Interaction of Human Replication Protein A with DNA Duplexes Containing Gaps of Varying Size

Replication protein A (RPA) is a heterotrimeric protein that has high affinity for single-stranded (ss) DNA and is involved in DNA replication, repair, and recombination in eukaryotic cells. Photoaffinity modification was employed in studying the interaction of human RPA with DNA duplexes containing various gaps, which are similar to structures arising during DNA replication and repair. A photoreactive dUMP derivative was added to the 3" end of a gap-flanking oligonucleotide with DNA polymerase , and an oligonucleotide containing a 5"-photoreactive group was chemically synthesized. The 5" end predominantly interacted with the large RPA subunit (p70) regardless of the gap size, whereas interactions of the 3" end with the RPA subunits depended both on the gap size and on the RPA concentration. Subunit p32 was mostly labeled in the case of a larger gap and a lower RPA concentration. The results confirmed the model of polar RPA–DNA interaction, which has been advanced earlier.