Purification and characterisation of dRP-A: a single-stranded DNA binding protein from Drosophila melanogaster.
Replication protein A (RP-A) is an essential single-stranded DNA binding protein (SSB) involved in the initiation and elongation phases of eukaryotic DNA replication. It has the ability to bind single-stranded DNA extremely tightly and possesses a characteristic hetero-trimeric structure. Here we present a method for the purification of RP-A from Drosophila melanogaster embryos. Drosophila RP-A (dRP-A) has subunits of about 66, 31 and 8 kDa, in line with analogues from other species. It binds single-stranded DNA very tightly via the large subunit. The complete protein has at least a 10- to 20-fold preference for single-stranded DNA over double-stranded DNA and it appears that binding is only weakly co-operative. Band shift experiments suggest that it has an approximate site covering the size of 16 nucleotides or less, however, it shows a greater affinity for long oligonucleotides than for short ones. We also demonstrate that dRP-A can stimulate the activity of its homologous DNA polymerase alpha in excess of 20 fold. Analysis of the protein's abundance during embryo development indicates that it varies in a manner akin to other replication proteins.
Marton
R F
RF
Department of Biochemistry, Imperial College of Science, Technology and Medicine, South Kensington, London, UK.
Thömmes
P
P
Cotterill
S
S
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D004257:DNA Polymerase II / Q000378:metabolism; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000254:growth & development / Q000378:metabolism*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D051716:Replication Protein A
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D009838:Oligodeoxyribonucleotides; D051716:Replication Protein A; C497166:RpA-70 protein, Drosophila; D004257:DNA Polymerase II