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Biochemical and Genetic Identifcation of Enzymes

Enzymes and other components involved in replication can be identified biochemically and genetically.

The summary of activities shown in Fig. 5.7 suggests several types of enzymes that might be expected at the replication fork. Obviously, at least one DNA polymerase should be present, but we would also expect to find enzymes that unwind DNA, initiate the assembly of nucleotides, and join Okazaki fragments. Experiments that identified the components needed at the replication fork proceeded along two avenues – biochemical fractionation and genetic analysis. The detailed understanding of the mechanism of synthesis at the replication fork has come from both approaches, as well as the powerful combination of them in a technique called in vitro complementation.

Biochemical methods

Biochemical purification requires an assay for the activity under investigation, which is usually a measurement of the product of the reaction being catalyzed by the enzyme. Because the reaction catalyzed by DNA polymerases is the fundamental step in DNA replication, we will examine it in a little detail. A fairly simple way to observe the activity of a DNA polymerase is to measure the incorporation of a radioactively labeled deoxyribonucleoside or deoxyribonucleotide into the high molecular weight polymer DNA. The latter can be precipitated with a strong acid, such as trichloroacetic acid, whereas unincorporated nucleotides or nucleosides do not precipitate. For instance, a crude cell extract can be incubated with dTTP labeled with a 32P atom in the a-phosphate (abbreviated [a32P] dTTP) plus other unlabeled nucleoside triphosphates, appropriate buffers and cofactors. The DNA synthesized in this reaction can be measured as the amount of 32P precipitated by acid (Figure 5.9A).

The DNA polymerases can be separated from other macromolecules in the crude cell extract by a series of steps. Most (but not all) enzymes are proteins, and the procedures used in enzyme purification are primarily methods for separating proteins. For example, a researcher may separate a mixture of proteins on a series of chromatographic columns to separate proteins by charge, then by size, and then by hydrophobicity. Each fraction from a chromatography column is assayed for the DNA polymerase; the aim is to separate the proteins with the desired activity from as many other proteins as possible with each column (Figure 5.9B). The fractionation procedures are continued until the enzyme is purified, which usually means that only one polypeptide (or set of polypeptides for a multi-subunit protein) is detected by gel electrophoresis. In principle, it should be possible to isolate enzymes that can carry out any process for which a reliable assay is available. However, several factors can make such purification difficult, such as very low abundance of the desired protein in the starting material, or the need for a multi-subunit complex to carry out a reaction, especially if such a complex is not very stable.

 

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B.

Figure 5.9. Biochemical assays for DNA polymerase.A.DNA synthesis can be assayed biochemically by the incorporation of a radioactive precursor, such as [a32P] dTTP, into acid-precipitable DNA as a function of time.B.Separation of a DNA polymerizing activity from other proteins by chromatography. Each fraction from a chromatographic column is assayed for total protein (e.g., the absorbance at 280 nm, gray line) and the ability to catalyze the incorporation of [a32P] dTTP into DNA (black line). In this hypothetical example, most of the proteins are in fractions other than the ones with the DNA polymerizing activity, and hence a substantial purification is achieved.

Many enzymes used in replication have been isolated by biochemical fractionation. These include not only the DNA polymerases, but also helicases, which unwind the parental DNA duplex to make two new templates, primase, which catalyzes the initial joining of nucleotides to start a DNA chain, DNA ligase, which joins fragments of DNA, and exonucleases, which can be used to remove incorrectly incorporated nucleotides. These and other enzymes have been used to reconstruct steps in DNA replication in the laboratory, and the activities described for these enzymes are used to build models for how replication can occur in living cells. Critical tests of such models can be made using genetic methods.

Genetic methods

Isolation and characterization of enzymes reveals proteins and RNAs that are capable of catalyzing reactions, and such activities can be used to postulate the events that occur in a biological pathway. Bi°Chemical fractionation and analysis are rich sources of insight into the chemical reactions within cells and cellular physiology. However, such results do not necessarily tell us whether an enzyme purified using its ability to catalyze a reaction in the test tube is actually used to catalyze that reaction inside the cell. Such a conclusion is best made with genetic evidence.

A genetic analysis begins with a screen or a selection for mutants that are defective in the process under investigation. Of course, cells that are no longer able to synthesize DNA will not grow, so we must isolate conditional mutants. You should recall from Chapter 1 that the product of a conditional allele retains function under a permissive culture condition (e.g., low temperature, 33°C), but it loses activity at a restrictiveculture condition (e.g., high temperature, 41°C). Other conditional mutants may be cold sensitive or salt sensitive. In the case of DNA synthesis, conditional mutants stop growing at the restrictive condition. Many temperature-sensitive mutants, i.e., those that do not grow at an elevated temperature such as 42 °C, were screened for the ability to synthesize DNA at the restrictive temperature. Such temperature-sensitive mutants in DNA synthesis were called dna mutants.

Once the conditional mutants have been isolated, they can be crossed to determine whether or not they complement at the restrictive temperature.  Results of this analysis allows the mutants to be placed into complementation groups, where each complementation group represents a gene whose product is required for DNA synthesis in growing cells. The genes represented by these complementation groups are called dnagenes, with each different gene given a different letter: dnaA, dnaB, etc. The aim of this genetic approach is to isolate a sufficiently large number of mutants such that at least one mutant is obtained in every gene needed for the process of interest, in this case DNA synthesis. If the genome were actually saturated with mutants, the number of complementation groups would be close to the number of genes encoding polypeptides carrying out the process under study. Studies in E. coli have revealed of the order of twenty dna genes. In order to find out what proteins and enzymatic activities are encoded by each of these genes, a method had to be developed to connect the genetically defined genes with a particular bi°Chemical activity. This is the subject of the next section.

Combining genetic and biochemical methods

The method of isolating dnagenes insures that their products are required for DNA synthesis. We would like to know exactly what enzymatic activity each gene encodes. For those activities for which a convenient in vitro assay is available, it is reasonably straightforward to find which mutants are defective in those activities at the restrictive condition. However, some dnagenes may encode a protein with an activity that is not expected or readily assayed. The proteins can still be isolated using the powerful approach of in vitro complementation. This technique allows the isolation of an enzyme simply from the knowledge that a gene needed for replication encodes it. Rather than assaying for a particular enzymatic activity, one assays for the ability of an extract or chromatographic fraction to restore DNA synthesis in an extract of a temperature-sensitive dnamutant at the restrictive temperature.

As illustrated in Figure 5.10 A, cell extracts of dnamutants will not synthesize DNA at 41o (the restrictive temperature), but addition of an extract of wild-type cells will restore DNA synthesis in vitro. This in vitrocomplementation assay can be used to purify the protein from wild type extracts, assaying fractions from chromatographic columns for the ability to complement extracts from the temperature sensitive (abbreviated ts) cells (Fig. 5.10 B). Many of the products of the dnagenes were isolated using this technique.

With this knowledge of the basic methods for identifying the enzymes needed for replication, we will proceed to a discussion of each of the major ones. We will cover DNA polymerases in considerable detail, whereas the other enzymes will be discussed less thoroughly.

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Figure 5.10. In vitro complementation to isolate enzymes needed for DNA replication. A. An extract of a temperature sensitive (ts) mutant defective in DNA synthesis cannot carry out this process at the restrictive temperature (dotted line). However, when a wild-type (wt) extract is added, synthesis is observed. This shows that the tsextract does not contain an inhibitor of synthesis, and thus a wild-type protein in vitro can complement it. B. The wild-type extract is fractionated by chromatographic methods, with each fraction assayed for the ability to restore in vitroDNA synthesis in the tsextract at the restrictive temperature. In this hypothetical illustration, the complementing activity (solid line) separates from many of the protein peaks (dotted line), showing a good purification at this step.