SUBUNITS OF HUMAN REPLICATION PROTEIN-A ARE CROSS-LINKED BY PHOTOREACTIVE PRIMERS SYNTHESIZED BY DNA-POLYMERASES

Human replication protein A (huRPA) is a multisubunit protein which is involved in DNA replication, repair and recombination processes, It e xists as a stable heterotrimer consisting of p70, p32 and p14 subunits , To understand the contribution of huRPA subunits to DNA binding we a pplied the photoaffinity labeling technique, The photoreactive oligonu cleotide was synthesized in situ by DNA polymerases, ans-3-aminopropen yl-1]deoxyuridine-5'-triphosphate (NABdUTP) was used as substrate for elongation of a radiolabeled primer template either by human DNA polym erase alpha primase (pol alpha), human DNA polymerase beta (pol beta) or Klenow fragment of Escherichia coli DNA polymerase I (KF). The poly merase was incubated with NABdUTP and radiolabeled primer-template in the presence or absence of huRPA, The reaction mixtures were then irra diated with monochromatic UV light (315 nm) and the crosslinked produc ts were separated by SDS-PAGE. The results clearly demonstrate crossli nking of the huRPA p70 and p32 subunits with DNA, The p70 subunit appe ars to bind to the single-stranded part of the DNA duplex, the p32 sub unit locates near the 3'-end of the primer, while the p14 subunit loca tes relatively far from the 3'-end of the primer. This approach opens new possibilities for analysis of huRPA loading on DNA in the course o f DNA replication and DNA repair.