Participation of the Fingers Subdomain of Escherichia coli DNA Polymerase I in the Strand Displacement Synthesis of DNA

Abstract

The replication of the genome requires the removal of RNA primers from the Okazaki fragments and their replacement by DNA. In prokaryotes, this process is completed by DNA polymerase I by means of strand displacement DNA synthesis and 5-nuclease activity. Here, we demonstrate that the strand displacement DNA synthesis is facilitated by the collective participation of Ser 769 , Phe 771 , and Arg 841 present in the fingers subdomain of DNA polymerase I. The steady and presteady state kinetic analysis of the properties of appropriate mutant enzymes suggest that: (a) Ser 769 and Phe 771 together are involved in the strand separation via the formation of a flap structure, and (b) Arg 841 interacts with the template strand to achieve the optimal strand separation and DNA synthesis. The amino acid residues Ser 769 and Phe 771 are constituents of the O1-helix, which together with O and O2 helices form a 3-helix bundle structure. We note that this 3-helix bundle motif also exists in prokaryotic RNA polymerase. Thus in both DNA and RNA polymerases, this motif may have been adopted to achieve the strand separation function.Strand displacement synthesis is an essential process in the removal and replacement of RNA primer moieties of Okazaki fragments. In prokaryotes, DNA polymerase I (pol I) 3 carries out this function by its 5Ј-nuclease and 5Ј-3Ј polymerase activities. Whereas early studies indicate a coordination of 5Ј-nuclease with the polymerase activity (1), the precise mechanism underlying this process is not clear. It appears that the participation of 5Ј-nuclease activity is not necessary for the strand displacement synthesis because the Klenow fragment of Escherichia coli DNA polymerase I has been known to catalyze strand displacement DNA synthesis (2). Thus, the strand separation activity resides in the polymerase domain of pol I.Despite the availability of numerous DNA-bound structures of polymerases (3-8), no significant information pertaining to strand displacement could be discerned because none of these crystal structures contained sufficiently long single-stranded template overhang or a downstream doublestranded DNA. One exception to this is the DNA-bound crystal structure of mammalian DNA polymerase ␤, where the enzyme-DNA complex contains gapped DNA (9). However, this enzyme has no strand displacement activity. In the DNA-bound crystal structures of pol I family DNA polymerases, the immediate unpaired template nucleotide assumes a flipped conformation (6) such that it cannot pair with the incoming dNTP substrate. The base moiety of the template (n ϩ 1) 4 nucleotide is positioned out of the DNA helical axis by more than 90°(4, 10, 11). In the enzyme-DNA-dNTP bound ternary complex, this nucleotide rearranges its conformation and pairs with the incoming dNTP substrate (6). In the crystal structures of polymerases, because of the short length of the single-stranded template overhang, only few interactions of downstream DNA with the enzyme protein could be discerned (3, 4, 6 -8, 11-13 4 The numbering scheme for...

Single molecule measurement of the “speed limit” of DNA polymerase

“…Although strand displacement synthesis and single stranded primer extension synthesis are expected to have substantial differences in their energetics, we compared our results to experiments that measured single stranded primer extension synthesis and found surprising agreement. Our observed rate of 14 nt sec Ϫ1 is consistent with bulk primer extension experiments (17,20). The rates measured here are 2-to 3-fold faster than a Pol I(KF) single molecule measurement (13), which was a low tension force spectroscopy study on a long template that did not have sufficient time resolution to separate pauses from bursts.…”

“…This is likely why our 29 rate is slightly less than the reported 29 bulk rates of 38 nt sec Ϫ1 (29). A recent bulk study of Pol I(KF) reported a strand displacement synthesis rate over an 18-bp template of 1.2 nt sec Ϫ1 (20), which is an order of magnitude slower than our time-averaged burst synthesis rate. However, the template used in the bulk study contained three Pyr-G-C pause motifs.…”

“…This bulk processive rate is twice as fast as that found by single molecule primer extension synthesis rate measurements (7 nt sec Ϫ1 ) (13). Primer extension synthesis is generally thought to be faster than strand-displacement synthesis, and this idea is supported by a bulk rapid quenching study which found that the strand displacement synthesis rate of Pol I(KF) was even slower at 1.2 nt sec Ϫ1 (20).…”

“…This assay allowed us to measure polymerase activity in real time at saturating nucleotide concentrations with near single base resolution, thereby enabling separation of burst synthesis rates from pausing effects. Our results show that the only literature strand displacement synthesis rate for Pol I(KF) (20) is not the true burst synthesis rate because it likely includes sequence specific pausing effects due to the template used in the experiments. These pausing events are not unique to Pol I(KF) and were also observed with 29 DNA polymerase.…”

“…Although the kinetics of the Klenow fragment of Escherichia coli DNA Polymerase I (Pol I(KF)) have been studied using a variety of approaches (13,(15)(16)(17)(18)(19)(20), there are conflicting reports of the synthesis rate and it has not yet been possible to dissect the interplay between pausing and burst synthesis. Rapid quenching and stopped-flow bulk techniques have determined that during primer extension synthesis, the first nucleotide is incorporated by Pol I(KF) with a rate ranging from 40-87 nt sec Ϫ1 (16)(17)19) while subsequent nucleotides are incorporated at a slower rate of 13-15 nt sec Ϫ1 (17,20). This bulk processive rate is twice as fast as that found by single molecule primer extension synthesis rate measurements (7 nt sec Ϫ1 ) (13).…”