Polarity of human replication protein A binding to DNA

Replication protein A (RPA), the nuclear single-stranded DNA binding protei n is involved in DNA replication, nucleotide excision repair (NER) and homo logous recombination, It is a stable heterotrimer consisting of subunits wi th molecular masses of 70, 32 and 14 kDa (p70, p32 and p14, respectively). Gapped DNA structures are common intermediates during DNA replication and N ER, To analyze the interaction of RPA and its subunits with gapped DNA we d esigned structures containing 9 and 30 nucleotide gaps with a photoreactive arylazido group at the 3'-end of the upstream oligonucleotide or at the 5' -end of the downstream oligonucleotide. UV crosslinking and subsequent anal ysis showed that the p70 subunit mainly interacts with the 5'-end of DNA ir respective of DNA structure, while the subunit orientation towards the 3'-e nd of DNA in the gap structures strongly depends on the gap size. The resul ts are compared with the data obtained previously with the primer-template systems containing 5'- or 3'-protruding DNA strands. Our results suggest a model of polar RPA binding to the gapped DNA.