DNA polymerase proofreading: active site switching catalyzed by the bacteriophage T4 DNA polymerase

 

proofreading by dna polymerase

DNA Polymerase Proofreading. It allows the enzyme to check each nucleotide during DNA synthesis and excise mismatched nucleotides in the 3´ to 5´ direction. The proofreading domain also enables a polymerase to remove unpaired 3´ overhanging nucleotides to create blunt ends. Protocols such as high-fidelity PCR, 3´ overhang polishing. DNA polymerase proofreading is a spell-checking activity that enables DNA polymerases to remove newly made nucleotide incorporation errors from the primer terminus before further primer extension and also prevents translesion synthesis. DNA polymerase proofreading improves replication fidelity ∼ fold, which is required by many organisms to prevent unacceptably high, life threatening mutation Cited by: DNA polymerase is an enzyme that synthesizes DNA molecules from deoxyribonucleotides, the building blocks of DNA. These enzymes are essential for DNA replication and usually work in pairs to create two identical DNA strands from a single original DNA habrnesq.tk: BRENDA entry.


“Proofreading” DNA | Biology for Majors I


DNA polymerases achieve high-fidelity DNA replication in part by checking the accuracy of each nucleotide that is incorporated and, if a mistake is made, the incorrect nucleotide is removed before further primer extension takes place. In order to proofread, the primer-end must be separated from the template strand and transferred from the polymerase to the exonuclease active center where the excision reaction takes place; then the trimmed primer-end is returned to the polymerase active center.

Thus, proofreading requires polymerase-to-exonuclease and exonuclease-to-polymerase active site switching. We have used a fluorescence assay that uses differences in the fluorescence intensity of 2-aminopurine 2AP to measure the rates of active site switching for the bacteriophage T4 DNA polymerase.

DNA polymerase proofreading removes misincorporated nucleotides at the primer-end 12which significantly improves the fidelity of DNA replication 3. The T4 DNA polymerase proofreading pathway has at least four steps 9 During chromosome replication, the proofreading pathway is initiated in the polymerase active center when an incorrect nucleotide is inserted step 1which hinders further primer elongation 2311 The terminal nucleotide is cleaved from the primer-end in the exonuclease active center step 3 and then the trimmed primer-end is returned to the polymerase active center where nucleotide incorporation can resume step 4.

Genetic studies indicate that four of the five protein domains of the T4 DNA polymerase are involved in the proofreading pathway The genetic studies are corroborated by structural studies, proofreading by dna polymerase, which find significant conformational differences for the exonuclease, palm and proofreading by dna polymerase domains in polymerase complexes compared to exonuclease complexes 1517— There are still unanswered questions about how the primer-end is shuttled back-and-forth between the polymerase and exonuclease active centers, which we address here.

The phage T4 clamp, the product of gene 45is also reported to stimulate proofreading 2122but is the clamp essential for processive transfer of the primer-end from the polymerase to the exonuclease active center and for transfer of the trimmed primer-end from the exonuclease back to the polymerase active center? We proposed that the clamp is essential for processive proofreading that initiates in the polymerase active center because greater intrinsic processivity in nucleotide incorporation is observed for mutant DNA polymerases that have reduced ability to initiate the proofreading pathway, while reduced processivity in primer extension is detected for mutant DNA polymerases that proofread more Coupled removal of an incorrect nucleotide and primer extension were observed under single turnover conditions in the presence of a heparin trap; however, proofreading by dna polymerase, it is not clear in proofreading by dna polymerase experiments if the T4 DNA polymerase first bound the DNA substrate in the polymerase or the exonuclease active center.

If the T4 DNA polymerase bound the mismatched DNA initially in the polymerase active center, then the entire proofreading pathway beginning from strand separation and transfer of the primer-end from the polymerase to the exonuclease active center can be carried out without enzyme dissociation. However, proofreading by dna polymerase, if the T4 DNA polymerase can form exonuclease complexes directly without first forming polymerase complexes, then just the steps of hydrolysis and transfer of the trimmed primer-end from the exonuclease to the polymerase active center have been demonstrated to be processive in the absence of the clamp.

Another outstanding question is the rate of active site switching. Proofreading during ongoing DNA replication is restricted primarily to incorrect nucleotides at the primer-end because the rate of primer extension for a matched primer terminus is much greater than the rate for initiation of the proofreading pathway, but replicative DNA polymerases have poor proofreading by dna polymerase to extend a mismatched primer terminus, which then tips the balance in favor of proofreading 39 Thus, there is a kinetic barrier to initiation of the proofreading pathway, which suggests that the rate of polymerase-to-exonuclease active site switching will be relatively slow.

In contrast, transfer of the trimmed primer-end from the exonuclease to polymerase active center could be rapid if the corrected primer-end returns to the polymerase active center unassisted We have developed a fluorescence assay using the fluorescent adenine base analog 2-aminopurine 2AP to examine shuttling of the primer-end between the polymerase and exonuclease active centers during the proofreading reaction catalyzed by the T4 DNA polymerase.

We performed this experiment with wild-type and exonuclease-deficient T4 DNA polymerases under pre-steady-state, single-turnover conditions in which heparin was used to trap any free T4 DNA polymerase Proofreading by dna polymerase results are discussed with respect to the overall proofreading reaction, active site switching, structural implications and replication fidelity of the wild-type and proofreading defective T4 DNA polymerases.

The substrates were prepared as described previously 1314 The 2AP phosphoramidite was purchased from Glen Research.

All oligonucleotides were purified by gel electrophoresis. The non-2AP containing DNA substrates used for Figure 2 were synthesized using standard procedures and purified by gel electrophoresis, proofreading by dna polymerase. The annealing conditions were the same as used for the 2AP-containing oligonucleotides. Coupled processive proofreading and nucleotide incorporation. A DNA substrates. The reaction conditions are described in Materials and Methods section.

B Test of the heparin trap under the conditions described by Reddy et al. Full exonuclease lane 2 and primer extension lane 4 activities were detected in the absence of heparin. The wild-type T4 DNA polymerase lanes 1 and 2 carried out processive primer extension with the matched DNA lane 1 and processive proofreading and primer extension reactions with the mismatched DNA lane 2.

The WS-DNA polymerase had less ability to fully extend the matched DNA lane 3 and almost no ability to carry out processive proofreading by dna polymerase and primer extension reactions with the mismatched DNA lane 4, proofreading by dna polymerase. A control reaction with no DNA polymerase is in lane 7.

Reddy et al. The same reaction conditions were used as described above for single-turnover reactions except that the heparin trap was omitted. Stopped-flow experiments were performed with the Applied Photophysics SX, proofreading by dna polymerase.

Excitation was at nm; a nm cutoff filter was used. The temperature in the sample-handling unit was maintained at The optimal DNA and enzyme concentrations to ensure full complex formation were determined by titration experiments Curves were fit either to single monophasic or double biphasic exponential equations. Six or more runs were performed with each set of reaction conditions; mean values were calculated, proofreading by dna polymerase.

In the absence of heparin, exonucleolytic degradation Figure 2 B, lane 2 and full primer extension Figure 2 B, lane 4 are observed, proofreading by dna polymerase. Although the wild-type T4 DNA polymerase has a potent exonuclease activity, only traces of products less than the length of the primer strand were observed Figure 2 C, lane 1.

Degradation products were detected because the only nucleotide provided in these reactions was dCTP, which means that if there was any primer degradation—first removal of the terminal dTMP, then another dTMP, proofreading by dna polymerase, etc. Figure 1 A the primer could not be resynthesized. Thus, the wild-type T4 DNA polymerase formed primarily polymerase complexes with the matched DNA substrate that were poised for nucleotide incorporation rather than exonuclease complexes poised for primer degradation.

Because the wild-type T4 DNA polymerase cannot efficiently extend a mismatched primer-end 21112the primer extension observed with the mismatched DNA substrate must have been preceded by removal of the incorrect terminal dTMP, which was followed by transfer of the trimmed primer-end from the exonuclease to the polymerase active center, incorporation of dAMP and then incorporation of two dCMPs.

All steps were performed without dissociation of the DNA polymerase since the heparin trap was present. We also tested the ability of the WS-DNA polymerase to carry out primer extension reactions of the matched and mismatched DNA substrates under single-turnover conditions.

Reactions with 25 nM single-stranded DNA and 25 nM or 50 nM enzyme are shown in lanes 1 and 3 and lanes 2 and 4, respectively. Primer extension reactions with the wild-type and WS-DNA polymerases are in lanes 6 and 7, respectively. Single-turnover experiments were performed as were done for the reactions shown in Figure 2 C except that the concentration of heparin was reduced from 1 to 0.

Thus, the processive primer extension reaction first required removal of the terminal 2AP nucleotide from the primer-end, then transfer proofreading by dna polymerase the trimmed primer-end to the polymerase active center and finally nucleotide incorporation. Proofreading of the 2AP-T terminal base pair occurs before primer extension; single turnover conditions. A control reaction with no enzyme is shown in lane 3.

The reactions shown in Figure 4 demonstrate that the terminal 2AP-T base pair with 2AP in the terminal position of the primer strand is recognized as a mismatch by the T4 DNA polymerase. The same is true if 2AP is in the n position in the template strand and T is in the terminal position of the primer strand 25proofreading by dna polymerase, The previous experiments demonstrate that the T4 DNA polymerase can proofread a T-T mismatch Figure 2 C and a 2AP-T terminal base pair Figure 4 and then incorporate nucleotides without dissociating from the DNA substrate; however, it is not possible to determine from these experiments if the proofreading pathway initiated in the polymerase or the exonuclease active center or in both.

These experiments also do not provide information about the rate of active site switching. If a single proofreading by dna polymerase is observed, then only one type of complex can carry out the proofreading reaction processively. The curve was best fit by a single exponential equation. Because a single rate was observed in the range of the reported hydrolysis rate, processive proofreading appears to be detected only for complexes in which the primer-end was bound initially in the exonuclease active center.

Time courses for conversion of exonuclease complexes to polymerase complexes. A Single turnover conditions, proofreading by dna polymerase. B Multiple turnover conditions. The experimental conditions described above were repeated except that heparin was omitted, proofreading by dna polymerase. The increase in fluorescence intensity was biphasic; the best curve fit was achieved using a double exponential equation. The above experiments were repeated without the proofreading by dna polymerase trap.

The increase in fluorescence intensity was biphasic in reactions with the wild-type T4 DNA polymerase. Proofreading and active site switching rates determined under single and multiple turnover conditions, proofreading by dna polymerase.

Details of the reactions and the calculation of reaction rates are described in the text. A single rate of 1. This slow rate is consistent with the severely reduced ability of this mutant DNA polymerase to degrade single- and double-stranded DNA Figure 3.

The T4 DNA polymerase and the closely related RB69 DNA polymerase can remove two incorrect nucleotides and then extend the primer terminus under single-turnover conditions in the presence of the heparin trap 12proofreading by dna polymerase, We repeated the above experiments with the DNA substrate illustrated in Figure 1 D, which has two incorrect G nucleotides at the end of the primer strand and 2AP is in the n position in the template strand.

Moderately, fluorescent exonuclease complexes are formed with this DNA substrate 25 The same rate of decrease in fluorescence intensity was also observed without the heparin trap, which indicates that none of the complexes formed during the process of removing two incorrect nucleotides are sensitive to the heparin trap.

Time course for removal of two incorrect nucleotides; single turnover conditions, proofreading by dna polymerase. The slower apparent rate for removal of the terminal nucleotide compared to the rate for removal of the second incorrect nucleotide suggests that there are extra steps for removal of the terminal nucleotide.

We propose that after removal of the terminal nucleotide, the trimmed primer-end proofreading by dna polymerase returned to the polymerase active center.

This proposal is reasonable since the correctness of the primer-end can only be examined in the polymerase active center where hydrogen bonding between the terminal base on the primer strand and the complementary template base can be evaluated as well as the geometry of the terminal base pair 29 Such a mechanism must exist in order to explain how exonucleolytic proofreading is limited primarily to the removal of incorrect nucleotides. If the primer-end is found to be incorrect, then the primer-end is returned to the exonuclease active center for a second cycle of excision, and then the further trimmed primer-end is returned to the polymerase active center.

We used this assay to confirm the results of Reddy et al, proofreading by dna polymerase. We 32 and others proofreading by dna polymerase proposed that the T4 DNA polymerase can form two types of complexes—[E-D] exo complexes that are active for hydrolysis of the terminal nucleotide and [E-D] proofreading by dna polymerase complexes that are inactive for hydrolysis.

This slow rate is not detected in the presence of the heparin trap, which indicates that conversion from an inactive to an active state involves enzyme dissociation. This point is discussed again later with respect to the clamped or tethered DNA polymerase. The 2AP fluorescence assay can also be used to determine the rates for active site switching.

Thus, proofreading by dna polymerase, once the hydrolysis reaction takes place, the trimmed primer-end is returned rapidly to the polymerase active center in position to resume nucleotide incorporation.

The efficient proofreading reaction that initiates in the exonuclease active center has several implications for understanding proofreading by the T4 DNA polymerase and Family B Proofreading by dna polymerase polymerases in general, proofreading by dna polymerase.

First, the template strand is likely bound in the polymerase active center when the primer-end is bound in the exonuclease active center. Intuitively, it makes sense for the template strand to be held in the polymerase active center during proofreading to ensure that the trimmed primer-end will be returned to the polymerase active center in correct alignment, otherwise frameshift mutations will be produced.

It is also important that proofreading be limited to only removing incorrect nucleotides proofreading by dna polymerase order to prevent gratuitous degradation of the newly synthesized DNA, proofreading by dna polymerase, which would slow DNA replication and waste dNTPs. Severely reduced DNA replication is observed in T4 infections with mutant DNA polymerases that catalyze excessive proofreading 23 These potential problems with proofreading can be reduced if the trimmed primer-end is returned to the polymerase active center in position to resume replication after an incorrect nucleotide is removed.

If the primer-end is matched, primer extension will be the favored reaction; however, if the primer-end is not correct or if the primer-end is misaligned, then another cycle of proofreading will be favored over primer extension.

 

Proofreading (biology) - Wikipedia

 

proofreading by dna polymerase

 

Have 3'-5' exonuclease; proofreading ability - keeps mutation rate below a certain level. Can only polymerize in a 5'-3' direction. Can only add nucleotides to an existing strand; can't start a new DNA strand. DNA Polymerase III. Holoenzyme, dimer of the core polymerase. Adds DNA nucleotides on to the end of the 3' primer. DNA polymerase is an enzyme that synthesizes DNA molecules from deoxyribonucleotides, the building blocks of DNA. These enzymes are essential for DNA replication and usually work in pairs to create two identical DNA strands from a single original DNA habrnesq.tk: BRENDA entry. DNA polymerase proofreading is a spell-checking activity that enables DNA polymerases to remove newly made nucleotide incorporation errors from the primer terminus before further primer extension and also prevents translesion synthesis. DNA polymerase proofreading improves replication fidelity ∼ fold, which is required by many organisms to prevent unacceptably high, life threatening mutation Cited by: