Single-molecule visualization of human RECQ5 interactions with single-stranded DNA recombination intermediates

Autor: Weixing Zhao, Lucia Molnarova, Chaoyou Xue, Mário Špírek, Ondrej Beláň, Kyle Kaniecki, Katerina Krejci, Lumir Krejci, Patrick Sung, Eric C. Greene, Simon J. Boulton, Justin B. Steinfeld, Chujian Ma, Youngho Kwon
Rok vydání: 2020
Předmět:
AcademicSubjects/SCI00010
Recombinant Fusion Proteins
genetic processes
Mutant
Mutation
Missense
RAD51
DNA
Single-Stranded
Genome Integrity
Repair and Replication
Microscopy
Atomic Force
Substrate Specificity
03 medical and health sciences
chemistry.chemical_compound
Adenosine Triphosphate
0302 clinical medicine
Replication Protein A
Genetics
Recombinase
Humans
Point Mutation
Homologous Recombination
Replication protein A
030304 developmental biology
0303 health sciences
RecQ Helicases
biology
Hydrolysis
Molecular Motor Proteins
Helicase
Recombinant Proteins
Single Molecule Imaging
Kinetics
enzymes and coenzymes (carbohydrates)
chemistry
030220 oncology & carcinogenesis
health occupations
biology.protein
Biophysics
Rad51 Recombinase
biological phenomena
cell phenomena
and immunity
Homologous recombination
DNA
Heteroduplex
Zdroj: Nucleic Acids Research
ISSN: 1362-4962
0305-1048
DOI: 10.1093/nar/gkaa1184
Popis: RECQ5 is one of five RecQ helicases found in humans and is thought to participate in homologous DNA recombination by acting as a negative regulator of the recombinase protein RAD51. Here, we use kinetic and single molecule imaging methods to monitor RECQ5 behavior on various nucleoprotein complexes. Our data demonstrate that RECQ5 can act as an ATP-dependent single-stranded DNA (ssDNA) motor protein and can translocate on ssDNA that is bound by replication protein A (RPA). RECQ5 can also translocate on RAD51-coated ssDNA and readily dismantles RAD51–ssDNA filaments. RECQ5 interacts with RAD51 through protein–protein contacts, and disruption of this interface through a RECQ5–F666A mutation reduces translocation velocity by ∼50%. However, RECQ5 readily removes the ATP hydrolysis-deficient mutant RAD51–K133R from ssDNA, suggesting that filament disruption is not coupled to the RAD51 ATP hydrolysis cycle. RECQ5 also readily removes RAD51–I287T, a RAD51 mutant with enhanced ssDNA-binding activity, from ssDNA. Surprisingly, RECQ5 can bind to double-stranded DNA (dsDNA), but it is unable to translocate. Similarly, RECQ5 cannot dismantle RAD51-bound heteroduplex joint molecules. Our results suggest that the roles of RECQ5 in genome maintenance may be regulated in part at the level of substrate specificity.
Databáze: OpenAIRE
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