Under or Over? Tracing Complex DNA Structures with High Resolution Atomic Force Microscopy DOI Creative Commons

Elizabeth P. Holmes,

Max C. Gamill, James I. Provan

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: June 28, 2024

ABSTRACT The topology of DNA plays a crucial role in the regulation cellular processes and genome stability. Despite its significance, remains challenging to determine due length conformational complexity individual topologically constrained molecules. We demonstrate unparalleled resolution complex topologies using Atomic Force Microscopy (AFM) aqueous conditions. present new high-throughput automated pipeline from raw AFM images, deep-learning methods trace backbone molecules identify crossing points. Our efficiently determines which segment passes over which, including handling crossings, where path each molecule may be harder resolve. wider applicability our tracing method by determining structure stalled replication intermediates Xenopus egg extracts, theta structures late products. By developing methodologies accurately through every crossing, we plasmids, knots catenanes E. coli Xer recombination system. In doing so uncover recurrent depositional effect reveal origins coarse-grained simulations. approach is broadly applicable range nucleic acid structures, those interact with proteins, opens avenues for understanding fundamental biological are regulated or affect topology.

Language: Английский

Leading and lagging strand abasic sites differentially affect vertebrate replisome progression but involve analogous bypass mechanisms DOI Open Access
Matthew T. Cranford,

Steven N Dahmen,

David Cortez

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 9, 2025

Abasic sites are one of the most frequent forms DNA damage that interfere with replication. However, abasic exhibit complex effects because they can be processed into other types damage. Thus, it remains poorly understood how affect replisome progression, which replication-coupled repair pathways elicit, and whether this is affected by template strand damaged. Using Xenopus egg extracts, we developed an approach to analyze replication containing a site-specific, stable site on leading or lagging template. We show robustly stall synthesis nascent strands but exert different when encountered At AP site, replisomes ∼100 bp from lesion until bypassed converging fork triggers termination. progression unaffected reprimed downstream, generating post-replicative gap, then bypassed. Despite both rely translesion for bypass. Our results detail similarities differences between vertebrate

Language: Английский

Citations

0
Use of Xenopus Egg Extracts to Study the Effects of Topoisomerase Poisons During Vertebrate DNA Replication DOI
Sabrina X. Van Ravenstein, James M. Dewar

Methods in molecular biology, Journal Year: 2025, Volume and Issue: unknown, P. 151 - 172

Published: Jan. 1, 2025

Language: Английский

Citations

0
Under or Over? Tracing Complex DNA Structures with High Resolution Atomic Force Microscopy DOI Creative Commons

Elizabeth P. Holmes,

Max C. Gamill, James I. Provan

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: June 28, 2024

ABSTRACT The topology of DNA plays a crucial role in the regulation cellular processes and genome stability. Despite its significance, remains challenging to determine due length conformational complexity individual topologically constrained molecules. We demonstrate unparalleled resolution complex topologies using Atomic Force Microscopy (AFM) aqueous conditions. present new high-throughput automated pipeline from raw AFM images, deep-learning methods trace backbone molecules identify crossing points. Our efficiently determines which segment passes over which, including handling crossings, where path each molecule may be harder resolve. wider applicability our tracing method by determining structure stalled replication intermediates Xenopus egg extracts, theta structures late products. By developing methodologies accurately through every crossing, we plasmids, knots catenanes E. coli Xer recombination system. In doing so uncover recurrent depositional effect reveal origins coarse-grained simulations. approach is broadly applicable range nucleic acid structures, those interact with proteins, opens avenues for understanding fundamental biological are regulated or affect topology.

Language: Английский

Citations

2