Premium
Purification and characterisation of dRP‐A: a single‐stranded DNA binding protein from Drosophila melanogaster
Author(s) -
Marton Richard F.,
Thömmes Pia,
Cotterill Sue
Publication year - 1994
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(94)80488-5
Subject(s) - dna replication , biology , dna , replication protein a , dna clamp , dna polymerase , oligonucleotide , hmg box , microbiology and biotechnology , drosophila melanogaster , dna binding protein , biochemistry , transcription factor , gene , rna , reverse transcriptase
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.