100
human- made artificial systems. Almost all enzymes are proteins with the exception
of ribozymes or RNAzymes, which are RNA molecules that are capable of catalyzing specific biological reactions. Molecular biology has benefited more from
enzymes than any other field in biosciences. Of various enzymes used in molecular
biology, restriction endonucleases are of utmost significance. These bacterial
enzymes serve as defense against bacteriophages by cleaving the phage DNA while
the host DNA is protected due to methylation. Type II restriction enzymes are the
most important among restriction enzymes, because of their capability to cut DNA
molecules at specific sites known as restriction sites. The discovery of restriction
endonucleases laid the foundation stone for rDNA technology. A vast number of
restriction enzymes from varying sources of bacteria are now available commercially. Danna and Nathans (1971) carried out the pioneer work in demonstrating the
possibilities of restriction enzymes in molecular biology, which paved the way for
the modern field of molecular biology as we see it today. Molecular biology currently employs a wide array of enzymes that are involved in the manipulation of
DNA, RNA, and protein molecules. In this current scenario, one must be familiar
with the specific properties and utilities of each enzyme to choose the best one for
application in a particular technique. Particulars regarding the most preferred
enzymes and their characteristics are discussed here.
4.2
DNA-Dependent DNA Polymerases
DNA polymerases are enzymes that catalyze the synthesis of DNA molecules from
their monomer units, deoxyribonucleotides. These enzymes are essential for DNA
replication and usually work in pairs to create two identical DNA strands from one
original DNA molecule. They are responsible for duplication and passing of genetic
data over generations. During cell division, DNA polymerases carry out semiconservative replication of DNA, where a daughter DNA strand is synthesized from
a single-stranded parent DNA template using four deoxyribonucleotides triphosphate monomers (dTTP, dCTP, dGTP, dATP) as precursors. DNA polymerases are
unable to carry out de novo synthesis of a polynucleotide molecule and can only
incorporate nucleotides to an existing 3′-OH end of a primer. DNA polymerization
occurs only in the 5′ → 3′ direction and each deoxynucleotide added are complementary to the one present on the template strand and thus the genetic information
is passed through generations. In addition, many DNA polymerases show proofreading via 3′ → 5′ exonuclease activity. If an error occurs during DNA synthesis,
the misincorporated nucleotide is excised out as a monophosphate, and the correct
base is introduced by the polymerase. Another catalytic property of DNA polymerases is its 5′ → 3′ exonuclease, nick translation activity which is required for the
elimination of RNA primers during replication. Some DNA polymerases possess
terminal transferase activity by which a single nucleotide (usually adenine) is added
to the 3′ termini of PCR products.
The type and number of DNA polymerases vary among different organisms.
They are all grouped together into A, B, C, X, and Y families based on amino acid
G. Valsala and S. Sugathan
human- made artificial systems. Almost all enzymes are proteins with the exception
of ribozymes or RNAzymes, which are RNA molecules that are capable of catalyzing specific biological reactions. Molecular biology has benefited more from
enzymes than any other field in biosciences. Of various enzymes used in molecular
biology, restriction endonucleases are of utmost significance. These bacterial
enzymes serve as defense against bacteriophages by cleaving the phage DNA while
the host DNA is protected due to methylation. Type II restriction enzymes are the
most important among restriction enzymes, because of their capability to cut DNA
molecules at specific sites known as restriction sites. The discovery of restriction
endonucleases laid the foundation stone for rDNA technology. A vast number of
restriction enzymes from varying sources of bacteria are now available commercially. Danna and Nathans (1971) carried out the pioneer work in demonstrating the
possibilities of restriction enzymes in molecular biology, which paved the way for
the modern field of molecular biology as we see it today. Molecular biology currently employs a wide array of enzymes that are involved in the manipulation of
DNA, RNA, and protein molecules. In this current scenario, one must be familiar
with the specific properties and utilities of each enzyme to choose the best one for
application in a particular technique. Particulars regarding the most preferred
enzymes and their characteristics are discussed here.
4.2
DNA-Dependent DNA Polymerases
DNA polymerases are enzymes that catalyze the synthesis of DNA molecules from
their monomer units, deoxyribonucleotides. These enzymes are essential for DNA
replication and usually work in pairs to create two identical DNA strands from one
original DNA molecule. They are responsible for duplication and passing of genetic
data over generations. During cell division, DNA polymerases carry out semiconservative replication of DNA, where a daughter DNA strand is synthesized from
a single-stranded parent DNA template using four deoxyribonucleotides triphosphate monomers (dTTP, dCTP, dGTP, dATP) as precursors. DNA polymerases are
unable to carry out de novo synthesis of a polynucleotide molecule and can only
incorporate nucleotides to an existing 3′-OH end of a primer. DNA polymerization
occurs only in the 5′ → 3′ direction and each deoxynucleotide added are complementary to the one present on the template strand and thus the genetic information
is passed through generations. In addition, many DNA polymerases show proofreading via 3′ → 5′ exonuclease activity. If an error occurs during DNA synthesis,
the misincorporated nucleotide is excised out as a monophosphate, and the correct
base is introduced by the polymerase. Another catalytic property of DNA polymerases is its 5′ → 3′ exonuclease, nick translation activity which is required for the
elimination of RNA primers during replication. Some DNA polymerases possess
terminal transferase activity by which a single nucleotide (usually adenine) is added
to the 3′ termini of PCR products.
The type and number of DNA polymerases vary among different organisms.
They are all grouped together into A, B, C, X, and Y families based on amino acid
G. Valsala and S. Sugathan