PURIFICATION AND CHARACTERIZATION OF DRP-A - A SINGLE-STRANDED-DNA BINDING-PROTEIN FROM DROSOPHILA-MELANOGASTER

Replication protein A (RP-A) is an essential single-stranded DNA bindi ng protein (SSB) involved in the initiation and elongation phases of e ukaryotic DNA replication. It has the ability to bind single-stranded DNA extremely tightly and possesses a characteristic hetero-trimeric s tructure. Here we present a method for the purification of RP-A from D rosophila melanogaster embryos. Drosophila RP-A (dRP-A) has subunits o f about 66, 31 and 8 kDa, in line with analogues from other species. I t binds single-stranded DNA very lightly via the large subunit. The co mplete protein has at least a 10- to 20-fold preference for single-str anded DNA over double-stranded DNA and it appears that binding is only weakly co-operative. Band shift experiments suggest that it has an ap proximate site covering the size of 16 nucleotides or less, however, i t shows a greater affinity for long oligonucleotides than for short on es. We also demonstrate that dRP-A can stimulate the activity of its h omologous DNA polymerase alpha in excess of 20 fold. Analysis of the p rotein's abundance during embryo development indicates that it varies in a manner akin to other replication proteins.