. Additionally, my results suggest NHEJ is more than a simple ligation machine but rather it is a sophisticated pathway suited to repair and join DSBs with associated nucleotide lesions.Doctor of Philosoph

Resolution of complex ends by Nonhomologous end joining - better to be lucky than good?

Abstract The Nonhomologous end joining pathway is essential for efficient repair of chromosome double strand breaks. This pathway consequently plays a key role in cellular resistance to break-inducing exogenous agents, as well as in the developmentally-programmed recombinations that are required for adaptive immunity. Chromosome breaks often have complex or “dirty” end structures that can interfere with the critical ligation step in this pathway; we review here how Nonhomologous end joining resolves such breaks

Nonhomologous end joining: A good solution for bad ends

Double strand breaks pose unique problems for DNA repair, especially when broken ends possess complex structures that interfere with standard DNA transactions. Nonhomologous end joining can use multiple strategies to solve these problems. It further uses sophisticated means to ensure the strategy chosen provides the ideal balance of flexibility and accuracy

Ku is a 5′-dRP/AP lyase that excises nucleotide damage near broken ends

Mammalian cells require Nonhomologous end joining (NHEJ) for efficient repair of chromosomal DNA double-strand breaks1. A key feature of biological sources of strand breaks is associated nucleotide damage, including base loss (abasic or AP sites)2. At single strand breaks, 5' terminal abasic sites are excised by pol β's 5'dRP lyase activity3,4,5,6: we show here in vitro and in cells that accurate and efficient repair by NHEJ of double-strand breaks with such damage similarly requires 5'dRP/AP lyase activity (Figure 1a). Classically defined NHEJ is moreover uniquely effective at coupling this end-cleaning step to joining in cells, helping distinguish this pathway from otherwise robust alternate NHEJ pathways. Surprisingly, the NHEJ factor Ku can be identified as an effective 5'dRP/AP lyase. Similar to other lyases7, Ku nicks DNA 3' of an abasic site by a mechanism involving a Schiff base covalent intermediate with the abasic site. We demonstrate using cell extracts that Ku is essential for efficient removal of AP sites near double-strand breaks and, consistent with this result, joining of such breaks is specifically reduced in cells complemented with a lyase-attenuated Ku mutant. Ku had previously been presumed only to recognize ends and recruit other factors that processed ends; our data supports an unexpected direct role for Ku in end processing steps as well

Prenatal exome sequencing in anomalous fetuses: new opportunities and challenges

We investigated the diagnostic and clinical performance of exome sequencing (ES) in fetuses with sonographic abnormalities with normal karyotype, microarray and, in some cases, normal gene specific sequencing

Organization and dynamics of the nonhomologous end-joining machinery during DNA double-strand break repair

Nonhomologous end-joining (NHEJ) is the main pathway for repair of DNA double-strand breaks (DSBs), the most cytotoxic form of DNA damage resulting from ionizing radiation, chemotherapeutics, and normal cellular processes. The mechanisms that control NHEJ play key roles in development, in immunity, and in response to cancer therapy; however, the current state of knowledge regarding the physical nature of the NHEJ repair process is limited. Here we used super-resolution microscopy to define the organization of NHEJ complexes in cells, showing that long filaments form at either side of the break. Single-molecule FRET revealed dynamic behavior in which breaks can pair in an adjacent, non–end-to-end configuration

The fidelity of the ligation step determines how ends are resolved during nonhomologous end joining

Nonhomologous end joining (NHEJ) can effectively resolve chromosome breaks despite diverse end structures, but it is unclear how the steps employed for resolution are determined. We sought to address this question by analyzing cellular NHEJ of ends with systematically mispaired and damaged termini. We show NHEJ is uniquely proficient at bypassing subtle terminal mispairs and radiomimetic damage by direct ligation. Nevertheless, bypass ability varies widely, with increases in mispair severity gradually reducing bypass products from 85% to 6%. End-processing by nucleases and polymerases is increased to compensate, though paths with the fewest number of steps to generate a substrate suitable for ligation are favored. Thus, both the frequency and nature of end processing are tailored to meet the needs of the ligation step. We propose a model where the ligase organizes all steps during NHEJ within the stable paired-end complex to limit end processing and associated errors

A semiquantitative metric for evaluating clinical actionability of incidental or secondary findings from genome-scale sequencing

As genome-scale sequencing is increasingly applied in clinical scenarios, a wide variety of genomic findings will be discovered as secondary or incidental findings, and there is debate about how they should be handled. The clinical actionability of such findings varies, necessitating standardized frameworks for a priori decision making about their analysis

A standardized, evidence-based protocol to assess clinical actionability of genetic disorders associated with genomic variation

Genome and exome sequencing can identify variants unrelated to the primary goal of sequencing. Detecting pathogenic variants associated with an increased risk of a medical disorder enables clinical interventions to improve future health outcomes in patients and their at-risk relatives. The Clinical Genome Resource, or ClinGen, aims to assess clinical actionability of genes and associated disorders as part of a larger effort to build a central resource of information regarding the clinical relevance of genomic variation for use in precision medicine and research

Evaluating the Clinical Validity of Gene-Disease Associations: An Evidence-Based Framework Developed by the Clinical Genome Resource

Supplemental Data Supplemental Data include 65 figures and can be found with this article online at http://dx.doi.org/10.1016/j.ajhg.2017.04.015. Supplemental Data Document S1. Figures S1–S65 Download Document S2. Article plus Supplemental Data Download Web Resources ClinGen, https://www.clinicalgenome.org/ ClinGen Gene Curation, https://www.clinicalgenome.org/working-groups/gene-curation/ ClinGen Gene Curation SOP, https://www.clinicalgenome.org/working-groups/gene-curation/projects-initiatives/gene-disease-clinical-validity-sop/ ClinGen Knowledge Base, https://search.clinicalgenome.org/kb/agents/sign_up OMIM, http://www.omim.org/ Orphanet, http://www.orpha.net/consor/cgi-bin/index.php With advances in genomic sequencing technology, the number of reported gene-disease relationships has rapidly expanded. However, the evidence supporting these claims varies widely, confounding accurate evaluation of genomic variation in a clinical setting. Despite the critical need to differentiate clinically valid relationships from less well-substantiated relationships, standard guidelines for such evaluation do not currently exist. The NIH-funded Clinical Genome Resource (ClinGen) has developed a framework to define and evaluate the clinical validity of gene-disease pairs across a variety of Mendelian disorders. In this manuscript we describe a proposed framework to evaluate relevant genetic and experimental evidence supporting or contradicting a gene-disease relationship and the subsequent validation of this framework using a set of representative gene-disease pairs. The framework provides a semiquantitative measurement for the strength of evidence of a gene-disease relationship that correlates to a qualitative classification: “Definitive,” “Strong,” “Moderate,” “Limited,” “No Reported Evidence,” or “Conflicting Evidence.” Within the ClinGen structure, classifications derived with this framework are reviewed and confirmed or adjusted based on clinical expertise of appropriate disease experts. Detailed guidance for utilizing this framework and access to the curation interface is available on our website. This evidence-based, systematic method to assess the strength of gene-disease relationships will facilitate more knowledgeable utilization of genomic variants in clinical and research settings