Investigation of the Role of FANCJ helicase in G-quadruplex Resolution in Human Cells

Abstract

DNA replication is challenged by intrinsic obstacles, such as secondary DNA structures, and by external factors, such as genotoxins inducing DNA damage. In this thesis, we explore how cells cope with different forms of DNA replication blocks to ensure faithful genome duplication. In the first part of this study, we investigated if the helicase FANCJ is involved in the resolution of DNA secondary structures, in particular G-quadruplexes (G4), during DNA replication in human cells. Previous studies have shown that C. elegans FANCJ (dog-1) mutants accumulate G4 deletions. However, it is not known if this phenomenon can be observed in human FANCJ-deficient cells. We generated human lymphoblastoid TK6 FANCJ knockout cell lines and analyzed their genomes using whole-genome sequencing. In the second part of this study, we investigate how cells tolerate replication-blocking DNA lesions induced by the alkylating agent temozolomide (TMZ). Our analysis focuses on 3-methyladenine adducts, which are primarily repaired through the base excision repair (BER) pathway. We examined if nucleotide excision repair (NER) can compensate for the lack of BER as a backup mechanism. In addition, we explored the role of the primase–polymerase PRIMPOL, which facilitates repriming downstream of replication-blocking lesions, thereby generating single-stranded DNA gaps that need to be filled by the translesion synthesis. Overall, this work highlights the coordination of DNA repair and damage tolerance pathways to support replication fork progression under conditions of replication stress. CONTRIBUTIONS The clonogenic survival experiments were conducted with the assistance of Ratih Khoirunnisa, David M. Samuel and Soyoung Park at the IBS Center for Genomic Integrity. The computational analysis of whole- genome sequencing (WGS) data was performed by Taejoo Hwang and Hyeyeon Won under the supervision of Professor Semin Lee at the Computational Biology Laboratory, Department of Biomedical Engineering, UNIST.