RESUMO
Generation of single-stranded DNA (ssDNA) is required for the template strand formation during DNA replication. Replication Protein A (RPA) is an ssDNA-binding protein essential for protecting ssDNA at replication forks in eukaryotic cells. While significant progress has been made in characterizing the role of the RPA-ssDNA complex, how RPA is loaded at replication forks remains poorly explored. Here, we show that the Saccharomyces cerevisiae protein regulator of Ty1 transposition 105 (Rtt105) binds RPA and helps load it at replication forks. Cells lacking Rtt105 exhibit a dramatic reduction in RPA loading at replication forks, compromised DNA synthesis under replication stress, and increased genome instability. Mechanistically, we show that Rtt105 mediates the RPA-importin interaction and also promotes RPA binding to ssDNA directly in vitro, but is not present in the final RPA-ssDNA complex. Single-molecule studies reveal that Rtt105 affects the binding mode of RPA to ssDNA These results support a model in which Rtt105 functions as an RPA chaperone that escorts RPA to the nucleus and facilitates its loading onto ssDNA at replication forks.
Assuntos
Genoma Fúngico , Instabilidade Genômica , Modelos Biológicos , Chaperonas Moleculares/metabolismo , Proteína de Replicação A/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , DNA Fúngico/genética , DNA Fúngico/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Carioferinas/genética , Carioferinas/metabolismo , Chaperonas Moleculares/genética , Proteína de Replicação A/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Single-stranded DNA (ssDNA) is widely generated during DNA metabolisms including DNA replication, repair and recombination and is susceptible to digestion by nucleases and secondary structure formation. It is vital for DNA metabolism and genome stability that ssDNA is protected and stabilized, which are performed by the major ssDNA-binding protein, and replication protein A (RPA) in these processes. In addition, RPA-coated ssDNA also serves as a protein-protein-binding platform for coordinating multiple events during DNA metabolisms. However, little is known about whether and how the formation of RPA-ssDNA platform is regulated. Here we highlight our recent study of a novel RPA-binding protein, Regulator of Ty1 transposition 105 (Rtt105) in Saccharomyces cerevisiae, which regulates the RPA-ssDNA platform assembly at replication forks. We propose that Rtt105 functions as an "RPA chaperone" during DNA replication, likely also promoting the assembly of RPA-ssDNA platform in other processes in which RPA plays a critical role.