Purification and characterisation of dRP‐A: a single‐stranded DNA binding protein from Drosophila melanogaster

FEBS Letters

Thömmes, Pia¹  Marton, Richard F.²  Cotterill, Sue¹ 

[1] Marie Curie Research Institute, The Chart, Oxted, Surrey, RH8 0TL, UK;Department of Biochemistry, Imperial College of Science, Technology and Medicine, South Kensington, London, SW7 2AZ, UK

关键词: Single-stranded DNA binding protein;    RP-A;    DNA replication;    Drosophila melanogaster;    ss;    single stranded;    ds;    double stranded;    SSB;    single-stranded DNA binding protein: RP-A;    replication protein A;    SV40;    simian virus 40;    T-ag;    SV40 large tumour antigen;   

DOI  :  10.1016/0014-5793(94)80488-5

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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 α 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.

【 授权许可】

Unknown