DNases proof reading

Although DNA proof-reading and repair mechanisms within cells are very effective, inevitably some errors in replication remain uncorrected, and as a result become perpetuated in the DNA of the organism. Such permanent changes are called mutations. 

E. coli DNA polymerase I requires all four deoxynucleoside 5 triphosphates (dNTPs) as precursors, Mg2+, a DNA template and a primer with a 3 -OH end. DNA synthesis occurs in a 5 - 3 direction. DNA polymerase I also has a 3 —>5 exonuclease (proof-reading) activity and a 5 —>3 exonuclease activity. E. coli DNA polymerases II and HI lack the 5 —>3 exonuclease activity. 

The 3 -+5 exonuclease activity plays an important role in polymerization in proof reading the base pair formed at each polymerization step. The enzyme checks the nature of each base-paired primer terminus before the polymerase proceeds to add the next nucleotide to the primer. It thus supplements the capacity of the polymerase to match the incoming nucleotide substrate to the template. A mismatched terminal nucleotide on the primer activates a site on the enzyme which results in the hydrolysis of the phosphodiester bond and the removal of the mismatched residue. The function of this 3 - 5 exonuclease activity is therefore to recognize and cleave incorrectly or non-base paired residues at the 3 -end of DNA chains. It will therefore degrade single stranded DNA and frayed or non-base paired residues at the ends of duplex DNA molecules provided they terminate in a 3 -hydroxyl group. 

In prokaryotes DNA polymerase I has a 5 — 3 DNA polymerase activity as well as a proof reading capacity to chop out nucleotides in either direction through a 5 — 3 and a 3 — 5 direction exonuclease activity DNA polymerases II and III have 5 — 3 ... 

DNA polymerases are ubiquitous enzymes required for replication, repair and recombination of DNA genomes [121], Most DNA polymerases are multi-functional enzymes which exhibit, in addition to the polymerase activity, a 3 to 5 exonuclease activity involved in the fidelity of DNA synthesis (proof-reading). 

The DNA polymerases of T. littoralis and P. furiosus have been marketed for use in DNA amplification by the polymerase chain reaction (PCR) method as the Vent and pfu DNA polymerases, respectively. These enzymes are more accurate in vitro than the Thermus aquaticus (Taq) DNA polymerase, both in classical fidelity tests [132] and in PCR [133,134]. Indeed, they have an associated 3 to 5 exonuclease activity involved in proof-reading, whereas the Taq polymerase is devoid of such activity. 

One of the early plant viruses used was the cauliflower mosaic virus (CaMV), a double stranded DNA virus that replicates through an RNA intermediate [38]. Using this virus extra DNA insertions are difficult to achieve, and often molecular recombinations rapidly eliminate the inserted transgene. In addition, replication of this virus requires an RNA step which can introduce errors because the inverse transcriptase does not have a proof-reading activity. Similarly, regarding RNA viruses, two viruses the tobacco mosaic virus (TMV) and the cowpea mosaic virus (CMV) have been successfully employed [39]. 

Contains a strong 3 5 proof-reading exonuclease activity. another proof-reading DNA polymerase exhibiting the lowest error rate of any thermostable DNA polymerase can also be used with an optimized Mg concentration and reaction buffer supplied by the manufacturer (Stratagene). 

The virtuosity of the DNA polymerase first isolated from E. coli still amazes me. Holding one or two turns of one chain of DNA helix as template, it assembles a complementary chain thousands of nucleotides long. It does so by Watson-Crick base pairing with an accuracy which exceeds chemical predictions. The enzyme achieves this fidelity of replication by a proof-reading exonuclease in its active center. This 3 5 exonuclease... 

DNA replication involves a highly developed enzymatic machinery which uses a proof reading mechanism in order to increase the single digit accuracy. Thereby, genomes as long as some million base pairs become possible. In procaryotes nature again seems to operate close to the error limit. Bacteria, nevertheless, have retained a certain flexibility to adjust mutation rates to the evolutionary needs of the moment. They do not seem to replicate as closely to the accuracy limit as viruses do. 

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