DNA Catalysis

2.3.1 DNA Catalysis - Mechanism

The mechanism of DNA synthesis is similar in all DNA polymerases and can be divided into five different steps, depicted in the scheme below (Figure 4).[23, 27-29] As KlenTaq DNA polymerase, a member of sequence family A, is in the focus of this work, the steps are mainly discussed with regard to the changes occurring in this enzyme.

The DNA is bound in a crevice formed by the thumb, palm and fingers subdomains of the DNA polymerase. For members of DNA polymerase family A, a conformational change of the thumb domain is observed upon binding of the primer/template duplex (step 1, E:P/T), which brings the tip of the thumb (a helix-loop-helix motif) in proximity to the DNA duplex.^[30]

Simultaneously, the 3’ terminus of the primer is aligned in the active site and the

single-The next step involves weak binding of the incoming dNTP by the finger subdomain and thereby progression from a binary to an open ternary complex (step 2, E:P/T:dNTP). The following step 3 corresponds to a conversion from an inactivated (E:P/T:dNTP) to an activated complex (E*:P/T:dNTP) in which all components are efficiently aligned in the active site, thus facilitating the chemical reaction.^{[29, 30]} The formation of the active complex was suggested to be the rate-limiting step in DNA catalysis and was thought to be connected to a conformational change of the protein finger domain. Crystal structure analysis of KlenTaq DNA polymerase revealed a reorientation of the tip of the finger domain by 46° rotation towards the active site from an open to a closed conformation. This conformational change affects the orientation of the O helix located in the finger domain. Whereas the orientation of the O helix in the open form resembles its conformation in a binary KlenTaq complex, it packs against the templating nucleobase and the incoming nucleotide in the closed state. This conformational change of the O helix releases a tyrosine residue at position 671 (Tyr671) from its stacking arrangement on top of the first base pair duplex and makes room for the templating base opposite the incoming dNTP. Recent studies, however, negate this conformational change of the fingers domain as the rate-limiting step, and suggest that conformational changes in the active site, such as the arrangement of side chains or the binding of metal ions, are responsible.^[28] With every component poised for catalysis, the chemical reaction (SN2) can occur (step 4, E*:P+1/T:PPi). Two metal ions, present in the active site, promote DNA catalysis and stabilize the trigonal bipyrimidal transition state of the reaction (Figure 5).

Figure 4. Kinetic pathway of nucleotide incorporation.

Depicted complexes as well as the kinetic steps are described in the text. Scheme was adapted from Rothwell and Waksman.^{[28, 29]}

Metal ion A supports deprotonation of the 3’ OH group of the primer and facilitates the nucleophilic attack of the primer on the -phosphate of the incoming nucleotide. Metal ion B interacts with the triphosphate moiety of the bound nucleotide and facilitates the release of the pyrophosphate (PPi). Both metal ions are further coordinated by catalytically active aspartic acid residues in the palm domain, which further stabilize the transition state. This

‘two-metal ion’ mechanism is conserved in every DNA polymerase sequence family (Figure 5).[23, 27, 31, 32]

The reaction is completed with the formation of the phosphodiester bond and the release of pyrophosphate (step 5, E:P/T). Next, the DNA polymerase either translocates for the next round of incorporation or dissociates from the primer/template duplex and DNA synthesis ends. Recently, it was shown that general acid catalysis is also part of the nucleotidyl transfer mechanism.^[33] An active site amino acid residue protonates the pyrophosphate leaving group, thus neutralizing the negative charge which is formed during the transition state and contributing to the release of pyrophosphate.

2.3.2 DNA Catalysis – Kinetic Analysis

The reaction pathway of an enzyme, in this study that of a DNA polymerase, can be investigated by single nucleotide incorporation experiments using either steady^{[34, 35]} or pre-steady^[36] state kinetic conditions. Steady state kinetic measurements provide information about KM and vmax of an enzymatic reaction, whereas KM is defined as the Michaelis-Menten constant and vmax represents the maximal velocity of the reaction. However, these paramters are determined based on the complete reaction pathway (see Figure 4) with the system in an equilibrium state. Steady state kinetics are conducted using ‘single completed hit’ conditions facilitated by an excess of primer/template complex compared to the amount of enzyme used and a maximal primer conversion of 20 %.^{[34, 35]} These conditions ensure that every DNA polymerase binds to a primer/template complex maximal once.

Pre-steady state kinetics allow the analysis of individual steps in the reaction pathway and can provide the affinity Kd and incorporation rate constant kpol. These constants are independent from association or dissociation events of the polymerase to the primer/template complex Figure 5. The ‘two-metal ion’ mechanism of

DNA polymerases. Two metal ions facilitate DNA synthesis and stabilize the trigonal bipyrimidal transition state of the reaction.

Scheme was adapted from Brautigam and Steitz^[27] showing the active site of E. coli DNA polymerase I. Water molecules are depicted as black spheres.

fluorescence.^{[39, 40]} However, not every system can be followed using an optical signal and it is often difficult to asign the signal to a particular step in the reaction pathway.^[37]

Quench-flow experiments are based on a quenching agent which is used to terminate the reaction after short reaction times which allows immediate quantification of substrate conversion. In general, radioactively labelled substrates are used to visualize the reaction products for subsequent quantification. Furthermore, rapid chemical quench flow instruments facilitate the measurement of substrate conversion after short time periods, which are not accessible in a manual set-up (see chapter VI 2.5.5). In this work, pre-steady state kinetic measurements were performed using a quench-flow approach.