ø29 DNA polymerase requires the N-terminal domain to bind terminal protein and DNA primer substrates (original) (raw)

1998, Journal of Molecular Biology

A 44 kDa C-terminal fragment of ù29 DNA polymerase has been separately expressed and puri®ed from Escherichia coli cells. As expected, the truncated protein lacked the 3 H-5 H exonuclease activity and strand-displacement capacity, previously mapped in the N-terminal domain of ù29 DNA polymerase. On the other hand, the 44 kDa C-terminal fragment retained polymerase activity when using Mn 2 as metal activator, although the catalytic ef®ciency was greatly reduced with respect to that of the complete enzyme. Moreover, and in contrast to the high processivity exhibited by ù29 DNA polymerase (>70 kb), polymerization by its C-terminal domain was completely distributive. All these polymerization defects were related to a strong impairment of DNA binding, suggesting that additional contacts present in the N-terminal domain are important for an optimal stabilization and translocation of the DNA during polymerization. Moreover, the C-terminal domain showed a very reduced capacity to initiate terminal protein (TP)-primed DNA replication, as a consequence of a weakened interaction with the TP primer, and a lack of activation by protein p6, the initiator of ù29 DNA replication. We conclude that the C-terminal portion of ù29 DNA polymerase (residues 188 to 575), although having a structural entity as the domain responsible for the synthetic activities, requires the N-terminal domain to provide important contacts for the two different substrates, DNA and TP, that prime DNA synthesis. These results support the hypothesis of a modular organization of enzymatic activities in DNA-dependent DNA polymerases, but emphasize the functional coordination required for coupling DNA synthesis and proofreading, and for the more speci®c functions (TP-priming, high processivity and strand-displacement) inherent to ù29 DNA polymerase.