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Interaction of the p70 subunit of RPA with a DNA template directs p32 to the 3′‐end of nascent DNA
Author(s) -
Kolpashchikov D.M.,
Weisshart K.,
Nasheuer H.-P.,
Khodyreva S.N.,
Fanning E.,
Favre A.,
Lavrik O.I.
Publication year - 1999
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/s0014-5793(99)00484-6
Subject(s) - protein subunit , primer (cosmetics) , heterotrimeric g protein , dna replication , dna , replication protein a , biochemistry , biology , specificity factor , microbiology and biotechnology , seqa protein domain , chemistry , dna binding protein , eukaryotic dna replication , g protein , polymerase , gene , receptor , organic chemistry , rna dependent rna polymerase , transcription factor
Human replication protein A is a heterotrimeric protein involved in various processes of DNA metabolism. To understand the contribution of replication protein A individual subunits to DNA binding, we have expressed them separately as soluble maltose binding protein fusion proteins. Using a DNA construct that had a photoreactive group incorporated at the 3′‐end of the primer strand, we show that the p70 subunit on its own is efficiently cross‐linked to the primer at physiological concentrations. In contrast, crosslinking of the p32 subunit required two orders of magnitude higher protein concentrations. In no case was the p14 subunit labelled above background. p70 seems to be the predominant subunit to bind single‐stranded DNA and this interaction positions the p32 subunit to the 3′‐end of the primer.