Rapid Long-distance Migration of RPA on Single Stranded DNA Occurs Through Intersegmental Transfer Utilizing Multivalent Interactions.
J Mol Biol
; 436(6): 168491, 2024 03 15.
Article
in En
| MEDLINE
| ID: mdl-38360091
ABSTRACT
Replication Protein A (RPA) is asingle strandedDNA(ssDNA)binding protein that coordinates diverse DNA metabolic processes including DNA replication, repair, and recombination. RPA is a heterotrimeric protein with six functional oligosaccharide/oligonucleotide (OB) domains and flexible linkers. Flexibility enables RPA to adopt multiple configurations andis thought to modulate its function. Here, usingsingle moleculeconfocal fluorescencemicroscopy combinedwith optical tweezers and coarse-grained molecular dynamics simulations, we investigated the diffusional migration of single RPA molecules on ssDNA undertension.The diffusioncoefficientDis the highest (20,000nucleotides2/s) at 3pNtension and in 100 mMKCl and markedly decreases whentensionor salt concentrationincreases. We attribute the tension effect to intersegmental transfer which is hindered by DNA stretching and the salt effect to an increase in binding site size and interaction energy of RPA-ssDNA. Our integrative study allowed us to estimate the size and frequency of intersegmental transfer events that occur through transient bridging of distant sites on DNA by multiple binding sites on RPA. Interestingly, deletion of RPA trimeric core still allowed significant ssDNA binding although the reduced contact area made RPA 15-fold more mobile. Finally, we characterized the effect of RPA crowding on RPA migration. These findings reveal how the high affinity RPA-ssDNA interactions are remodeled to yield access, a key step in several DNA metabolic processes.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
DNA, Single-Stranded
/
Replication Protein A
Language:
En
Journal:
J Mol Biol
Year:
2024
Document type:
Article
Affiliation country:
Estados Unidos
Publication country:
HOLANDA
/
HOLLAND
/
NETHERLANDS
/
NL
/
PAISES BAJOS
/
THE NETHERLANDS