BIOCHEMISTRY - L. Stryer - 1984

VOLUME 3

Part IV INFORMATION

CHAPTER 24 DNA: THE GENETIC ROLE, STRUCTURE, AND REPLICATION

24.13. DNA Polymerase Receives Instructions from the Template

DNA polymerase catalyzes The formation of a phosphodiester bond only if the Base of the incoming nucleotide is complementary to the corresponding base of the template strand. If the base of the incoming nucleotide does not form a Watson-Crick pair with the corresponding base of the template strand, the probability of covalent bond formation is very low. Consequently, DNA polymerase is a template-directed enzyme. It was the first such enzyme to be discovered.

A series of experiments demonstrates that DNA polymerase receives instructions from the template.

1. The first evidence supporting this assertion was the fact that significant DNA Synthesis occurs only in the presence of all four deoxynucleoside triphosphates and template DNA.

2. DNA polymerase can incorporate certain base analogs into DNA. For example, uracil or 5-bromouracil can substitute for thymine. Hypoxanthine can substitute for guanine. The ability of analogs to substitute for bases is highly specific. According to the substitution rule, a given analog can be incorporated only if it is capable of forming a Watson-Crick pair with a base complementary to the replaced base. Thus, hypoxanthine can substitute for G (but not A, T, or C) because it forms a suitable base pair with cytosine.

3. The Nucleotide Composition of newly synthesized DNA depends on The Nature of the template, rather than on the relative amounts of the four precursor NUCLEOTIDES. The resulting DNA has the same nucleotide composition as the double-helical template DNA. This implies that both strands of the template DNA are replicated under the action of DNA polymerase.

4. The most compelling evidence comes from data showing that DNA of phage ɸX174 replicated in vitro by DNA polymerase I is fully infectious. Consequently, the error rate of this enzyme is extremely low.

24.14. DNA Polymerase I Corrects Errors in DNA

DNA polymerase possesses another type of enzymatic activity: under certain conditions, it is capable of cleaving DNA strands. DNA polymerase progressively hydrolyzes a DNA strand from the 3'-hydroxyl end, releasing mononucleotides in the process. Thus, DNA polymerase I also Functions as a 3' → 5' exonuclease (Fig. 24.28). The removed nucleotide must have a free 3'-OH end and must not be part of a double helix. Is this exonuclease activity an undesirable side effect of the enzyme, or does it somehow participate in the biological action of DNA polymerase? Experiments using chemically synthesized polynucleotides with a non-complementary residue at the primer terminus have shown that the 3' → 5' exonuclease activity performs an editing function during polymerization. Consider the polymer shown in Fig. 24.28, in which a sequence of dT residues forms a double helix with a longer dA polymer. At the 3' end of this poly(dT) sequence, There is a single dC residue that does not form Hydrogen Bonds because it is not complementary to dA. Upon The addition of DNA polymerase and dTTP, this non-complementary dC residue is excised before the addition of dT residues begins. Experiments using a variety of different synthetic polymers have shown that DNA polymerase I always removes non-complementary residues at the primer terminus before continuing polymerization. If a complementary base is located at the terminus and activated precursors are present in the medium, virtually no Hydrolysis occurs. Polymerization prevents 3'-end hydrolysis.

Class="center">Fig. 24.28. 3' → 5' exonuclease activity of DNA polymerase I

In all likelihood, METABOLISM/36.html">DNA Replication proceeds with high fidelity because base pairing is checked twice. In cases where a base pair does not fit within The Double Helix, polymerization generally does not proceed. However, if an error does occur at this stage, it can be corrected before the next nucleotide is attached. Thus, DNA polymerase I checks the result of each completed polymerization event before moving on to the next.

In addition, DNA polymerase I can hydrolyze DNA starting from the 5' end of the strand. This 5' → 3' nuclease activity (Fig. 24.29) differs significantly from the 3' → 5' exonuclease activity discussed above. First, the bond to be cleaved must be located within a double-helical region. Second, Cleavage can occur both at the terminal phosphodiester bond and at a bond located several nucleotides away from the 5' end (which may have a free or phosphorylated hydroxyl group). Third, the 5' → 3' nuclease activity is enhanced when DNA synthesis is concurrently underway. Fourth, the Active Site for the 5' → 3' nuclease activity is distinctly separate from the active sites for polymerization and 3' → 5' hydrolysis. DNA polymerase I can be cleaved by Proteolytic Enzymes into a 36 kDa fragment containing all the 5' → 3' nuclease activity and a 75 kDa fragment containing all the polymerase and 3' → 5' exonuclease activities. Thus, DNA polymerase I contains at least two distinct enzymes within a single polypeptide chain. The 5' → 3' nuclease complements the 3' → 5' exonuclease activity by correcting Different types of errors. For example, the 5' → 3' nuclease participates in the excision of pyrimidine dimers formed upon UV irradiation of DNA (Sec. 24.24). Moreover, the 5' → 3' nuclease activity plays a key role in DNA replication itself (Sec. 24.19).

Fig. 24.29. 5' → 3' nuclease activity of DNA polymerase I

24.15. DNA Ligase Joins DNA Fragments

DNA polymerase I can add deoxynucleotides to a primer strand, but it is incapable of catalyzing the joining of two DNA strands or the cyclization of a single DNA strand. The discovery of circular DNA indicated that such an enzyme must exist. In 1967, DNA ligase—an enzyme catalyzing the formation of a phosphodiester bond between two DNA strands—was discovered simultaneously in several laboratories (Fig. 24.30). This enzyme is active in the presence of a free OH group at the 3' end of One DNA strand and a phosphate group at the 5' end of another. The formation of a phosphodiester bond between these groups is an endergonic reaction. Consequently, an energy source is required for the strand-joining reaction. In E. coli and other Bacteria, this role is fulfilled by NAD+, whereas in animal Cells and bacteriophage-infected cells, this reaction is driven by the energy of ATP.

Fig. 24.30. DNA ligase catalyzes the joining of two DNA strands belonging to the same double-helical molecule

Another notable aspect of DNA ligase function is that it cannot join two single-stranded DNA molecules. The DNA strands joined by DNA ligase must be part of a double-stranded DNA molecule. Studies on model systems suggest that DNA ligase forms a phosphodiester bond only if at least a few Base Pairs are present near the nick. In fact, DNA ligase seals single-strand breaks in the backbone of double-helical DNA. This process is essential for normal DNA synthesis, for the Repair of Damaged DNA, and for the splicing of DNA strands During genetic recombination.

Let us consider The Mechanism of this reaction, elucidated by Robert Lehman. ATP or NAD+ react with DNA ligase to form a covalent enzyme-AMP complex, in which AMP is linked to the ε-amino group of a Lysine residue of the enzyme via a phosphoamide bond (Fig. 24.31). The AMP residue activates the phosphate group at the 5' end of the DNA. The final step is a nucleophilic attack on the activated phosphorus atom by the 3'-OH group. As a result, a phosphodiester bond is formed and AMP is released. The driving force behind this sequence of reactions is the hydrolysis of pyrophosphate, which is split off during the Formation of the enzyme-adenylate complex. Thus, the formation of a phosphodiester bond in the DNA backbone consumes two high-energy phosphate bonds if ATP serves as the energy source.

Fig. 24.31. Covalent enzyme-AMP intermediate complex