Biochemical analysis of DNA synthesis blockage by G-quadruplex structure and bypass facilitated by a G4-resolving helicase

Sommers, Joshua A.; Estep, Katrina N; Maul, Robert W.; Brosh, Robert M.

doi:10.1016/j.ymeth.2021.12.005

Cited by 2 publications

(2 citation statements)

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“…Stalling or blockage of replication has been shown to cause DNA damage such as double-stranded DNA breaks, chromosomal rearrangement, and eventual cell death. , A 2001 study by Kamath-Loeb et al employed a primer-extension assay to demonstrate that the activity of the three replicative polymerases operating in humans (α, δ, and ε) was inhibited by the presence of a qDNA structure . Since then, a vast array of ensemble in vitro studies have demonstrated qDNA structures can stall and even completely block the replication fork from progressing. − In contrast, there are far fewer studies which use single-molecule techniques to study the impact of qDNA formation on the dynamics of DNA replication. This is due to the difficulty in replicating a dynamic replication fork/replisome system with a qDNA structure and observing its interaction with DNA polymerases in real time using such methods within a heterogeneous sample.…”

Section: G-quadruplex Biology: Ally or Enemy To Genomic Maintenance?mentioning

confidence: 99%

Understanding G-Quadruplex Biology and Stability Using Single-Molecule Techniques

Kusi-Appauh,

Ralph,

van Oijen

et al. 2023

J. Phys. Chem. B

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The link between the chemical stability of Gquadruplex (qDNA) structures and their roles in eukaryotic genomic maintenance processes has been an area of interest now for several decades. This Review seeks to demonstrate how single-molecule force-based techniques can provide insight into the mechanical stabilities of a variety of qDNA structures as well as their ability to interconvert between different conformations under conditions of stress. Atomic force microscopy (AFM) and magnetic and optical tweezers have been the primary tools used in these investigations and have been used to examine both free and ligand-stabilized Gquadruplex structures. These studies have shown that the degree of stabilization of G-quadruplex structures has a significant effect on the ability of nuclear machinery to bypass these roadblocks on DNA strands. This Review will illustrate how various cellular components including replication protein A (RPA), Bloom syndrome protein (BLM), and Pif1 helicases are capable of unfolding qDNA. Techniques such as single-molecule fluorescence resonance energy transfer (smFRET), often in conjunction with the aforementioned force-based techniques, have proven extremely effective at elucidating the factors underpinning the mechanisms by which these proteins unwind qDNA structures. We will provide insight into how single-molecule tools have facilitated the direct visualization of qDNA roadblocks and also showcase results obtained from experiments designed to examine the ability of G-quadruplexes to limit the access of specific cellular proteins normally associated with telomeres.

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Section: G-quadruplex Biology: Ally or Enemy To Genomic Maintenance?mentioning

confidence: 99%

Understanding G-Quadruplex Biology and Stability Using Single-Molecule Techniques

Kusi-Appauh,

Ralph,

van Oijen

et al. 2023

J. Phys. Chem. B

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“…The common denominator of defective G‐rich lagging strand synthesis in cells deficient in WRN or BRCA2 discussed above raises the possibility that the two proteins act to protect replication forks at telomeres in parallel pathways. One of the provocative DNA structures formed by the G‐rich telomeric strand is G4, which has been shown to interfere with smooth DNA synthesis by various DNA polymerases in vitro (for review, see [ 31 ] ). A useful tool to investigate G4 DNA metabolism in cells has been the pharmacologically active G4 binding ligands.…”

Section: Wrn's Role In Telomere Replication and Potential Interplay W...mentioning

confidence: 99%

WRN rescues replication forks compromised by a BRCA2 deficiency: Predictions for how inhibition of a helicase that suppresses premature aging tilts the balance to fork demise and chromosomal instability in cancer

Datta

Brosh

2022

BioEssays

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Hereditary breast and ovarian cancers are frequently attributed to germline mutations in the tumor suppressor genes BRCA1 and BRCA2. BRCA1/2 act to repair double-strand breaks (DSBs) and suppress the demise of unstable replication forks. Our work elucidated a dynamic interplay between BRCA2 and the WRN DNA helicase/exonuclease defective in the premature aging disorder Werner syndrome. WRN and BRCA2 participate in complementary pathways to stabilize replication forks in cancer cells, allowing them to proliferate. Whether the functional overlap of WRN and BRCA2 is relevant to replication at gaps between newly synthesized DNA fragments, protection of telomeres, and/or metabolism of secondary DNA structures remain to be determined.Advances in understanding the mechanisms elicited during replication stress have prompted the community to reconsider avenues for cancer therapy. Insights from studies of PARP or topoisomerase inhibitors provide working models for the investigation of WRN's mechanism of action. We discuss these topics, focusing on the implications of the WRN-BRCA2 genetic interaction under conditions of replication stress.

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Biochemical analysis of DNA synthesis blockage by G-quadruplex structure and bypass facilitated by a G4-resolving helicase

Cited by 2 publications

References 44 publications

Understanding G-Quadruplex Biology and Stability Using Single-Molecule Techniques

Understanding G-Quadruplex Biology and Stability Using Single-Molecule Techniques

WRN rescues replication forks compromised by a BRCA2 deficiency: Predictions for how inhibition of a helicase that suppresses premature aging tilts the balance to fork demise and chromosomal instability in cancer

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