Analysis of phi 29 DNA polymerase by partial proteolysis: Binding of terminal protein in the double-stranded DNA channel

phi 29 DNA polymerase, which belongs to the family of the eukaryotic type D NA polymerases, is able to use two kinds of primers to initiate DNA replica tion: DNA and terminal protein (TP). By partial proteolysis we have studied the regions of phi 29 DNA polymerase involved in primer binding. With prot einase K, no change in the proteolytic pattern was observed upon DNA bindin g, suggesting that it does not induce a global conformational change in phi 29 DNA polymerase. Conversely, two of the three main cleavage sites obtain ed by partial digestion of free phi 29 DNA polymerase with endoproteinase L ysC were protected upon DNA binding, indicating that the IDNA could be occl uding these cleavage sites to the protease either directly by itself and/or indirectly by induction of local conformational changes affecting their ex posure. Partial proteolysis with endoproteinase LysC of phi 29 DNA polymera se/TP heterodimer resulted in a protection and digestion pattern similar to that obtained with DNA, suggesting that both primers, DNA and TP, fit in t he same double-stranded DNA-binding channel and protect the same regions of phi 29 DNA polymerase. (C) 2000 Academic Press.