G.E. ToolsAmritha m. s2010-09-115

DNA POLYMERASES

DNA polymerases are enzymes that catalyze the formation of polymers made by the assembly of multiple structural units (dNTPs).

None of the DNA polymerases can direct de novo synthesis of a poly nucleotidic molecule from individual nucleotides.

The DNA polymerases only add nucleotides to the 3’--OH end of a pre-existing primer containing a 5’-phosphate group.

Most polymerases are originate from bacteria or their infecting viruses (bacteriophages or phages).

RibozymesRNA molecules that act as

enzymes are called ribozymes.  This property of some RNAs was discovered by

Sidney Altman and Thomas Czech, who were awarded the

Nobel Prize in Chemistry in 1989.  

Sidney AltmanThomas Czech

RibozymesRNA molecules capable of catalyzing biochemical

reactions Earliest known examples:

RNase PGroup I and II intronsRibosomeshammerhead ribozymes

Principal reactions:RNA transesterificationRNA cleavage (hydrolysis of phosphodiester bonds)

Substrate aligned into the active site using a guide sequence which is complimentary to the substrate

All ribozymes depend absolutely on the assumption of correct 3-dimensional structure for activity

Transesterification

Guide sequence

Target sequence

Cleavage

site

Transesterification is the process in which an ester group is exchanged with that of another, alcohol to form a new ester.

RNA cleavage at alkaline pH

alkaline pH

RNA undergoes spontaneous hydrolytic cleavage about one hundred times faster than DNA. This is believed due to intramolecular attack of the 2'-hydroxyl group on the neighboring phosphate diester, yielding a 2',3'-cyclic phosphate

Major types of endoribonucleases•RNase A is an RNase that is commonly used in research. •RNase H is a ribonuclease that cleaves the RNA in a DNA/RNA duplex •RNase III is a type of ribonuclease that cleaves rRNA (16s rRNA and 23s rRNA) •RNase L is an interferon-induced nuclease which, destroys all RNA within the cell

RNase P is a ribozyme – a ribonucleic acid that acts as a catalyst. Its function is to cleave off an extra on tRNA molecules•RNase PhyM is sequence specific for single-stranded RNAs. •RNase T1 is sequence specific for single-stranded RNAs.

RNase P

Ribosome is a Ribozymes

The three-dimensional structure of the large (50S) subunit shows that formation of the peptide bond is catalyzed by the 23S RNA (& 28S RNA) molecule in the large subunit. The 31 proteins in the subunit probably provide the scaffolding needed to maintain the tertiary structure of the RNA.

hammerhead ribozyme

The hammerhead ribozyme is a RNA module that catalyzes reversible cleavage and joining reactions at a specific site within an RNA molecule

The minimal catalytic sequence active consists of three base-paired stems flanking a central core of 15 conserved nucleotides.

Hammerhead ribozymes play an important role as • therapeutic agents• biosensors, and • its applications in functional genomics and gene discovery

Hairpin ribozyme It can cleave itself internally, or,

can cleave other RNA strands in a transesterification reaction. 

The structure consists of two domains, stem A required for binding (self or other RNA molecules) and stem B, required for catalysis.

Self-cleavage in the hairpin ribozyme occurs in stem A between an A and G bases when the 2' OH on the A attacks the phosphorous in the phosphodiester bond connecting A and G.

Application of ribozyme

•Ribozyme based therapeutics RNA containing short EGS injected into host cell for destruction of RNA of mumps virus, influenza, human papilloma virus etc•Inverse Genomics to find out the function of gene•Ribozyme as biosensor: oligonucleotide-regulated ribozymes, also known as aptazymes

LIGATION

DNA ligases connect DNA fragments by catalyzing the formation of a phosphodiester bond between a 3’-OH and a 5’-phosphate group at a single-strand break in double-stranded DNA (Lehman 1974).

In cells, they are essential for joining Okazaki fragments during replication, and in the last step of DNA repair process.

used in molecular biology to join DNA fragments with blunt or sticky ends such as those generated by restriction enzyme digestion, add linkers or adaptors to DNA, or repair nicks.

ACTION

DNA ligases operate in a three-step reaction. The fist step involves the creation of a ligase adenylate intermediate, in which a phospho amide bond is created between a lysine residue and one AMP molecule of the enzyme cofactor (ATP or NAD+).

Second, the AMP is transferred to the 5’-phosphate end of the DNA nick to form a DNA-adenylate (AppDNA).

Finally, a nucleophilic attack from the 3’ end of the DNA nick directed to the AppDNA results in joining of the two polynucleotides and release of AMP.

Ligation efficiency depends on the DNA ends in the reactionComplementary “sticky” ends

• Ligation is efficient• annealing of complementary overhangs brings

5’P and 3’OH into close proximity

“Blunt” ends• Ligation is inefficient• need high concentrations of ligase and DNA• molecular crowding reagents (like PEG 8000)

improve intermolecular ligation, then dilute to promote intramolecular ligation

NON-THERMOSTABLE DNA LIGASES

The smallest known DNA ligases are the ATP-dependant DNA ligase from Chlorella virus and bacteriophage T7.

The ligase the most frequently used in molecular biology is the bacteriophage T4 DNA ligase.

T4 DNA ligase can connect blunt and cohesive ends, or repair single stranded nicks in duplex DNA, RNA,or DNA/RNA hybrids.

The E. Coli DNA ligase works preferentially on cohesive double-stranded DNA ends.However, it is also active on blunt ends DNA in the presence of Ficoll or polyethylene glycol.

THERMOSTABLE DNA LIGASES

Thermostable DNA ligases can perform ligation of duplex molecules and repair of single stranded nicks at temperatures ranging from 45 to 80°C.

Thermostable DNA ligases are isolated from:- Thermus thermophilus ,

Bacillus stearothermophilus , Thermus scotoductus , Rhodothermus marinus.

methylation

methylation denotes the addition of a methyl group to a substrate or the substitution of an atom or group by a methyl group.

Methylation is a form of alkylation with a methyl group, rather than a larger carbon chain, replacing a hydrogen atom.

DNA methylation is a biochemical process involving the addition of a methyl group to the cytosine or adenine DNA nucleotides.

DNA methylation stably alters the expression of genes in cells 

The resulting change is normally permanent and unidirectional

DNA methylation is typically removed during zygote formation and re-established through successive cell divisions during development.

DNA methyltransferase responsible for methylation

Applications Cloning of restriction enzyme generated DNA

fragments. Cloning of PCR products. Joining of double-stranded oligonucleotide

linkers or adaptors to DNA. Site-directed mutagenesis. Amplified fragment length polymorphism (AFLP). Ligase-mediated RNA detection (3). Nick repair in duplex DNA, RNA or DNA/RNA

hybrids. Self-circularization of linear DNA.