The Replication of DNA

Introduction

Deoxyribonucleic acid (DNA) is a nucleic acid. It is the genetic material constituting the genes, which in eukaryotic cells are found in chromosomes. The genes control the traits of living organisms. DNA contains the genetic code which is present in the form of the sequence of nitrogenous bases found within in the molecule. It is responsible for coding the structure of proteins and as a result for determining much of the structure of our bodies and by determining the structure of the class of proteins known as enzymes, controlling metabolic reactions in living organisms.

Components of DNA

DNA is composed of four nitrogenous bases: Adenine, Thymine, Cytosine, and Guanine.

The 5-carbon sugar in DNA is called Deoxyribose.

DNA also contains phosphate groups.

Note that Adenine and Guanine are large double-ring molecules. These are known as purines. Cytosine and Thymine are smaller single-ring molecules known as pyrimidines.

DNA is composed of basic units known as nucleotides. A nucleotide consists of a nitrogenous base, joined to a 5-carbon sugar, joined to a phosphate group.

In the deoxyribose sugar of DNA the carbon atoms are numbered 1′ to 5′ proceeding clockwise from the oxygen atom.

The DNA molecule is composed of two strands, wound around one another in a helix, this structure is known as the double helix.

Imagine that the helical molecule is flattened out. If an analogy is made between DNA and a ladder, the outer rails of the ladder are composed of alternating units of deoxyribose sugar and phosphate. The sugar and phosphate units are joined by phosphodiester bonds. The rungs of the ladder consist of pairs of nitrogenous bases, which are joined to one another by hydrogen bonds.

Hydrogen bond- a bond between a positively-charged hydrogen atom and a negatively-charged oxygen or nitrogen atom.

Importance of Hydrogen bonds being weak bonds: Because the hydrogen bonds holding the two strands of DNA are weak bonds, it allows the two strands of DNA to separate easily. This must happen when the DNA molecule is replicated. The two strands separate and a new complementary strand of DNA is built upon each unwound strand. The two strands must also separate when DNA is transcribed and a molecule of messenger RNA is built upon one of the unwound strands.

In DNA the nitrogenous base Adenine is joined to Thymine and Cytosine is joined to Guanine. (It can be Thymine first then Adenine or Guanine first then Cytosine, as long as Adenine is together with Thymine, and Cytosine is together with Guanine.)

The pairing relationship between Adenine and Thymine and Cytosine and Guanine is known as the base pairing rule.

Note that when the bases pair, a large double-ring purine is always connected to a smaller single-ring pyrimidine. As a result, the distance across the DNA molecule between the two strands is always consistent. If two small pyrimidines, for example Thymine and Cytosine were connected together, the distance between the two strands would be shorter than it should be. If two large purines, for example Adenine and Guanine were connected together would be longer than it should be. In either case the structure of the molecule would be compromised.

Note that in the DNA molecule, if we know the sequence of bases along one side of the molecule we can automatically predict the sequence of the bases on the other side. The sequence of bases on the opposite strand is not the same as in the first strand, instead it is said to be complementary. For example, if the sequence of bases on one side is ATCTGC, the sequence on the complementary strand would be TAGACG.

DNA contains genetic information that is used to determine the structure of RNA and Proteins and to replicate DNA. The information in DNA consists of the sequence of nitrogenous bases found along the length of the molecule.

Comparison of the two strands in the DNA molecule reveals that they are running in opposite directions. That is they are antiparallel. One strand is running in a 5′ to 3′ direction and the other strand is running in a 3′ to 5′ direction.

Francis Crick codified the key functions of DNA in a concept he called the Central Dogma illustrated by the following diagram:

Replication is the process in which the two strands comprising a DNA molecule unwind and a new complementary strand of DNA is built upon each unwound strand.

Transcription is the process in which the molecule of DNA unwinds and a complementary molecule of messenger RNA is produced upon one of the unwound strands.

Translation is the assembly of a protein on the ribosomes using the genetic code in mRNA to specify the order of amino acids.

RIBONUCLEIC ACID

Ribonucleic acid (RNA) is a nucleic acid that helps DNA to construct a protein.

RNA is a single-stranded molecule. It contains the following components:

1. Nitrogenous bases

RNA contains the nitrogenous bases adenine, uracil, cytosine, and guanine. Note that in RNA, the nitrogenous base uracil replaces the thymine found in DNA.

2. 5-carbon sugar

The 5-carbon sugar found in RNA is ribose (not Deoxyribose as in DNA).

3. Phosphate group

Types of RNA

There are 3 types of RNA:

1. Messenger RNA- carries the code for the construction of a protein from the DNA in the nucleus to the ribosomes in the cytoplasm where the protein is made.

2. Transfer RNA- picks up amino acids in the cytoplasm and transfers them to the ribosomes.

3. Ribosomal RNA- makes up the structure of the ribosomes.

REPLICATION OF DNA

Replication is the process in which a molecule of DNA unwinds and a new complementary strand of DNA is built upon each unwound strand.

DNA replication can be described as Semi-conservative; that is each newly formed double-stranded DNA molecule contains one strand conserved intact from the parent molecule and one newly built complementary strand.

The enzyme DNA Polymerase is needed for the replication of DNA. During replication it links free nucleotides as they line up on the template formed by the original strand of the parent molecule.

The Meselson-Stahl Experiment

Watson and Crick found that the two strands of DNA were complementary. Based on this finding they predicted a copying mechanism for DNA. The predicted that replication of DNA produces two daughter duplex DNA molecules, each of which contains one strand from the parental DNA and one new strand. This process is called semiconservative replication. Meselson and Stahl confirmed that DNA replication is semiconservative in an experiment they conducted in 1958. At this time, biologists were considering three possible mechanisms by which two complementary strands of the parental duplex DNA could yield daughter DNA duplexes identical to those of the parent. In addition to semiconservative replication mechanism postulated by Watson and Crick, two other schemes, conservative and dispersive replication had to be considered.

In conservative replication of DNA, each of the two strands of the parent DNA is replicated to yield the unchanged parent DNA and a newly synthesized DNA. The F2 generation consists of the parental DNA and three new DNAs. In semiconservative replication of DNA, each F1 duplex contains one parent strand. The F2 generation consists of two hybrid DNAs and two totally new DNAs. In dispersive replication, the parent chains break at intervals, and the parental segments combine with new segments to form the daughter strands.

In order to determine which mechanism was responsible for the replication of DNA, Meselson and Stahl grew E. coli cell in a medium in which the sole nitrogen source is ammonium chloride, whose nitrogen was nearly pure 15N, the “heavy” nitrogen isotope, instead of the normal N14. The 15N-DNA of these cells (heavy DNA) has a significantly greater density than normal 14N-DNA; the heavy and light forms of DNA can be separated by equilibrium centrifugation in a cesium chloride density gradient. To separate DNA based on density, a cesium chloride density gradient is prepared. This is done by mixing DNA with CsCl and centrifuging it at very high speeds (e.g., 50,000 rpm) in an ultracentrifuge for many hours. Centrifugation produces a stable, linear gradient of CsCl with the lightest density at the top and the heaviest density at the bottom. Within the CsCl gradient, the DNA comes to equilibrium in the gradient where its density equals the density of the surrounding CsCl.

Meselson and Stahl carried out the crucial experiment on the mechanism of DNA replication in the following way. The growth of the “heavy” 15N-containing E. coli cells was continued by placing them in a “light” medium, which contained normal 14N-NH4Cl as the sole nitrogen source. The cells were allowed to grow for several generations and samples were harvested at intervals. From these samples the DNA was extracted and its buoyant density determined by centrifugation in CsCl density gradients.

The experimental group was compared to a control group which was grown on a medium which contains the normal nitrogen source (14N).

Control tubes and experimental tubes were examined and the location of DNA containing bands was determined. The number of bands, their relative position, and composition with respect to 15N and 14N was determined.

If DNA replication is semiconservative, after exactly one generation time in the 14N medium, which yields doubling of the number of cells, the DNA isolated should show but a single band in the density gradient, midway in density between the normal or light 14N-DNA and the heavy 15N-DNA of cells grown exclusively on 15N.

Results of the experiment. (Left) The positions of the DNA bands after density – gradient centrifugation to equilibrium. (Right) The forms of DNA in controls and after three cell divisions in the 14N-containing medium. “Heavy” refers to 15N-DNA; “light” refers to 14N-DNA. The results conclusively support semiconservative replication.

This result is what might be expected if the duplex DNA of the daughter cells contained one 15N strand and one newly synthesized 14N strand. After two generations on 14N-NH4Cl, the DNA isolated exhibited two bands, one having a density equal to that of normal light DNA and the other equal to that of the hybrid DNA observed after one generation. These results, are exactly those expected from the hypothesis of semiconservative replication proposed by Watson and Crick, but they are not consistent with the alternative hypotheses of conservative or dispersive replication.

DNA Replication Requires a Template, Nucleotides, and a Polymerase Enzyme

DNA polymerase matches the existing DNA bases with complementary nucleotides and then links the nucleotides together to make the new strand. DNA polymerases add new bases to the 3′end of existing strands. That is, they synthesize in a 5′to 3′direction. DNA polymerases also require a primer to begin synthesis. They cannot begin synthesizing without a strand of RNA or DNA base-paired to the template. RNA polymerases do not require a primer, so they usually synthesize the primers.

Replication is Continuous on the Leading Strand, Discontinuous on the Lagging Strand

Recall that the two strands in a DNA molecule are antiparallel; one of the strands runs in a 5′to 3′ direction and the other strand runs in a 3′to 5′ direction. Because of this, the polymerase that is working on one strand must be moving in the opposite direction to the polymerase working on the opposite strand. As the DNA molecule is opened up and replication begins, an RNA primer is added to the end of the leading strand and new bases are added to the 3′ end of the existing bases to extend the strand. Because synthesis in the 5′to 3′direction is proceeding in the direction toward the opening replication fork, synthesis in this direction is continuous. New bases are continually added as the DNA is unwound. However, this situation does not exist on the lagging strand. As this strand is opened up, multiple primers must be added as opening proceeds. Because the DNA polymerase can only synthesize in a 5′ to 3′ direction, synthesis has to proceed in a direction away from the opening fork; that is synthesis is discontinuous on this strand. Synthesis on this strand must be delayed until a sufficiently long stretch of the lagging strand is opened up. Synthesis then progresses from the primers, producing short fragments of the new complementary strand known as Okazaki fragments.

DNA, RNA, and Protein Synthesis

The information in the DNA molecule (sequence of codons) specifies the sequence of amino acids in polypeptides. In a eukaryotic cell, proteins are made only in the cytoplasm. The sites of protein synthesis are the ribosomes, tiny granules composed of RNA and protein. During protein synthesis DNA stays in the nucleus. In order for the information to get from the nucleus to the cytoplasm where proteins are made, DNA must first transfer its information to a molecule of messenger RNA. This molecule then carries the information from the nucleus to the ribosomes.

Transcription is the process in which a molecule of DNA unwinds and a molecule of mRNA is produced on one of the unwound strands.

The Genetic Code

A codon is a set of three consecutive nucleotides that codes for a single amino acid.

An anticodon is a set of three unpaired bases on a tRNA molecule that binds to a complementary codon on mRNA

Protein Synthesis

Translation is the assembly of a protein on the ribosomes using the genetic code in mRNA to specify the order of amino acids.

Codon-Anticodon Bonding

The key to protein synthesis is codon-anticodon binding. Codons are sequences of three consecutive nitrogenous bases on a messenger RNA molecule. Each three-base sequence is a code that specifies either a start signal, a stop signal, or codes for a particular amino acid in a protein. During the synthesis of a protein, a codon in mRNA determine the placement of particular amino acid in the protein by joining with a complementary 3-base sequence on a transfer RNA molecule known as an anticodon. For example, if the codon is UGG, the complementary anticodon is ACC. This anticodon is found on the transfer RNA molecule that is carrying the amino acid leucine. In this way, the codon on the messenger RNA specifies that leucine be placed in that specific location in the polypeptide that is being constructed.

Proteins are composed of long chains of amino acids. There are 20 different kinds of amino acids. The amino acids of a polypeptide are joined together by peptide bonds.

The genetic information in the DNA molecule, which is in the form of the sequence of nitrogenous bases along the length of the molecule, specifies of the sequence of amino acids that are found along the length of the polypeptide chain. DNA directs the synthesis of proteins.

Overview of Protein Synthesis

During protein synthesis in the Eukaryotic Cell, DNA remains in the nucleus. However, the proteins are constructed outside the nucleus in the cytoplasm of the cell at the ribosomes. Somehow the genetic information that is found in the DNA in the nucleus must travel out of the nucleus to the ribosomes so that it can direct the synthesis of the protein. In order to accomplish this, the DNA molecule must first be transcribed, that is, the code for the construction of a protein that is found within the DNA molecule must be transferred to a messenger RNA molecule. In the process of transcription, the DNA builds a molecule of messenger RNA. The molecule of DNA unwinds and a complementary strand of messenger RNA is built upon one of the unwound strands. In this way, the code for the construction of a protein is transferred from the DNA, to a messenger— a molecule of messenger RNA. The messenger RNA then leaves the nucleus through the large pores in the nuclear membrane and goes out into the cytoplasm, carrying the code for the construction of a protein from the DNA in the nucleus to the ribosomes where the protein is made.

Transcription

In the process of transcription, the two strands of DNA unwind. On one of the strands, called the template strand, a complementary strand of messenger RNA is produced. The enzyme RNA Polymerase is necessary for the transcription of DNA. The synthesis of the messenger RNA molecule is directed by the base-pairing rule, however, in the mRNA molecule the nitrogenous base uracil replaces the thymine found in DNA and the 5-carbon sugar in mRNA is ribose rather than deoxyribose.

Codons

A codon is a sequence of three nitrogenous bases on a messenger RNA molecule. A codon can specify a “start" signal, code for an amino acid, or specify a “stop” signal. The codons as well as the amino acids they code for and the “start” and “stop” signals are listed in the Genetic Code table below.

In the genetic code, there are 43 = 64 possible codons. Three of these 64 mRNA codons (UAA, UAG, and UGA) are stop codons. One of the codons, AUG codes for the amino acid methionine in eukaryotic cells and also serves as a “start” signal. These codons terminate the translation of a polypeptide by causing a protein called a release factor to join to the mRNA on the ribosome. When this happens, the completed polypeptide is released. This would leave 61 codons available for coding for amino acids. Since there are only 20 amino acids, there are a lot of codons left over. This means the code is degenerate, meaning that some codons code for more than one codon. If the Genetic Code table listing the codons is examined, it can be seen that most of the codons can code for more than one amino acid. The first two bases in the 3-base codon are specific for a particular amino acid. However, less precise base pairing can occur between the 3rd base of the codon and the 1st base of the anticodon. This is called wobble base pairing. As a result, multiple codons can code for a single amino acid.

The Genetic Code

Transfer RNA

Transfer RNA molecules pick up amino acids in the cytoplasm and bring them to the mRNA at the ribosome. Originally, it was believed that there were 20 different kinds of amino acids, one for each of the 20 different amino acids. Because the genetic code contains multiple codons that specify the same amino acid, there are several tRNA molecules bearing different anticodons which carry the same amino acid. As mentioned, there are 64 possible codons. Eliminating the three codons that code for “stop signals” rather than amino acids (AUG codes for a “start signal” but also codes for the amino acid methionine) that leaves 61 codons. However, because of wobble base pairing, multiple codons can code for a single amino acid. In order to recognize 61 codons, a minimum of 31 tRNAs is required. The maximum number of tRNAs observed is 41.

Protein synthesis involves four general steps:

1. Amino acid activation

2. Initiation

3. Polypeptide elongation

4. Termination

Amino Acid Activation

Before synthesis actually begins, amino-acid activation takes place; amino acids are activated or joined to tRNAs, by high energy bonds. The energy for this reaction comes from ATP. In the first step of the reaction, the amino acid reacts with ATP forming an intermediate with AMP attached to the carboxyl end of the amino acid. The two terminal phosphates (pyrophosphates) are removed from ATP during the reaction. The tRNA then binds to the enzyme holding its amino acid-AMP complex. In the next step of the reaction, the amino acid is transferred from AMP to the tRNA and AMP is released. This produces a charged tRNA consisting of a specific amino acid attached to the 3′acceptor stem of its RNA.

Initiation

Next, initiation of protein synthesis in prokaryotic cells occurs when a ribosome, a mRNA, and an initiator tRNA bearing N-formylmethionine bind together. First, a small ribosomal unit attaches to the initiator tRNAfMet. The small ribosomal unit which is carrying the initiator tRNAfMet then attaches to messenger RNA. The coding information in the mRNA is read off in groups of three nucleotides each. Each group of three nucleotides is called a codon. The first group of three nucleotides is AUG. This is a start codon that codes for the first amino acid in the protein methionine. In eukaryotic cells methionine is always the first amino acid in a polypeptide, although during later processing of the protein it may be removed. In prokaryotic cells, the first amino acid is a chemically modified methionine, N-formylmethionine. Notice that the codon AUG on the mRNA molecule codes for the complementary codon UAC which is found on the tRNAfMet molecule. In addition to the initiator tRNAfMet, the initiation complex also includes the small ribosomal subunit and the mRNA strand. Initiation factors assist in positioning the small ribosomal subunit, the initiator tRNAfMet, and the mRNA. When these units are in place, the large ribosomal subunit attaches to the mRNA molecule. The ribosome contains three binding sites: a P site, an A site, and an E site. The P site (peptidyl) binds to the tRNA attached to the growing peptide chain. The A site (aminoacyl) binds to the tRNA carrying the next amino acid to be added. The E site (exit) binds the tRNA that carried the previous amino acid added. At the time of initiation the initiator tRNA carrying N-formylmethionine is occupying the P site with the A site empty.

Elongation

Elongation takes place as the code in the mRNA molecule is read out and amino acids are added one by one to the growing polypeptide chain. Elongation begins when a new charged tRNA molecule is brought to the ribosome bound to the initiator tRNA and messenger RNA. The initiator tRNAfMet is occupying the P site and the A site is empty. The next codon on the mRNA then codes for the next amino acid to be positioned in the polypeptide. It does this by binding to the complementary anticodon on a tRNA molecule that is carrying the amino acid specified by the codon. For example, suppose the next codon is CUG. Referring to the genetic code shows that the amino acid specified by this codon is leucine. The codon on the messenger RNA molecule CUG, would code for the complementary codon GAC on the incoming tRNA molecule carrying leucine. As it enters the A site, leucine is positioned next to N-formylmethionine. The enzyme peptidyl transferase then breaks the bond between the first amino acid, N-formylmethionine, and its tRNA and forms a peptide bond immediately between the N-formylmethionine and the second amino acid (leucine). This results in the transfer of the growing chain to the tRNA in the A site. After the peptide bond has been formed the ribosome moves to the next codon. The next codon is now in the A site, and the tRNA with the growing chain moves to the P site. The uncharged tRNA formerly in the P site is now in the E site, and it will be ejected in the next cycle. This process is repeated over and over until the polypeptide chain is completed and a stop codon is reached during the termination step.

Termination

Elongation continues until a stop codon is reached. Instead a new tRNA carrying an amino acid being put into place, a protein molecule called a release factor is attached. The release factor binds to the stop codon and causes the release of the completed polypeptide. The ribosomal subunits dissociate from the mRNA.

Study Guide DNA The Structure and Function of the Double Helix Draft 5

Corrected 12/22/2018

1