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Master's Thesis
Analysis of DNA polymerase theta-exclusive mutation signatures in the context of proximal and
distal end joining repair
Andrew Aujin Kim
Department of Biological Sciences
Ulsan National Institute of Science and Technology
Andrew Aujin Kim
Department of Biological Sciences
Ulsan National Institute of Science and Technology
Analysis of DNA polymerase theta-exclusive mutation signatures in the context of proximal and
distal end joining repair
A thesis/dissertation submitted to
Ulsan National Institute of Science and Technology in partial fulfillment of the
requirements for the degree of Master of Science
Andrew Aujin Kim
Analysis of DNA polymerase theta-exclusive mutation signatures in the context of proximal and
distal end joining repair
12.13.2021 of submission
Approved by
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- to
Abstract
DNA polymerase theta (POLQ)-mediated end joining (TMEJ) is a distinct DNA double-strand break (DSB) repair pathway besides homologous recombination (HR) and non-homologous end joining (NHEJ). Because HR- and/or NHEJ-defective tumors predictively exhibit hyper-sensitivity to POLQ inhibition, identification of tumors that rely on TMEJ for DSB repair is a pivotal task for screening eligibility for therapeutics exploiting synthetic lethality of POLQ. TMEJ mediates the joining of two resected 3′ ends harboring microhomology (MH) and generates distinctive genomic scars. While studies have identified mutation signatures generated by MH-mediated end-joining (MMEJ), distinction of MMEJ performed by TMEJ or NHEJ has not been clearly defined. Since NHEJ is able to anneal a few protruding nucleotides at a broken DNA end and join, the sequence end-joined by TMEJ or MMEJ is almost identical. In this study, to directly address the question whether present of functional POLQ influence behavior of EJ, we have examined deletion signatures of proximal EJ (direct joining of a DSB) and distal EJ (end joining of two separated DSBs) products generated by POLQ+/+ and POLQ-/- with the hypothesis that if POLQ affects EJ, then we can observe POLQ-exclusive mutation patterns that are not present in POLQ-KO cell lines. My study demonstrated that POLQ+/+ generated distinct deletion signature landscape from POLQ-/-. A significantly larger variety of MH-mediated end joined products associating with longer DNA deletion were identified in POLQ+/+ than in POLQ-/- in distal EJ. The result suggests that TMEJ may stimulate DNA end resection to expose MH for MMEJ. Similarly, POLQ-KO cell lines demonstrated overall attenuated frequency of deletions and shorter MH-mediated deletions in proximal EJ. The better understanding of TMEJ mutation signatures as described here will help to develop more effective pipelines to detect TMEJ-dependent mutation signatures in genome sequencing data.
Table of Contents
Abstract ... i
Table of Contents ... ii
List of Tables and Figures ... iii
List of Abbreviations ... iv
I. Introduction ... 1
II. Materials and Methods ... 4
2-1. Datasets ... 4
2-2. Bioinformatics ... 4
2-3. Visualization ... 5
III. Results ... 6
3-1. POLQ partially attenuates ID6 and ID8 mutational signatures ... 6
3-2. Knocking down 53BP1 or DNA-PKcs enhances deletions with longer MHs ... 7
3-3.POLQ enhances distal EJ of longer deletions with longer MHs ... 8
IV. Discussion ... 11
References ... 13
Acknowledgement ... 15
iii
List of Tables and Figures
Figure 1. Three major DNA DSB repair pathways ... 1
Figure 2. Domain locations of POLQ and Mus308 ... 2
Figure 3. COSMIC3 mutational signatures enriched by POLQ ... 3
Figure 4. Brief overview of bioinformatics pipeline to characterize MH-mediated deletions ... 4
Figure 5. Proximal EJ in comparison to COSMIC3 mutational signatures ID6 and ID8 ... 6
Figure 6. Mis-categorization of MH-mediated deletion verified via IGV ... 7
Figure 7. Two-dimensional grid characterizing unique proximal EJ products ... 8
Figure 8. Characterization of distal EJ products by MH and deletion lengths ... 9
List of Abbreviations
DSB Double strand break
POLQ DNA polymerase theta (θ)
EJ End joining
TMEJ Theta-mediated end joining
MH Microhomology
NHEJ Non-homologous end joining
MMEJ Microhomology-mediated end joining altEJ Alternative end joining
53BP1 p53-binding protein 1
DNA-PKcs DNA-dependent protein kinase, catalytic subunit
CSR Class switch recombination
COSMIC Catalogue Of Somatic Mutations In Cancer
SBS Single Base Substitution
ID Insertion/deletion
OFP Orange Fluorescent Protein
GFP Green Fluorescent Protein
MRN Mre11-Rad50-Nbs1
IGV Integrative Genomics Viewer
