The catalytic mechanism of DNA polymerases involves multiple steps that precede and follow the transfer of a nucleotide to the 3 -hydroxyl of the growing DNA chain. Here we report a singlemolecule approach to monitor the movement of E. coli DNA polymerase I (Klenow fragment) on a DNA template during DNA synthesis with single base-pair resolution. As each nucleotide is incorporated, the single-molecule Fö rster resonance energy transfer intensity drops in discrete steps to values consistent with single-nucleotide incorporations. Purines and pyrimidines are incorporated with comparable rates. A mismatched primer/template junction exhibits dynamics consistent with the primer moving into the exonuclease domain, which was used to determine the fraction of primer-termini bound to the exonuclease and polymerase sites. Most interestingly, we observe a structural change after the incorporation of a correctly paired nucleotide, consistent with transient movement of the polymerase past the preinsertion site or a conformational change in the polymerase. This may represent a previously unobserved step in the mechanism of DNA synthesis that could be part of the proofreading process.Klenow Fragment ͉ polymerase and exonuclease site ͉ single molecule fluorescence ͉ single nucleotide resolution ͉ structural dynamics T he catalytic mechanism of Escherichia coli DNA polymerase I has been rigorously studied for more than 40 years (1, 2). The E. coli DNA polymerase I (Klenow fragment [KF]), an active truncated form of polymerase I, is composed of two domains: a polymerase domain that incorporates nucleotides, and a 3Ј-5Ј exonuclease domain that excises misincorporated nucleotides. The polymerase domain consists of three subdomains: the fingers, the palm, and the thumb. The fingers subdomain is primarily involved in interactions with the singlestranded region of the DNA template and the incoming nucleotide; the palm forms the active site of the polymerase upon interaction with the incoming dNTP; and the thumb is responsible for binding double-stranded DNA. The exonuclease domain, located Ϸ30 Å from the polymerase domain, binds to the 3Ј-terminus of the primer when a mismatched base is incorporated (3).Like other high-fidelity polymerases, KF achieves its extraordinary accuracy through a series of steps that discriminate between a correct and incorrect dNTP. A minimal reaction pathway for KF has been proposed with much of the data obtained from chemical quench experiments (4, 5) (Fig. 1A). The rate-limiting step (k 3 ) that precedes the phosphoryl-transfer step (k 4 ) had been tentatively attributed to a conformational change of the fingers domain (6). A comparison of the crystal structures of the binary polymerase-DNA complexes with those of the ternary polymerase-DNA-dNTP complexes reveals a substantial movement upon nucleotide binding, supporting the model that fingers closing was the rate-limiting step (7). However, recent results have shown this step is much too fast to be rate limiting, suggesting additional noncovalent steps th...

The binding of plasminogen activator inhibitor-1 (PAI-1) to serine proteinases, such as tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), is mediated by the exosite interactions between the surface-exposed variable region-1, or 37-loop, of the proteinase and the distal reactive center loop (RCL) of PAI-1. Although the contribution of such interactions to the inhibitory activity of PAI-1 has been established, the specific mechanistic steps affected by interactions at the distal RCL remain unknown. We have used protein engineering, stopped-flow fluorimetry, and rapid acid quenching techniques to elucidate the role of exosite interactions in the neutralization of tPA, uPA, and ␤-trypsin by PAI-1. Alanine substitutions at the distal P4 (Glu-350) and P5 (Glu-351) residues of PAI-1 reduced the rates of Michaelis complex formation (k a ) and overall inhibition (k app ) with tPA by 13.4-and 4.7-fold, respectively, whereas the rate of loop insertion or final acyl-enzyme formation (k lim ) increased by 3.3-fold. The effects of double mutations on k a , k lim , and k app were small with uPA and nonexistent with ␤-trypsin. We provide the first kinetic evidence that the removal of exosite interactions significantly alters the formation of the noncovalent Michaelis complex, facilitating the release of the primed side of the distal loop from the active-site pocket of tPA and the subsequent insertion of the cleaved reactive center loop into ␤-sheet A. Moreover, mutational analysis indicates that the P5 residue contributes more to the mechanism of tPA inhibition, notably by promoting the formation of a final Michaelis complex.Plasminogen activator inhibitor-1 (PAI-1) 1 functions as the primary regulator of the fibrinolytic system by inhibiting the conversion of plasminogen into plasmin via its action on tissuetype plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) (1). This inhibitor also plays a vital role in other physiological processes, including tumor invasion and tissue remodeling (2). PAI-1 belongs to the serpin superfamily, which shares several unique structural features, including a five-stranded ␤-sheet motif and a flexible reactive center loop (RCL). The conformational changes associated with these structures have been linked to the inhibitory function of PAI-1 (3-9). Notably, the mechanism of inhibition depends on the structural changes accompanying the S (stressed) to R (relaxed) transition that results in complete insertion of the Nterminal (proximal) part of the RCL into ␤-sheet A as an additional ␤-strand, s4A. The proteinase, tethered by a covalent bond with the P1 residue of the serpin, is displaced from the initial docking site to the opposite end of the serpin molecule, separating the P1Ј and P1 residues by ϳ70 Å (10 -12). This mechanism efficiently distorts and inactivates the catalytic triad of the proteinase, stabilizing the serpin-proteinase complex at the acyl-enzyme intermediate stage.Unique to the PAI-1 inhibitory mechanism are the exosite i...

We have previously observed that stepwise replacement of amino acid residues in the Nascent base-pair Binding Pocket (NBP) of RB69 DNA polymerase (RB69pol) with Ala or Gly expanded the space in this pocket resulting in a progressive increase in misincorporation. However, results in vivo with the similar RB69 pol NBP mutants, showed that mutation rates, as determined by the T4 phage rI forward and rII reversion assays, were significantly lower for the RB69pol S565G/ Y567A double mutant than for the Y567A single mutant, the opposite of what we would have predicted. To investigate the reasons for this unexpected result we have determined the pre-steadystate kinetic parameters and crystal structures of the relevant ternary complexes. We found that the S565G/Y567A mutant generally had greater base selectivity than the Y567A mutant and that the kinetic parameters for dNMP insertion, excision of the 3′ terminal nucleotide residue as well as primer extension beyond a mispair, differed not only between these two mutants but also between the two highly mutable sequences in the T4 rI complementary strand. Comparison of the crystal structures of these two mutants with correct and incorrect incoming dNTPs provides insight into the unexpected increase in fidelity of the S565G/Y567A double mutant. Taken together, the kinetic and structural results provide a basis for integrating and interpreting the in vivo and in vitro observations.

The adenine base analog 2-aminopurine (2AP) is a potent base substitution mutagen in prokaryotes because of increased ability to form a mutagenic base pair with an incoming dCTP. Despite more than 50 years of research, the structure of the 2AP-C base pair remains unclear. We report the structure of the 2AP-dCTP base pair formed within the polymerase active site of the RB69 Y567A-DNA polymerase. A modified wobble 2AP-C base pair was detected with one H-bond between the N1 of 2AP and a proton from the C4 amino group of cytosine and an apparent bifurcated H-bond between a proton on the 2-amino group of 2-aminopurine and the ring N3 and O2 of cytosine. Interestingly, a primer-terminal region rich in AT base pairs, compared to GC base pairs, facilitated dCTP binding opposite template 2AP. We propose that increased flexibility of the nucleotide binding pocket formed in the Y567A-DNA polymerase and increased ‘breathing’ at the primer-terminal junction of A+T-rich DNA facilitates dCTP binding opposite template 2AP. Thus, interactions between DNA polymerase residues with a dynamic primer-terminal junction play a role in determining base selectivity within the polymerase active site of RB69 DNA polymerase.

Minor groove hydrogen bonding (HB) interactions between DNA polymerases and N3 of purines or O2 of pyrimidines have been proposed to be essential for DNA synthesis from results obtained using various nucleoside analogues lacking the N3 or O2 contacts that interfered with primer-extension. Since there has been no direct structural evidence to support this proposal, we decided to evaluate the contribution of minor groove HB interactions with family B pols. We have used RB69 DNA pol and 3-deaza-2’-deoxyadenosine (3DA), an analog of 2-deoxyadenosine, which has the same HB pattern opposite T but with N3 replaced with a carbon atom. We then determined pre-steady state kinetic parameters for the insertion of dAMP opposite dT using primer/template (P/T) containing 3DA. We also determined three structures of ternary complexes with 3DA at various positions in the duplex DNA substrate. We found that the incorporation efficiency of dAMP opposite dT decreased 102–103 fold even when only one minor groove HB interaction was missing. Our structures show that the HB pattern and base-pair geometry of 3DA/dT is exactly the same as dA/dT, which makes 3DA an optimal analogue for probing minor groove HB interactions between a DNA polymerase and a nucleobase. In addition, our structures provide a rationale for the observed 102–103 fold decrease in nucleotide incorporation. The minor groove HB interactions between the n-2 position of the primer strand and RB69pol fixes the rotomer conformations of the K706 and D621 side chains, as well as the position of metal ion A and its coordinating ligands so that they are in the optinal orientation for DNA synthesis