Recombinant DNA technology- Restriction and modification enzymes -1

Recombinant DNA Technology (RDT) is the set of techniques that enables DNA from different sources to be identified, isolated and recombinant so that new character can be introduced into an organism. Nowadays, RDT has various purpose in genetic engineering like DNA cloning. The cutting and joining manipulations that underlie gene cloning are carried out by enzymes termed restriction endonucleases (for cutting) and ligases (for joining).

DNA manipulative enzymes can be grouped into four broad classes, depending on the type of reaction that they catalyse: 

• Nucleases are enzymes that cut, shorten, or degrade nucleic acid molecules. 
• Ligases join nucleic acid molecules together. 
• Polymerases make copies of molecules. 
• Modifying enzymes remove or add chemical groups.

Before considering in detail each of these classes of enzyme, two points should be made. The first point is that, although most enzymes can be assigned to a particular class, a few display multiple activities that span two or more classes. Most importantly, many polymerases combine their ability to make new DNA molecules with an associated DNA degradative (i.e. nuclease) activity.

Second, it should be appreciated that, as well as the DNA manipulative enzymes, many similar enzymes able to act on RNA are known. The ribonuclease used to remove contaminating RNA from DNA preparations is an example of such an enzyme.

Nucleases

There are two different kinds of nuclease: 

Exonucleases remove nucleotides one at a time from the end of a DNA molecule. Examples are;

• Bal31 (purified from the bacterium Alteromonas espejiana) is an example of an exonuclease that removes nucleotides from both strands of a double-stranded molecules. 
• E. coli exonuclease III degrade just one strand of a double-stranded molecule, leaving single-stranded DNA as the product

Endonucleases are able to break internal phosphodiester bonds within a DNA molecule. Examples are,

• S1 endonuclease (from the fungus Aspergillus oryzae) only cleaves single strands.
• Deoxyribonuclease I (DNase I), which is prepared from cow pancreas, cuts both single- and double-stranded molecules.
• Restriction endonuclease cleave at specific sites.

 Ligases

Within the cell, the function of DNA ligase is to repair single-stranded breaks (‘discontinuities’) that arise in double-stranded DNA molecules during, for example, DNA replication.

DNA ligases from most organisms can also join together two individual fragments of double-stranded DNA. The role of these enzymes in construction of recombinant DNA molecules.

 Polymerases

 DNA polymerases are enzymes that synthesize a new strand of DNA complementary to an existing DNA or RNA template. Most polymerases can function only if the template possesses a double-stranded region that acts as a primer for the initiation of polymerization. Four types of DNA polymerase are used routinely in genetic engineering : 

• The first is DNA polymerase I, which is usually prepared from E. coli. This enzyme attaches to a short, single-stranded region (or nick) in a mainly double-stranded DNA molecule, and then synthesizes a completely new strand, degrading the existing strand as it proceeds. DNA polymerase I is therefore an example of an enzyme with a dual activity, namely DNA polymerization and DNA degradation. 
• The polymerase and nuclease activities of DNA polymerase I are controlled by different parts of the enzyme molecule. The nuclease activity is contained in the first 323 amino acids of the polypeptide, so removal of this segment leaves a modified enzyme that retains the polymerase function but is unable to degrade DNA. This modified enzyme, called the Klenow fragment, can still synthesize a complementary DNA strand on a single-stranded template, but as it has no nuclease activity it cannot continue the synthesis once the nick is filled in.
• The Taq DNA polymerase used in the polymerase chain reaction (PCR) is the DNA polymerase I enzyme of the bacterium Thermus aquaticus. This organism lives in hot springs, and many of its enzymes – including the Taq DNA polymerase – are thermostable. This is the special feature of Taq DNA polymerase that makes it suitable for PCR.
• The final type of DNA polymerase that is important in genetic engineering is reverse transcriptase, an enzyme involved in the replication of several kinds of virus. Reverse transcriptase is unique in that it uses as a template not DNA but RNA. The ability of this enzyme to synthesize a DNA strand complementary to an RNA template is central to the technique called complementary DNA (cDNA) synthesis

DNA-modifying enzymes

Numerous enzymes exist that modify DNA molecules by the addition or removal of specific chemical groups. There are: 

• Alkaline phosphatase (from E. coli, calf intestinal tissue, or arctic shrimp), which removes the phosphate group present at the 5′ terminus of a DNA molecule.
• Polynucleotide kinase (from E. coli infected with T4 phage), which has the reverse effect to alkaline phosphatase, adding phosphate groups onto free 5′ termini.
• Terminal deoxynucleotidyl transferase (from calf thymus tissue), which adds one or more deoxyribonucleotides onto the 3′ terminus of a DNA molecule.