Biomedical subjects

Sara K Binz

Publications and source records attributed to Sara K Binz.

5 recordsLinked to original sources

PubMed2006

Replication protein A directs loading of the DNA damage checkpoint clamp to 5'-DNA junctions.

The heterotrimeric checkpoint clamp comprises the Saccharomyces cerevisiae Rad17, Mec3, and Ddc1 subunits (Rad17/3/1, the 9-1-1 complex in humans). This DNA damage response factor is loaded onto DNA by the Rad24-RFC (replication factor C-like complex with Rad24) clamp loader and ATP. Although Rad24-RFC alone does not bind to naked partial double-stranded DNA, coating of the single strand with single-stranded DNA-binding protein RPA (replication protein A) causes binding of Rad24-RFC via interactions with RPA. However, RPA-mediated binding is abrogated when the DNA is coated with RPA containing a rpa1-K45E (rfa1-t11) mutation. These properties allowed us to determine the role of RPA in clamp-loading specificity. The Rad17/3/1 clamp is loaded with comparable efficiency onto naked primer/template DNA with either a 3'-junction or a 5'-junction. Remarkably, when the DNA was coated with RPA, loading of Rad17/3/1 at 3'-junctions was completely inhibited, thereby providing specificity to loading at 5'-junctions. However, Rad17/3/1 loaded at 5'-junctions can slide across double-stranded DNA to nearby 3'-junctions and thereby affect the activity of proteins that act at 3'-termini. These studies show a unique specificity of the checkpoint loader for 5'-junctions of RPA-coated DNA. The implications of this specificity for checkpoint function are discussed.

Adenosine Triphosphatases↗

PubMed2006

Functional assays for replication protein A (RPA).

Replication protein A (RPA) is a heterotrimeric, single-stranded DNA-binding protein. RPA is conserved in all eukaryotes and is essential for DNA replication, DNA repair, and recombination. RPA also plays a role in coordinating DNA metabolism and the cellular response to DNA damage. Assays have been established for many of these reactions. This chapter provides an overview of the methods used for analyzing RPA-DNA interactions, RPA-protein interactions, and functional activities of RPA. Methods are also discussed for visualizing RPA in the cell and analyzing the effects of RPA function on cell cycle progression in mammalian cells.

Chromatography, Affinity↗

PubMed2003

Replication protein A interactions with DNA: differential binding of the core domains and analysis of the DNA interaction surface.

Human replication protein A (RPA) is a heterotrimeric (70, 32, and 14 kDa subunits), eukaryotic single-stranded DNA (ssDNA) binding protein required for DNA recombination, repair, and replication. The three subunits of human RPA are composed of six conserved DNA binding domains (DBDs). Deletion and mutational studies have identified a high-affinity DNA binding core in the central region of the 70 kDa subunit, composed of DBDs A and B. To define the roles of each DBD in DNA binding, monomeric and tandem DBD A and B domain chimeras were created and characterized. Individually, DBDs A and B have a very low intrinsic affinity for ssDNA. In contrast, tandem DBDs (AA, AB, BA, and BB) bind ssDNA with moderate to high affinity. The AA chimera had a much higher affinity for ssDNA than did the other tandem DBDs, demonstrating that DBD A has a higher intrinsic affinity for ssDNA than DBD B. The RPA-DNA interface is similar in both DBD A and DBD B. Mutational analysis was carried out to probe the relative contributions of the two domains to DNA binding. Mutation of polar residues in either core DBD resulted in a significant decrease in the affinity of the RPA complex for ssDNA. RPA complexes with pairs of mutated polar residues had lower affinities than those with single mutations. The decrease in affinity observed when polar mutations were combined suggests that multiple polar interactions contribute to the affinity of the RPA core for DNA. These results indicate that RPA-ssDNA interactions are the result of binding of multiple nonequivalent domains. Our data are consistent with a sequential binding model for RPA, in which DBD A is responsible for positioning and initial binding of the RPA complex while DBD A together with DBD B direct stable, high-affinity binding to ssDNA.

Conserved Sequence↗

PubMed2003

The phosphorylation domain of the 32-kDa subunit of replication protein A (RPA) modulates RPA-DNA interactions. Evidence for an intersubunit interaction.

Replication protein A (RPA) is a heterotrimeric (subunits of 70, 32, and 14 kDa) single-stranded DNA-binding protein that is required for DNA replication, recombination, and repair. The 40-residue N-terminal domain of the 32-kDa subunit of RPA (RPA32) becomes phosphorylated during S-phase and after DNA damage. Recently it has been shown that phosphorylation or the addition of negative charges to this N-terminal phosphorylation domain modulates RPA-protein interactions and increases cell sensitivity to DNA damage. We found that addition of multiple negative charges to the N-terminal phosphorylation domain also caused a significant decrease in the ability of a mutant form of RPA to destabilize double-stranded (ds) DNA. Kinetic studies suggested that the addition of negative charges to the N-terminal phosphorylation domain caused defects in both complex formation (nucleation) and subsequent destabilization of dsDNA by RPA. We conclude that the N-terminal phosphorylation domain modulates RPA interactions with dsDNA. Similar changes in DNA interactions were observed with a mutant form of RPA in which the N-terminal domain of the 70-kDa subunit was deleted. This suggested a functional link between the N-terminal domains of the 70- and 32-kDa subunits of RPA. NMR experiments provided evidence for a direct interaction between the N-terminal domain of the 70-kDa subunit and the negatively charged N-terminal phosphorylation domain of RPA32. These findings suggest that phosphorylation causes a conformational change in the RPA complex that regulates RPA function.

Animals↗

PubMedDate not supplied

Replication protein A phosphorylation and the cellular response to DNA damage.

Defects in cellular DNA metabolism have a direct role in many human disease processes. Impaired responses to DNA damage and basal DNA repair have been implicated as causal factors in diseases with DNA instability like cancer, Fragile X and Huntington's. Replication protein A (RPA) is essential for multiple processes in DNA metabolism including DNA replication, recombination and DNA repair pathways (including nucleotide excision, base excision and double-strand break repair). RPA is a single-stranded DNA-binding protein composed of subunits of 70-, 32- and 14-kDa. RPA binds ssDNA with high affinity and interacts specifically with multiple proteins. Cellular DNA damage causes the N-terminus of the 32-kDa subunit of human RPA to become hyper-phosphorylated. Current data indicates that hyper-phosphorylation causes a change in RPA conformation that down-regulates activity in DNA replication but does not affect DNA repair processes. This suggests that the role of RPA phosphorylation in the cellular response to DNA damage is to help regulate DNA metabolism and promote DNA repair.

Animals↗