Presence of 18-Å Long Hydrogen Bond Track in the Active Site of Escherichia coli DNA Polymerase I (Klenow Fragment)

Complexes formed between DNA polymerase and genomic DNA at the replication fork are key elements of the replication machinery. We used sedimentation velocity, fluorescence anisotropy, and surface plasmon resonance to measure the binding interactions between bacteriophage T4 DNA polymerase (gp43) and various model DNA constructs. These results provide quantitative insight into how this replication polymerase performs template-directed 5 3 3 DNA synthesis and how this function is coordinated with the activities of the other proteins of the replication complex. We find that short (single-and double-stranded) DNA molecules bind a single gp43 polymerase in a nonspecific (overlap) binding mode with moderate affinity (K d ϳ150 nM) and a binding site size of ϳ10 nucleotides for single-stranded DNA and ϳ13 bp for double-stranded DNA. In contrast, gp43 binds in a site-specific (nonoverlap) mode and significantly more tightly (K d ϳ5 nM) to DNA constructs carrying a primer-template junction, with the polymerase covering ϳ5 nucleotides downstream and ϳ6 -7 bp upstream of the 3-primer terminus. The rate of this specific binding interaction is close to diffusion-controlled. The affinity of gp43 for the primer-template junction is modulated specifically by dNTP substrates, with the next "correct" dNTP strengthening the interaction and an incorrect dNTP weakening the observed binding. These results are discussed in terms of the individual steps of the polymerase-catalyzed single nucleotide addition cycle and the replication complex assembly process. We suggest that changes in the kinetics and thermodynamics of these steps by auxiliary replication proteins constitute a basic mechanism for protein coupling within the replication complex.Major cellular processes, such as DNA replication, DNA transcription, RNA translation, and protein degradation, are carried out by multiprotein assemblies (sometimes called macromolecular machines) (1), within which the constituent proteins act in a coordinated manner to perform the specific functions of the complex at issue. Furthermore, these protein machines interact within the cell and are functionally coupled to additional complexes. For example, in eukaryotes, the transcription machinery is coupled to the RNA processing complex (or spliceosome), which is itself coupled to the nuclear mRNA export system (2). Similarly, the rescue of a stalled DNA replication fork by recombination proteins illustrates the functional interactions that occur between the DNA replication, recombination, and repair assemblies (3). As a consequence, a coordination or coupling of protein activities exists, not only within a given multiprotein complex, but also between such complexes within the cell. Attaining a molecular understanding of this protein-protein coupling requires characterization of the activities and structures of the proteins as they function in isolation and also as they operate in the presence of one or more protein and/or nucleic acid interaction partners.The DNA replication machinery is a model of ...

Section: Figsupporting

confidence: 72%

Function and Assembly of the Bacteriophage T4 DNA Replication Complex

Delagoutte

Hippel

2003

“…The plasmids confirmed for mutation at the desired site were transformed into the expression strain CJ376 also obtained from C. Joyce. Expression and purification of the WT and of mutant enzymes R821A, R822A, L823A, Y824A, and R822A/Y824A was carried out by methods described previously (9,10,17).…”

Section: Methodsmentioning

confidence: 99%

Identification of a New Motif Required for the 3′–5′ Exonuclease Activity of Escherichia coli DNA Polymerase I (Klenow Fragment)

Kukreti

Singh

Ketkar

et al. 2008

Self Cite

The Klenow fragment of Escherichia coli DNA polymerase I houses catalytic centers for both polymerase and 3 -5 exonuclease activities that are separated by about 35 Å . Upon the incorporation of a mismatched nucleotide, the primer terminus is transferred from the polymerase site to an exonuclease site designed for excision of the mismatched nucleotides. The structural comparison of the binary complexes of DNA polymerases in the polymerase and the exonuclease modes, together with a molecular modeling of the template strand overhang in Klenow fragment, indicated its binding in the region spanning residues 821-824. Since these residues are conserved in the "A" family DNA polymerases, we have designated this region as the RRRY motif. The alanine substitution of individual amino acid residues of this motif did not change the polymerase activity; however, the 3 -5 exonuclease activity was reduced 2-29-fold, depending upon the site of mutation. The R821A and R822A/ Y824A mutant enzymes showed maximum cleavage defect with single-stranded DNA, mainly due to a large decrease in the ssDNA binding affinity of these enzymes. Mismatch removal by these enzymes was only moderately affected. However, data from the exonuclease-polymerase balance assays with mismatched template-primer suggest that the mutant enzymes are defective in switching mismatched primer from the polymerase to the exonuclease site. Thus, the RRRY motif provides a binding track for substrate ssDNA and for nonsubstrate single-stranded template overhang, in a polarity-dependent manner. This binding then facilitates cleavage of the substrate at the exonuclease site.The faithful synthesis of DNA by E. coli DNA polymerase I (pol I) 2 is accomplished by a combination of two processes: (a) correctly incorporating matched nucleotides and (b) removing incorrectly incorporated mismatched nucleotides with its 3Ј-5Ј exonuclease activity (1). The incorporation of a mismatched nucleotide on a growing primer strand stalls further DNA synthesis and decreases the affinity of the polymerase for the template-primer. This results in shuttling of the primer to the exonuclease active site (exo site). Thus, error correction by DNA polymerases that contain both the polymerase and the exonuclease activities involves at least the following four steps: (a) sensing of the misincorporated nucleotide at the polymerase site (pol site), (b) transfer of the mismatched primer from the pol to the exo site, (c) excision of the incorrect nucleotide, and (d) transfer of the corrected primer terminus to the pol site for further nucleotide incorporation. The mechanistic details of these steps are not fully understood. However, the availability of DNA polymerase structures, with DNA bound in either the polymerase or the exonuclease mode, provides some clues to the possible structural alterations of the polymerase protein during DNA synthesis.The active site of DNA polymerases catalyzing the polymerase reaction has a configuration that allows non-sequence-specific contacts with both DNA and the incom...

“…Plasmid DNA from mutant clones, after confirming desired mutation by DNA sequencing, was used to transfect an expression strain, E. coli CJ376 (19). Isolation and purification of S769A, F771A, R841A, and S769A/F771A (double mutant) enzymes were carried out as described before (20). The WT pol I and the double mutant S769A/ F771A were expressed and purified using the procedure described by Joyce and Derbyshire (21) reaction, the rates of single nucleotide incorporation were determined under single turnover conditions by rapid mixing of enzyme 32 P-radiolabeled template primer blocker (32/ 14/14-mer; see Scheme 1) complex with Mg⅐dNTP, followed by quenching of the reaction by EDTA.…”

Section: Methodsmentioning

confidence: 99%

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

Singh

Srivastava

Patel³

et al. 2007

Self Cite

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...