DAIRY BIOTECHNOLOGY

Module 2. Fundamental biological principles

Lesson 5
DNA REPLICATION AND GENETIC CODE

5.1 Introduction

During the cell division the hereditary information is transferred from parent cell to the daughter cell by the faithful duplication of parental DNA molecule. This process of duplication of DNA is called replication. Three possible modes of DNA replication have been proposed which include conservative, semiconservative and dispersive replication (Fig. 5.1). However, semi conservative mode of replication was experimentally proved by Meselson and Stahl in 1958 which perfectly fits into the double helical model of DNA as proposed by Watson and Crick. In this mode of replication two strands of DNA are separated and each one acts as a template for the synthesis of daughter strand. The first generation progeny DNA produced after replication will constitute one parental and one newly synthesized DNA strand.

The process of replication of DNA is described as below:

5.2 Components of DNA Replication Machinery

Several enzymes, protein factors and ATP molecules are required during the replication process of DNA.

5.2.1 Template

Double stranded DNA is used as template in DNA replication. It is generally circular in prokaryotes and linear in eukaryotes.

5.2.2 Enzymes

The key enzymes that take part in DNA replication are as follows:

DNA helicases bind to the double stranded DNA and catalyze the separation of the two strands.

RNA Primase synthesizes short RNA primers that provide free 3′ hydroxyl group for the action of DNA polymerases.

DNA polymerase polymerizes the newly synthesized DNA strand. There are two main DNA polymerases i.e. DNA polymerase-I and DNA polymerase-III that participate in the replication process. DNA polymerase-III a multi subunit complex is the main replicative enzyme that polymerizes DNA in 5′ to 3′ direction by its 5′ - 3′ polymerization activity and proof reads the newly synthesized DNA by its 3′ - 5′ exonuclease activity. DNA polymerase-I catalyses the filling of gaps created during the RNA primer removal.

The DNA gyrase enzyme catalyzes the formation of negative supercoils that helps in the unwinding process to separate the two strands of the duplex DNA.

DNA Ligase catalyses the formation of phosphodiester bonds between the DNA fragments and fills the gaps.

5.2.3 Deoxyribo nucleotide triphosphates (dNTPs)

Four dNTPs i.e. dATP, dCTP, dGTP and dTTP are used as precursors by DNA polymerases to extend the growing chain of DNA.

5.2.4 Single strand binding proteins

Single strand binding proteins stabilize the single stranded structure of DNA by preventing the folding of the single strand of DNA on itself. They also protect the single strand DNA from nucleases.

5.3 DNA Replication Process

DNA replication is a bidirectional process that starts from a specific site on the DNA molecule known as origin of replication. There is a single of origin of replication in prokaryotic DNA (Fig. 5.2A) whereas eukaryotic genomes replicate through multiple origins of replications.

The process of replication involves the following steps:

DNA helicases bind the DNA duplex at the region of replication and leads to unwinding of DNA molecule and in the process creates replication fork. The replication fork is further extended by DNA helicases in opposite directions resulting in twists of DNA molecule offering resistance to the unwinding process at the unopened regions. This super coiling problem is solved with the help of enzymes called DNA topoisomerases, which create nicks in the DNA molecule to release the twisting or coiling of the DNA molecule as the replication process proceeds forward. The separated single strands have the tendency to fold back and form secondary structures. To prevent this, the single strand DNA binding proteins attach to the single strands and allow the replication process to continue without any constraint.

A short strand of RNA is synthesized by RNA primase using one of the strands as template, which provides a free 3′ hydroxyl group for the synthesis of DNA strand. RNA primers are required during replication because DNA polymerases cannot initiate DNA synthesis de novo i.e. without a pre formed substrate. Once RNA primers are synthesized DNA polymerase-III is loaded on to the single strand. It extends RNA primers adding nucleotides to the 3′ hydroxyl group of growing DNA strand using information available in the form of nucleotide sequence in DNA template. Deoxyribonucleotide triphosphates are first paired with their corresponding, complementary partner on the original, or template strand followed by formation of phosphodiester bond with the previously added nucleotide on the growing DNA strand. The specific base pairing as per the Watson and Crick model is strictly followed during DNA replication. Adenine (A) is always paired with Thymine (T) and Guanine (G) with Cytosine (C).

One strand of DNA is continuously synthesized in 5′ to 3′ direction using the DNA template having 3′ - 5′ polarity. This is called leading strand. The other strand being anti-parallel i.e. having 5′ - 3′ polarity, has to be synthesized in short stretches of ~1000 bases called Okazaki fragments. This discontinuously synthesized strand is called lagging strand (Fig 5.3).

After completion of the replication of the whole genome it is processed to remove RNA primers and replace it with the complimentary missing DNA bases. RNA primers are removed by the 5' to 3' exonuclease activity of DNA Polymerase-I. The gaps thus created are filled by incorporating the deoxyribonucleotides by 5' - 3' polymerization activity of DNA polymerase-I. Finally DNA ligase closes the nicks by the formation of phosphodiester bonds between Okazaki fragments with the hydrolysis of ATP molecule.

5.4 Genetic Code

Genetic code is defined as the set of rules by which information is encoded on the genetic material. Precisely it is ordering of nucleotides in DNA molecules to incorporate amino acids during protein synthesis in correct order to make it biologically active to express a specific function in the cell. It reflects the relationship between the nucleotide triplets called codons located on a mRNA molecule and the 20 amino acids which are the building blocks of proteins. These triplet codons formed from adjacent nucleotides on mRNA molecule represent different amino acids with few exceptions and are made up of 4 different nitrogen bases namely adenine, guanine, cytosine and uracil.

There are 20 amino acids involved in the synthesis of proteins. Each amino acid is coded by one or more codons. Hence, there is a need of at least twenty different codons for synthesis of all the amino acids. Moreover, the start and stop signals in the synthesis of polypeptide chain are also coded by different codons. Since each codon is made of three nucleotides, the minimum possible number of codons by utilizing four available nitrogen bases could be 64 (4x4x4).

Salient features of genetic code:

• It is nearly universal code as it is used by all biological systems

• Sixty-one codons code for 20 amino acids

• Genetic code is degenerate due to redundancy of the codons. Many of the amino acids have more than one codon (redundancy) but no single codon codes for more than one amino acid (no ambiguity). For example phenylalanine is coded by two codons (UUU and UUC) while Leucine is coded by six codons (UUA, UUG, CUU, CUC, CUA and CUG). However, tryptophan (UGG) and methionine (AUG) are coded by only a single codon.

• The first codon for initiating the protein synthesis is always AUG (start or initiation codon) and codes for methionine

• UAG, UAA, and UGA are the Stop codons for termination of protein synthesis

All the possible codons constituting the genetic code and their corresponding amino acids are listed in Table 5.1.

Table 5.1 Triplet codons

5.4.1 Anticodon

Anti-codon comprises of a sequence of 3 bases that can base pair with a codon sequence in the mRNA. Anticodon is present on one of the four regions of tRNA and help in the incorporation of correct amino acid in the growing polypeptide chain.

Further Reading

Books

Berg J, Tymoczko JL, Stryer L (2006). Biochemistry (6th ed.). San Francisco: W. H. Freeman. ISBN 0716787245

Fundamental Bacterial Genetics, Nancy Trun, Janine Trempy (Eds), Wiley-Blackwell, 2003, ISBN: 978-0-632-04448-1

Molecular Biology of the Gene, Sixth Edition, James D. Watson (Editor) Cold Spring Harbour Press and Benjamin Cummings, ISBN 978-080539592-1

Molecular Biotechnology - Second Edition, S. B. Primrose, Blackwell Science Inc., ASIN: 0632030534

DNA and Biotechnology, Fitzgerald-Hayes, M. And Reichsman, F. 2^nd Amsterdam : Elsevier, 2010. ISBN : 0-12-048930-5

Internet Resources

http://en.wikipedia.org/wiki/DNA_replication

http://en.wikipedia.org/wiki/Genetic_code