Mechanism of the idling-turnover reaction of the large (Klenow) fragment of Escherichia coli DNA polymerase I.

Abstract

The mechanism of the idling-turnover reaction catalyzed by the large (Kienow) fragment ofEscherichia coli DNA polymerase I has been investigated. The reaction cycle involved is one of excision/incorporation, in which the 3' deoxynucleotide residue of the primer DNA strand is partitioned into its 5'-monoand 5'-triphosphate derivatives, respectively. Meanic studies suggest the 5'-monophosphate product is formed in the first step by simple 3' --5' exonucleolytic cleavage. Rapid polymerization follows with the concomitant release of inorganic pyrophosphate. In the second step, the 5'-triphosphate product is generated by a pyrophosphorolysis reaction, which, despite the low concentration of pyrophosphate that has accumulated, occurs at a rate that is comparable with that of the parallel 3' -*5' hydrolysis reaction.The multifunctional DNA polymerase I of Escherichia coli has served as the widely studied model for describing, at the molecular level, certain enzymatic processes involved in the replication of DNA (1). In addition to its polymerase activity, the enzyme also catalyzes DNA degradation by distinct 5' --3' and 3' -* 5' exonuclease activities, as well as by net pyrophosphorolysis. Extensive kinetic (2, 3) and stereochemical (4, 5) studies of the various activities have elucidated the important underlying features of the phosphodiester bondforming and bond-breaking reactions. In addition, the availability of a 3-A resolution x-ray structure of the large proteolytic (Klenow) fragment of DNA polymerase I (6) has generated considerable interest in the area of structure-function assignment (7).The conversion ofa fraction ofthe available deoxynucleoside 5'-triphosphate (dNTP) pool into a corresponding monophosphate pool has provided evidence in support of alternating polymerase and 3' -> 5' exonuclease expression during the course of DNA synthesis (8,9). In view of the convincing evidence implicating the 3' -*5' exonuclease activity in ensuring fidelity (10-13), the extent of the dNTP -* dNMP conversion may thus reflect the degree of proofreading accompanying replication by DNA polymerases possessing such an exonuclease activity (14). In the absence of the following complementary dNTP that is required for normal polymerization, the turnover process is exaggerated since the enzyme is constrained to "idle" at the pnimer terminus until depletion of the available dNTP pool is complete (15). Comparative kinetic studies ofthe turnover reaction with the enzyme held in such an idling mode have allowed the evaluation of base misinsertion frequencies, thus demonstrating an important application ofthe idling-turnover process (16).In this paper, we report our results on the idling-turnover reaction catalyzed by the Klenow fragment (KF), which bear on the general problem of describing a unified mechanism of the interrelated activities of this enzyme. MATERIALS AND METHODSThe Klenow fragment was purified from E. coli CJ155 according to Joyce and Grindley (17 (20): The sample was loaded onto a 2.5-ml syringe column of Sep...

Mechanism of DNA polymerase I: exonuclease/polymerase activity switch and DNA sequence dependence of pyrophosphorolysis and misincorporation reactions.

“…Previous studies demonstrated that the accumulation of PP1 during idling-turnover results in a partitioning of the excision pathway into hydrolysis and pyrophosphorolysis of the 3'-tDirect comparison of the level of incorporation observed in the switch experiment with that observed in the first control assumes that the concentration of E poly(dA)*(dT) 16 EvidenceforMisincorporation/ExcisionDuringIdling-Turnover. The terminal residue of 3'-end-labeled EcoRI-or BamHI-digested pBR322 is not protected from excision during idling-turnover, suggesting that the reaction involves an excision/incorporation cycle rather than one of misincorporation/excision (7). In order to directly investigate the possible involvement of the latter pathway, the products of the reactions of BamHI-and EcoRI-digested pBR322 with [a-32P]dGTP and [a-32P]dATP, respectively, were subjected to a gel electrophoretic analysis.…”

“…The first step in excision/incorporation idling-turnover involves a 3' -) 5' exonucleolytic removal of the terminal residue, followed by a rapid incorporation step to reinsert the correct nucleotide from the available dNTP pool (7). The conversion of the enzyme from an exonuclease to a polymerase mode, when presented with a complementary dNTP pool, suppresses further exonuclease activity (10 poly(dA).…”

“…DE-81 and GF/C filters (2.5 cm) were from Whatman. TLC was performed as described (7). Scintiverse II scintillation fluid was from Fisher.…”

“…Restriction digestion reactions were carried out by standard procedures (9 3'-end-labeling. BamHI-or EcoRI-digested pBR322 was 3'-end-labeled as described (7).…”

“…Extensive kinetic (2,3), stereochemical (4,5), and structural (6) studies of Pol I and its large proteolytic (Klenow) fragment have provided detailed mechanistic insight into the enzymology of DNA replication. Recent studies aimed at attempting to describe a unified mechanism of the interrelated activities of the Klenow fragment (KF) focused upon the nature of the dNTP -* dNMP turnover reaction that is observed when the enzyme is constrained to idle at the 3' terminus of the DNA template-primer (7). The idling-turnover reaction was found to involve an alternating excision/ incorporation cycle in which the 3'-deoxynucleotide residue of the primer strand is partitioned via exonucleolytic and pyrophosphorolytic degradation into its 5'-mono-and 5'-triphosphate derivatives, respectively.…”