End Of Primer Strand Research Articles

Time Dependence of Partitoning between Polymerization and Editing Sites by Klenow Polymerase

Klenow polymerase is the large fragment of DNA polymerase I from E. coli. Klenow possesses 5'-3' polymerase and intrinsic 3'-5' exonuclease activities with two distinct active sites that are located ∼30 Ǻ apart. During DNA replication, proofreading activity enhances replication fidelity by excising misincorporated nucleotides from the 3' end of primer strand. The first step of the proofreading process is sending the 3'-primer terminus to the proofreading site, which requires separation of the primer terminus from the template strand. Here we examined the binding of Klenow to matched and mismatched primed-template DNA (pt-DNA) by monitoring the steady state fluorescence intensity change of a single 2-aminopurine base site-specifically substituted in the template strand within the duplex part of pt-DNA. The changes in fluorescence intensity allow us to follow shuttling of the primer terminus between the polymerization and proofreading sites.We have found that the rate of partitioning of the primer between the two active sites depends on: 1) the number of mismatched bases at the primer-template junction, 2) the presence or absence of divalent ions, and 3) the type of divalent ions. Magnesium and calcium ions have opposite effects on the direction of the shift between the pol and exo sites. Substitution of the normal phosphodiester linkage between the last two bases of the primer strand with a non-hydrolysable phosphorothioate linkage also has significant effects on the partitioning between sites.

Time Dependence of Switching Between Polymerization and Editing Mode DNA Binding by Klenow Polymerase

Klenow polymerase is the large fragment of DNA polymerase I from E. coli. Klenow possesses 5’-3’ polymerase and intrinsic 3’-5’ exonuclease activities with two distinct active sites that are located ∼30 angstroms apart. During DNA replication, proofreading activity enhances replication fidelity by excising misincorporated nucleotides from the 3’ end of primer strand. The first step of the proofreading process is sending the 3’-primer terminus to the proofreading site, which requires separation of the primer terminus from the template strand. Here we examined the binding of Klenow to matched and mismatched primed-template DNA (pt-DNA) by monitoring the steady state fluorescence intensity change of a single 2-aminopurine base site-specifically substituted in the template strand within the duplex part of pt-DNA. The changes in fluorescence intensity allow us to follow shuttling of the primer terminus between the polymerization and proofreading sites. We have found that partitioning of the primer between the two active sites depends on: 1) the number of mismatched bases at the primer-template junction, 2) the presence or absence of divalent ions, 3) the type of divalent ions, and 4) the time in the presence of the divalent ions. A significant time dependence of the partitioning between the two sites was observed with the matched and single mismatched pt-DNA in the presence of Mg2+. The slow kinetic dependence of the primer partitioning between the polymerization and proofreading sites in Klenow polymerase helps explain previous conflicting reports of the equilibrium partitioning between sites.