sara-binz@uiowa.edu
Mentor: Marc Wold, Ph.D.
Lab Phone: 335-7851

Phosphorylation of Replication Protein A (RPA) Regulates RPA Structure and Function

The Wold Lab researches a heterotrimeric protein, Replication Protein A (RPA) consisting of 70- 32- and 14-Kda subunits, required for DNA replication, recombination and repair. RPA interacts with ssDNA and facilitates the separation of partially duplexed DNA. RPA also interacts with a number of proteins involved in DNA metabolism.

I’m investigating the role of RPA phosphorylation on RPA function. Phosphorylation occurs on the N-terminus of RPA32 during the cell cycle and after DNA damage. My studies have indicated that the negatively charged N-terminus of RPA32 (phosphorylation domain), interacts with a basic cleft in the N-terminal domain of RPA70, DNA binding domain F (DBD F) which is required for optimal interactions with dsDNA. This proposed interaction reduces the ability of RPA to interact with dsDNA without a concurrent loss of ssDNA binding. In the absence of DBD F, the negatively charged phosphorylation domain causes additional defects in RPA-DNA interactions. These results suggest that in the absence of DBD F, the negatively charged phosphorylation domain interacts with a secondary site to alter RPA function. My current research seeks to identify the properties of DBD F necessary for interactions with the negatively charged phosphorylation domain. Furthermore, I am interested in determining if these intersubunit interactions are conserved in the yeast RPA homologue.

Bartos JD, Willmott LJ, Binz SK, Wold MS, Bambara RA. Catalysis of strand annealing by replication protein A derives from its strand melting properties. J Biol Chem. 2008 Aug 1;283(31):21758-68. Epub 2008 Jun 3. PubMed PMID: 18522944; PubMed Central PMCID: PMC2490778.

Binz SK, Wold MS. Regulatory functions of the N-terminal domain of the 70-kDa subunit of replication protein A (RPA). J Biol Chem. 2008 Aug 1;283(31):21559-70. Epub 2008 May 30. PubMed PMID: 18515800; PubMed Central PMCID: PMC2490791.

Haring SJ, Mason AC, Binz SK, Wold MS. Cellular functions of human RPA1. Multiple roles of domains in replication, repair, and checkpoints. J Biol Chem.2008 Jul 4;283(27):19095-111. Epub 2008 May 9. PubMed PMID: 18469000; PubMed Central PMCID: PMC2441558.

Majka J, Binz SK, Wold MS, Burgers PM. Replication protein A directs loading of the DNA damage checkpoint clamp to 5'-DNA junctions. J Biol Chem. 2006 Sep 22;281(38):27855-61. Epub 2006 Jul 24. PubMed PMID: 16864589.

Binz SK, Dickson AM, Haring SJ, Wold MS. Functional assays for replication protein A (RPA). Methods Enzymol. 2006;409:11-38. PubMed PMID: 16793393.

Binz SK, Sheehan AM, Wold MS. Replication protein A phosphorylation and the cellular response to DNA damage. DNA Repair (Amst). 2004 Aug-Sep;3(8-9):1015-24. Review. PubMed PMID: 15279788.

Wyka IM, Dhar K, Binz SK, Wold MS. Replication protein A interactions with DNA: differential binding of the core domains and analysis of the DNA interaction surface. Biochemistry. 2003 Nov 11;42(44):12909-18. PubMed PMID: 14596605.

Binz SK, Lao Y, Lowry DF, Wold MS. The phosphorylation domain of the 32-kDa subunit of replication protein A (RPA) modulates RPA-DNA interactions. Evidence for an intersubunit interaction. J Biol Chem. 2003 Sep 12;278(37):35584-91. Epub 2003 Jun 20. PubMed PMID: 12819197.