Enzymes that replicate DNA using a DNA template are called DNA polymerases. However, there are also enzymes that synthesize DNA using an RNA template (reverse transcriptases) and even enzymes that make DNA without using a template (terminal transferases). Most organisms have more than one type of DNA polymerase (for example, E. coli has five DNA polymerases), but all work by the same basic rules.
1. Polymerization occurs only 5' to 3'
2. Polymerization requires a template to copy: the complementary strand.
3. Polymerization requires 4 dNTPs: dATP, dGTP, dCTP, dTTP (TTP is sometimes not designated with a 'd' since there is no ribose containing equivalent)
4. Polymerization requires a pre-existing primer from which to extend. The primer is RNA in most organisms, but it can be DNA in some organisms; very rarely the primer is a protein in the case of certain viruses only.
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DNA Polymerase I from E. coli was the first DNA polymerase characterized. There are approximately 400 molecules of the enzyme per cell. E. coli DNA polymerase I is abbreviated pol I. The enzyme is a single large protein with a molecular weight of approximately 103 kDa (103,000 grams per mole). The enzyme requires a divalent cation (Mg++) for activity and has three enzymatic activities associated with it:
1. 5'-to-3' DNA Polymerase activity
2. 3'-to-5' exonuclease (Proofreading activity)
3. 5'-to-3' exonuclease (Nick translation activity)
The location of the three enzymatic activities
within the protein is known, and it is possible to remove the
5'-to-3' exonuclease activity using an enzyme called a protease
to cut DNA pol I into two protein fragments (see Klenow fragment
of DNA pol I in the Table above; both the polymerization and 3'-to-5'
exonuclease activities are on the large Klenow fragment of DNA
pol I, and the 5'-to-3' exonuclease activity is on the small fragment).
Like all known DNA polymerases, DNA polymerase I requires a primer
from which to initiate replication and polymerizes deoxyribonucleotides
into DNA in the 5' to 3' direction using the complementary strand
as a template. Newly synthesized DNA is covalently attached to
the primer, but only hydrogen-bonded to the template. The template
provides the specificity according to Watson-Crick base pairing
(Figure 24.2). Only
the alpha phosphate of the dNTP is incorporated into newly
synthesized DNA
The rate of DNA synthesis by pol I is only 20 nucleotides/second, much slower than the rate of 1,000 nucleotides/second measured for the replication of E. coli DNA. In 1969, John Cairns isolated a viable mutant (polA) lacking DNA polymerase I activity, an indication that polI is NOT the main enzyme used to replicate DNA. Interestingly, polA mutants are defective in repairing DNA damage.
DNA polymerase I also has two additional activities: a 3'-to-5' exonuclease activity and a 5'-to-3' exonuclease activity. The role of the 3'-to-5' exonuclease is to EDIT DNA (remove incorrectly polymerized nucleotides; see Figure 24.44). This was first discovered in "mutator" mutants of phage T4 DNA polymerase; these mutants had diminished 3'-to-5' exonuclease activity. "Antimutator" mutants had increased 3'-to-5' exonuclease activity. In general, the 3'-to-5' exonuclease increases the accuracy or fidelity of DNA synthesis by a factor of 10 to 1000. Thus, when the 5' to 3' polymerization of the enzyme accidentally puts the wrong base into DNA, the 3'- to 5' exonuclease proofreading activity immediately removes it. Thus errors due to incorporation of the wrong bases by the DNA polymerase are low because it must escape screening by two systems: the base pairing rules recognized by the 5' to 3' catalytic site as well as the 3' to 5' exonuclease proofreading site.
Why don't DNA polymerases elongate chains in the 3' to 5' direction?
IF a DNA polymerase could synthesize DNA in the 3' to 5' direction, then nucleotides would add to the primer terminus which has a 5'-triphosphate: (a) The 3'-OH of each incoming deoxyribonucleoside triphosphate would attack the 5'-triphosphate of the growing chain. (b) The editing removal of an incorrect 5'-terminal nucleoside triphosphate would prevent the DNA chain from being further extended because the primer terminus would now be a 5'-monophosphate, not a 5'-triphosphate.
Interestingly, E. coli temperature-sensitive (conditional lethal) mutants lacking the 5'-to-3' exonuclease activity of DNA polymerase I at non-permissive temperatures (40 degrees C) are NOT viable (remember, the polymerizing activity of DNA polymerase I is dispensible!). This enzymatic activity plays an essential role by removing RNA primers during replication. This process is similar to nick translation (here translation means "movement" not "protein synthesis"), the simultaneous polymerization in the 5'-to-3' direction from a nick with concurrent removal of nucleotides ahead by the 5'-to-3' exonuclease activity (Figure 24.5).
DNA sequencing with dideoxyribonucleotides (ddNTPs, chain terminators) is described in Figure G4. The following points should be noted:
The fastest method to sequence the entire genome of an organism involves sequencing of all possible randomly cloned genomic DNA fragments (Figure G6). This is called shotgun sequencing.
The polyerase chain reaction (PCR) is
a technique widely used in biotechnology to amplify trace amounts
of DNA. A few uses of PCR are: (a) amplification of desired genomic
DNA sequences with or without subsequent cloning, (b) direct sequencing
of any DNA without the need to clone the molecule, (c) medical
or criminal diagnostics (DNA fingerprinting), (d) mutating DNA
molecules, and (e) analysis of ancient DNAs. Kary
Mullis, the inventor of the technique was awarded the Nobel prize
for discovering the process. Since the introduction of the technique
in the late 1980s, the practice of molecular biology has been
markedly transformed. The PCR process is based on the inherent
doubling of material built into each round of DNA replication.
By annealing specific oligonucleotide primers to DNA and performing
multiple rounds of replication directed by the primers, desired
DNA sequences can be amplified millions of times in several hours.
Materials needed for PCR
1. DNA (typically genomic DNA)
2. Thermostable DNA polymerase (for example, Taq polymerase)
3. Primers (oligonucleotides complementary to specific sequences in DNA)
4. dATP, dCTP, dGTP, dTTP
Diagram of PCR
(Figure G7)
Limitations of PCR
1. Requires two primers
2. Amplifies only sequences between two primers. Surrounding
sequences are not amplified.
3. In vitro DNA replication is 100 to 10,000 fold more inaccurate
(error prone) than in vivo DNA replication.
Cloned eukaryotic genes cannot be expressed directly in prokaryotic organisms because prokaryotes do not have the ability to splice out introns (We will learn about introns latter). It is possible, however, to make a DNA copy of messenger RNA, a so called cDNA. A cloned cDNA can be expressed in bacteria if the cDNA is constructed next to a bacterial promoter. The synthesis of cDNA requires the enzyme reverse transcriptase. This enzyme has the very useful property of making DNA from an RNA template (as described above).
Eukaryotic messenger RNAs (mRNAs) are usually present in cells in low amounts, and must be purified to remove the more abundant ribosomal RNAs (rRNAs) and transfer RNAs (tRNAs). Most eukaryotic mRNAs are polyadenylated; that is, there is the post-transcriptional addition of several hundred A nucleotides to the 3'-ends of eukaryotic mRNA molecules. The polyA tail provides a "handle" to purify the mRNA. Total RNA is extracted from the eukaryotic cell and hybridized to oligo dT (short synthetic DNA made entirely of T nucleotides) that has been immobilized by chemical attachment to a support matrix (usually Sepharose beads) (Figure G8). The polyA tails of the mRNAs will base pair with the oligo dT, and "stick" the mRNAs to the column. The rRNAs and tRNAs have no long stretches of A that can base pair with the oligo dT, and therefore do not stick to the column. After the rRNAs and tRNAs are washed through the column, the purified mRNAs are finally removed by breaking the hydrogen bonding between the polyA and oligo dT.
Purified mRNAs can now be converted to cDNAs using reverse transcriptase and oligo dT as a primer (Figure G9). The oligo dT base pairs to the polyA tails of the mRNAs, and reverse transcriptase synthesizes a complementary DNA (cDNA) strand using the RNA as a template. The DNA/RNA duplexes are denatured with NaOH which also destroys the RNA. The single-stranded DNA can be converted to double-stranded DNA using random hexamer primers (synthetic DNA strands containing all possible combinations of six nucleotides) and DNA polymerase.
There are several directions that can be taken at this point in the procedure. The cDNA molecules can be cloned to create a cDNA library. A cDNA encoding a particular protein can be identified by screening the cDNA library with a "gene-specific" probe. Alternatively, the cDNA mixture can be amplified by PCR using "gene-specific" primers. The amplified cDNA product will encode a particular protein, and can be cloned for further recombinant DNA manipulations. This last method is called RT-PCR (reverse transcriptase mediated PCR).