DNases 3 -»5 exonuclease activity

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... 

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. 

The HPLC-MS/MS assay was also successfully applied to the measurement of UV-induced dimeric pyrimidine photoproducts [123, 124]. The latter lesions were released from DNA as modified dinucleoside monophosphates due to resistance of the intra-dimer phosphodiester group to the exonuclease activity during the hydrolysis step [125, 126]. The hydrolyzed photoproducts exhibit mass spectrometry and chromatographic features that allow simultaneous quantification of the three main classes of photolesions, namely cyclobutane dimers, (6-4) photoproducts, and Dewar valence isomers, for each of the four possible bipyrimidine sequences. It may be added that these analyses are coupled to UV detection of normal nucleosides in order to correct for the amount of DNA in the sample and obtain a precise ratio of oxidized bases or dimeric photoproducts to normal nucleosides. 

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. 

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.)...

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

Answer E. The 3 to 5 exonuclease activity of DNA pol 8 represents the proofreading activity of an enzyme required for the replication of human chromosomal DNA. DNA pol y (mitochondrial) and DNA pol III (prokaryotic) do not participate in this process, short RNA primers are replaced with DNA during replication, and new DNA strands are always... 

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. 

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... 

FIGURE 25-15 Final steps in the synthesis of lagging strand segments. RNA primers in the lagging strand are removed by the 5 —>3 exonuclease activity of DNA polymerase I and replaced with DNA by the same enzyme. The remaining nick is sealed by DNA ligase. The role of ATP or NAD+ is shown in Figure 25-16. 

Like bacteria, eukaryotes have several types of DNA polymerases. Some have been linked to particular functions, such as the replication of mitochondrial DNA. The replication of nuclear chromosomes involves DNA polymerase a, in association with DNA polymerase S. DNA polymerase a is typically a multisubunit enzyme with similar structure and properties in all eukaryotic cells. One subunit has a primase activity, and the largest subunit (Afr -180,000) contains the polymerization activity. However, this polymerase has no proofreading 3 —>5 exonuclease activity, making it unsuitable for high-fidelity DNA replication. DNA polymerase a is believed to function only in the synthesis of short primers (containing either RNA or DNA) for Okazaki fragments on the lagging strand. These primers... 

The fidelity of DNA replication is maintained by (1) base selection by the polymerase, (2) a 3 —>5 proofreading exonuclease activity that is part of most DNA polymerases, and (3) specific repair systems for mismatches left behind after replication. 

Exonuclease activity enables DNA polymerase III to proofread the newly synthesized DNA strand. 

Exonuclease activity In addition to having the 5 —>3 po ) merase activity that synthesizes DNA, and the 3 ->5 exonucleas activity that proofreads the newly synthesized DNA chain lik DNA polymerase III, DNA polymerase I also has a 5 - 3 exon clease activity that is able to hydrolytically remove the RN primer. [Note These activities are exonucleases because the remove one nucleotide at a time from the end of the DNA chaii rather than cleaving it internally as do the endonucleases (Figui 29.18).] First, DNA polymerase I locates the space ("nick between the 3 -end of the DNA newly synthesized by DNA pol] merase III and the 5 -end of the adjacent RNA primer. Next, DN... 

Differences between 5 - 3 and 3 - 5 exonucleases The 5 - 3 exonuclease activity of DNA polymerase I differs from the 3 - 5 exonuclease used by both DNA polymerase I and III in two important ways. First, 5 3 exonuclease can remove one nucleotide at a time from a region of DNA that is properly base-paired. The nucleotides it removes can be either ribonucleotides or deoxyribonucleotides. Second, 5 —>3 exonuclease can also remove groups of altered nucleotides in the 5 —>3 direction, removing from one to ten nucleotides at a time. This ability is important in the repair of some types of damaged DNA. 

DNA chain elongation is catalyzed by DNA polymerase III using 5 -deoxyribonucleoside triphosphates as substrates. The enzyme "proofreads" the newly synthesized DNA, removing terminal mismatched nucleotides with its 3 —>5 exonuclease activity. 

RNA primers are removed by DNA polymerase I using its 5 —>3 exonuclease activity. The resulting gaps are filled in by this enzyme which can also proofread. The final phosphodiester linkage is catalyzed by DNA ligase. 

There are at least five classes of eukaryotic DNA polymerases. Pol a is a multisubunit enzyme, one subunit of which performs the primase function. Pol a 5 ->3 polymerase activity adds a short piece of DNA to the RNA primer. Pol 8 completes DNA synthesis on the leading strand and elongates each lagging strand fragment, using 3 ->5 exonuclease activity to proofread the newly synthesized DNA. Pol p and pol e are involved in carrying out DNA "repair," and pol y replicates mitochondrial DNA. 

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