Revealing SMC5/6's Dynamic Behavior on Diverse DNA Topologies Through Single-Molecule Methods

<p>The structural maintenance of chromosomes (SMC) protein complexes serve as critical molecular motors for maintaining genome stability and ensuring faithful genome propagation. As one of the three eukaryotic SMC complexes, the Smc5/6 complex maintains chromosomal stability by coordinating DNA repair processes, promoting successful genome duplication, and silencing viral extrachromosomal DNA. Although these main functionalities have been identified, there is little understanding of how Smc5/6 operates as a versatile molecular machine capable of undertaking different functionalities. And despite being intimately involved with DNA processing reactions, little is known about Smc5/6's dynamic behavior on various DNA substrates, including double-stranded DNA (dsDNA), stretches of single-stranded DNA (ssDNA), chromatinized DNA, forked/junction DNA, and recombination intermediates. In this thesis, our approach was to utilize correlative single-molecule fluorescence and force microscopy, which combined optical tweezers, automated microfluidics, and multi-color confocal microscopy, to investigate the S. cerevisiae Smc5/6 complex's dynamic behavior on a diverse array of DNA topologies. Firstly, we uncovered dynamic modes of association for Smc5/6 on linear dsDNA. For the octameric holo-complex that contains the Nse5-6 subunit, Smc5/6 demonstrates both an ATP-independent dsDNA association mode and an ATP- dependent mode that exhibits greatly enhanced dsDNA association. This is likely due to a conformational change in the ATP-bound state that results in a 'clamped' configuration around dsDNA, making Smc5/6 resistant to high-salt challenges on dsDNA. However, the Smc5/6 hexamer complex lacking Nse5-6 exhibited dramatically increased dsDNA association independently of ATP, and this hexamer binding mode was resistant to high salt without requiring ATP. This indicates a novel mode of dsDNA association that may play a role in Smc5/6's ability to loop extrude. Additionally, the Smc5/6 complex showed a striking capacity to accumulate onto free ssDNA stretches that can inhibit reannealing of complementary ssDNA strands, which could contribute to Smc5/6's roles in stabilizing replication forks and preventing spurious strand invasion during homologous recombination. We also show that Smc5/6 can stably bind to DNA junctions resembling replication forks as well as linear ss-dsDNA junctions—both at the 5' and 3' end junctions. However, ATP and the RPA-ssDNA complex can confer a polarity preference for Smc5/6. We show that different subunits of Smc5/6 contribute to different functions as the Nse1-3-4 subunit constitutively contributes to DNA binding while the Nse5-6 subunit performs a modulatory role. Lastly, Smc5/6 displays the surprising interaction of stably associating with nucleosomes, while exhibiting more competitive behaviors with PCNA on DNA. Overall, our findings on the intrinsic properties of the Smc5/6 complex contribute to a framework for understanding how Smc5/6 may target and associate with DNA in a diverse array of cellular contexts ranging from DNA repair and stressed replication forks, to telomeric heterochromatin and extrachromosomal viral DNA.</p>

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Thesis

Year

2024

Author dc:creator

• Chang, Jeremy Tzu-Huai

Contributors dc:contributor

• Shixin Liu

• Smc5/6 complex
• DNA binding
• single-molecule microscopy
• DNA repair
• replication fork stabilization
• genome stability
• Life Sciences

Repository record dc:identifier

https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/755

OAI identifier oai:identifier

oai:digitalcommons.rockefeller.edu:student_theses_and_dissertations-1759