Interaction of the p70 subunit of RPA with a DNA template directs p32 to the 3'-end of nascent DNA.
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.
Kolpashchikov
D M
DM
Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences, Novosibirsk.
Weisshart
K
K
Nasheuer
H P
HP
Khodyreva
S N
SN
Fanning
E
E
Favre
A
A
Lavrik
O I
OI
D001386:Azides / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics; D004261:DNA Replication / Q000235:genetics; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D058619:Maltose-Binding Proteins; D015394:Molecular Structure; D019920:Photoaffinity Labels; D011487:Protein Conformation; D016601:RNA-Binding Proteins / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D051716:Replication Protein A; D013698:Templates, Genetic; D014544:Uridine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism
C110508:5-(N-(2-nitro-5-azidobenzoyl)-3-aminopropen-1-yl)deoxyuridine-5'-triphosphate; D001386:Azides; D002352:Carrier Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D058619:Maltose-Binding Proteins; D019920:Photoaffinity Labels; D016601:RNA-Binding Proteins; C497167:RPA1 protein, human; D011993:Recombinant Fusion Proteins; D051716:Replication Protein A; D014544:Uridine Triphosphate