Distinct RPA functions promote eukaryotic DNA replication initiation and elongation.
Nucleic Acids Res
; 51(19): 10506-10518, 2023 10 27.
Article
in En
| MEDLINE
| ID: mdl-37739410
ABSTRACT
Replication protein A (RPA) binds single-stranded DNA (ssDNA) and serves critical functions in eukaryotic DNA replication, the DNA damage response, and DNA repair. During DNA replication, RPA is required for extended origin DNA unwinding and DNA synthesis. To determine the requirements for RPA during these processes, we tested ssDNA-binding proteins (SSBs) from different domains of life in reconstituted Saccharomyces cerevisiae origin unwinding and DNA replication reactions. Interestingly, Escherichia coli SSB, but not T4 bacteriophage Gp32, fully substitutes for RPA in promoting origin DNA unwinding. Using RPA mutants, we demonstrated that specific ssDNA-binding properties of RPA are required for origin unwinding but that its protein-interaction domains are dispensable. In contrast, we found that each of these auxiliary RPA domains have distinct functions at the eukaryotic replication fork. The Rfa1 OB-F domain negatively regulates lagging-strand synthesis, while the Rfa2 winged-helix domain stimulates nascent strand initiation. Together, our findings reveal a requirement for specific modes of ssDNA binding in the transition to extensive origin DNA unwinding and identify RPA domains that differentially impact replication fork function.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
DNA-Binding Proteins
/
DNA Replication
/
Replication Protein A
Type of study:
Prognostic_studies
Language:
En
Journal:
Nucleic Acids Res
Year:
2023
Document type:
Article
Affiliation country:
Estados Unidos
Publication country:
ENGLAND
/
ESCOCIA
/
GB
/
GREAT BRITAIN
/
INGLATERRA
/
REINO UNIDO
/
SCOTLAND
/
UK
/
UNITED KINGDOM