Phosphorylation and cellular function of the human Rpa2 N-terminus in the budding yeast Saccharomyces cerevisiae.

Maintenance of genome integrity is critical for proper cell growth. This occurs through accurate DNA replication and repair of DNA lesions. A key factor involved in both DNA replication and the DNA damage response is the heterotrimeric single-stranded DNA (ssDNA) binding complex Replication Protein A (RPA). Although the RPA complex appears to be structurally conserved throughout eukaryotes, the primary amino acid sequence of each subunit can vary considerably. Examination of sequence differences along with the functional interchangeability of orthologous RPA subunits or regions could provide insight into important regions and their functions. This might also allow for study in simpler systems. We determined that substitution of yeast Replication Factor A (RFA) with human RPA does not support yeast cell viability. Exchange of a single yeast RFA subunit with the corresponding human RPA subunit does not function due to lack of inter-species subunit interactions. Substitution of yeast Rfa2 with domains/regions of human Rpa2 important for Rpa2 function (i.e., the N-terminus and the loop 3-4 region) supports viability in yeast cells, and hybrid proteins containing human Rpa2 N-terminal phospho-mutations result in similar DNA damage phenotypes to analogous yeast Rfa2 N-terminal phospho-mutants. Finally, the human Rpa2 N-terminus (NT) fused to yeast Rfa2 is phosphorylated in a manner similar to human Rpa2 in human cells, indicating that conserved kinases recognize the human domain in yeast. The implication is that budding yeast represents a potential model system for studying not only human Rpa2 N-terminal phosphorylation, but also phosphorylation of Rpa2 N-termini from other eukaryotic organisms.

Characterization of the interaction between Rfa1 and Rad24 in Saccharomyces cerevisiae.

Maintaining the integrity of the genome requires the high fidelity duplication of the genome and the ability of the cell to recognize and repair DNA lesions. The heterotrimeric single stranded DNA (ssDNA) binding complex Replication Protein A (RPA) is central to multiple DNA processes, which are coordinated by RPA through its ssDNA binding function and through multiple protein-protein interactions. Many RPA interacting proteins have been reported through large genetic and physical screens; however, the number of interactions that have been further characterized is limited. To gain a better understanding of how RPA functions in DNA replication, repair, and cell cycle regulation and to identify other potential functions of RPA, a yeast two hybrid screen was performed using the yeast 70 kDa subunit, Replication Factor A1 (Rfa1), as a bait protein. Analysis of 136 interaction candidates resulted in the identification of 37 potential interacting partners, including the cell cycle regulatory protein and DNA damage clamp loader Rad24. The Rfa1-Rad24 interaction is not dependent on ssDNA binding. However, this interaction appears affected by DNA damage. The regions of both Rfa1 and Rad24 important for this interaction were identified, and the region of Rad24 identified is distinct from the region reported to be important for its interaction with Rfc2 5. This suggests that Rad24-Rfc2-5 (Rad24-RFC) recruitment to DNA damage substrates by RPA occurs, at least partially, through an interaction between the N terminus of Rfa1 and the C terminus of Rad24. The predicted structure and location of the Rad24 C-terminus is consistent with a model in which RPA interacts with a damage substrate, loads Rad24-RFC at the 5' junction, and then releases the Rad24-RFC complex to allow for proper loading and function of the DNA damage clamp.