DNA polymerase exonuclease

Klenow polymerase T4-DNA polymerase Exonuclease III Restriction enzymes... 

Zang, H., Harris, T.M., and Guengerich, F.P. (2005) Kinetics of nucleotide incorporation opposite DNA bulky guanine N2 adducts by processive bacteriophage T7 DNA polymerase (exonuclease-) and HIV-1 reverse... 

Another class of DNA-binding proteins are the polymerases. These have a nonspecific interaction with DNA because the same protein acts on all DNA sequences. DNA polymerase performs the dual function of DNA repHcation, in which nucleotides are added to a growing strand of DNA, and acts as a nuclease to remove mismatched nucleotides. The domain that performs the nuclease activity has an a/P-stmcture, a deep cleft that can accommodate double-stranded DNA, and a positively charged surface complementary to the phosphate groups of DNA. The smaller domain contains the exonuclease active site at a smaller cleft on the surface which can accommodate a single nucleotide. 

DNA polymerase I is a nonessential enzyme, since viable E. coli mutants lack it (pol A). This conclusion is complicated, however, since the enzyme catalyzes three separate chemical reactions. It polymerizes deoxyribonucleoside triphosphates, and it has two exonucleolytic activities, a 3 to 5 activity and a 5 to 3 activity. The pol A - mutants lack only the polymerization activity. Other mutants lacking both the polymerase and the 5 to 3 exonuclease activity are lethal. Thus the exonuclease function is the more important one. This fits with the role of this enzyme in removing damaged DNA segments (DNA repair) and in removing covalently attached RNA from DNA chains. We will later see that small RNAs serve as primers of DNA synthesis. 

DNA polymerase I has been purified to homogeneity. When the pure enzyme is treated with subtilisin, a proteolytic enzyme from Bacillus subtilis, the polymerase is cleaved into two pieces. The small fragment retains the 5 to 3 nuclease activity, whereas the larger piece, called a Klenow fragment, has both polymerase activity and the 3 to 5 exonuclease activity. The Klenow fragment is sold commercially for use in labeling DNA for use in detecting recombinant DNA. 

Under certain circumstances DNA has both primer and template activities. For example, the addition of mononucleotides is to the 3 end of the growing DNA primer. This presents a problem with regard to how the other strand is synthesized. Biochemists have looked hard but unsuccessfully for an enzyme that can add deoxyribonucleotides onto the 5 end of DNA primers. Such a primer should contain a triphosphate on the hydroxyl group of the 5 end. Although a very active 5 -exonuclease, actually part of DNA polymerase I, has made the search for such an activated 5 end extremely difficult, investigators conclude that a polymerase able to use such a primer probably does not exist. On the contrary, good evidence suggests that the synthesis of both strands is by the known DNA poly-merases. 

Tabor, S., and Richardson, C.C. (1987) Selective oxidation of the exonuclease domain of bacteriophage T7 DNA polymerase./. Biol. Chem. 262, 15330-15333. 

Figure 7. Reaction mechanism of the 3, 5 -exonuclease subunit DNA polymerase I.

Figure 10.14 The proposed transition state in the mechanism of the 3 -5 exonuclease activity of DNA polymerase I. (From Steitz and Steitz, 1993. Copyright (1993) National Academy of Sciences, USA.)...

Moser, M. J., Holley, W. R., Chatterjee, A., and Mian, I. S. (1997). The proofreading domain of Escherichia coli DNA polymerase land other DNA and/ or RNA exonuclease domains. Nucleic Acids Res. 25, 5110-5118. 

DNA polymerases all synthesize new DNA using a template and make the new DNA in a 5 3 direction (new nucleotides are added to the 3 end). In addition to making DNA, some of the DNA polymerases can also hydrolyze it. An exonuclease works only on the ends of the DNA (or RNA), and like everything else about DNA, exonuclease activity has a direction too. The 3 5 exonuclease activity removes nucleotides from the 3 end (by hydrolyzing the phosophodi-ester bond). Since the chain grows in the 5 3 direction, polymerases that have a 3 5 exonuclease activity can look back over their work... 

Biolabs (Pickering, ON), and Promega (Madison, WI). It is important to note that restriction enzymes producing 3 -overhangs should be avoided if possible (e.g., Psfl, Sfil, Kpnl). The use of such enzymes has been reported to result in the production of additional, nonspecific transcripts (Schenborn and Mierendorf, 1985). If these enzymes must be used, an exonuclease such as DNA Polymerase 1 Large (Klenow) Fragment can be utilized to convert the overhang to a blunt end before the template is transcribed. 

DNA polymerases can correct mistakes ( proofreading ), whereas RNA polymerases cannot. DNA polymerases have 3 —> 5 exonuclease activity for proofreading. 

Comparison between DNA repair and phospholipid repair The processes that can lead to DNA damage and the type of damage are described in Chapter 9 and Appendix 9.6. The repair processes involve removal of the specific nucleotide(s) by an exonuclease and replacement of the nucleotide by a DNA polymerase. Since the strand must be broken to remove the damage (by an endonuclease) these parts of the strand must be repaired by a ligase. The process is known as excision-repair. Of interest, there is a degree of similarity between the removal of damaged polyunsaturated fatty acids from phospholipids in membranes and replacement with a new fatty acid by two enzymes, a deacylase and an acyltransferase (see above and Chapter 11), and excision-repair of DNA. 

Beese LS, Steitz . Structural basis for the 3 -5 exonuclease activity of Escherichia //DNA polymerase I a two metal ion mechanism. EMBO J 1991 10 25-33. 

DNA polymerase I (E. coli) Reverse transcriptase Polynucleotide kinase Terminal transferase Exonuclease III... 

The mispaired 3 -OH end of the growing strand blocks further elongation. DNA polymerase slides back to position the mispaired base in the 3 —>5 exonuclease active site. 

FIGURE 25-7 An example of error correction by the 3 —>5 exonuclease activity of DNA polymerase I. Structural analysis has located the exonuclease activity ahead of the polymerase activity as the enzyme is oriented in its movement along the DNA. A mismatched base (here, a C-A mismatch) impedes translocation of DNA polymerase I to the next site. Sliding backward, the enzyme corrects the mistake with its 3 —>5 exonuclease activity, then resumes its polymerase activity in the 5 —>3 direction. 

DNA polymerase I, then, is not the primary enzyme of replication instead it performs a host of clean-up functions during replication, recombination, and repair. The polymerase s special functions are enhanced by its 5 —>3 exonuclease activity. This activity, distinct from the 3 —>5 proofreading exonuclease (Fig. 25-7), is located in a structural domain that can be separated from the enzyme by mild protease treatment. When the 5 —>3 exonuclease domain is removed, the remaining fragment (Afr 68,000), the large fragment or Klenow fragment (Fig. 25-8), retains the polymerization and... 

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