Biophysical Society Thematic Meeting| Lima 2019

Revisiting the Central Dogma of Molecular Biology at the Single-Molecule Level

Saturday Speaker Abstracts

SUPER-RESOLUTION IMAGING OF REPLICATION-ASSOCIATED G4 DNA IN HUMAN CELLS Wei Ting Chelsea Lee 1 ; Yandong Yin 1 ; Peter Tonzi 1 ; Sharon B Cantor 2 ; Tony Huang 1 ; Eli Rothenberg 1 ; 1 New York University School of Medicine, Department of Biochemistry and Molecular Pharmacology, New York, NY, USA 2 University of Massachusetts Medical School, Department of Molecular, Cell and Cancer Biology, Worcester, MA, USA DNA G-quadruplex (G4) structures are noncanonical secondary structures formed within guanine-rich DNA that play important roles in various genomic metabolic processes such as DNA replication. Despite their importance, the specific impacts of DNA G4 formation and regulation during replication remain elusive in part due to the limitations of conventional in vitro and ensemble experimental techniques. To address this knowledge gap, we developed a platform to quantitatively visualize the spatial associations between DNA G4 structures and individual replisome complexes in human cells, by coupling multi-color single-molecule localization fluorescence microscopy (SMLM) with robust triple-correlation (TC) analysis. We showed that during normal replication, DNA G4 predominantly form at newly unwound single-stranded DNA (ssDNA) in between MCM helicase and nascent DNA. By measuring nascent DNA signals at individual replication fork level, we observed a reduced nascent DNA incorporation at G4- associated forks (RF-G4) compared to regular replication forks, indicating that RF-G4s can block replication progression locally. Interestingly, the loading of RPA onto ssDNA is also restricted specifically at RF-G4s but not in regular forks, suggesting that G4 accumulation at forks may result in the suppression of RPA-mediated replication stress response. Importantly, we found that the FANCJ helicase is required to maintain normal replication fork progression by counteracting the abundance of RF-G4s, and the deficiency of FANCJ results in an increased amount of gH2AX signal at RF-G4s, indicating local replication fork collapse and formation of DNA double strand breaks. These findings suggest an important regulatory role of FANCJ in the processing of DNA G4 structures during replication in order to maintain normal replication and preserve genomic integrity.

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