ENZYMES USED IN ENZYMES USED IN rDNA TECHNOLOGYrDNA TECHNOLOGY

prepared by:Ashish Kumar & yogesh kumarC.C.S.University Meerut

INTRODUCTIONINTRODUCTION The major tools for the Rdna The major tools for the Rdna

technology are the enzymes used for technology are the enzymes used for recombinant DNA technology have recombinant DNA technology have also been overproduced by also been overproduced by genetically engineered bacteria and genetically engineered bacteria and their use is still increasing.their use is still increasing.

ADVANTAGESADVANTAGES First, they are significantly less First, they are significantly less

expensive.expensive. Second, purification of enzymes is Second, purification of enzymes is

easier and more rapid, resulting in easier and more rapid, resulting in more homogeneous enzymes with more homogeneous enzymes with higher specific activities.higher specific activities.

Restriction Restriction EnzymesEnzymes

Restriction Enzymes scan the DNA codeRestriction Enzymes scan the DNA code Find a very specific set of nucleotidesFind a very specific set of nucleotides Make a specific cutMake a specific cut

HaHaeeIIIIIIHaHaeeIII is a restriction enzyme that III is a restriction enzyme that

searches the DNA molecule until searches the DNA molecule until it finds this sequence of four it finds this sequence of four

nitrogen bases.nitrogen bases.5’ TGACGGGTTCGAGGCCAG 3’3’ ACTGCCCAAGGTCCGGTC 5’

5’ TGACGGGTTCGAGGCCAG 3’3’ ACTGCCCAAGGTCCGGTC 5’

Once the recognition site was found Once the recognition site was found HaHaeeIII could go to work cutting III could go to work cutting

(cleaving) the DNA(cleaving) the DNA

5’ TGACGGGTTCGAGGCCAG 3’3’ ACTGCCCAAGGTCCGGTC 5’

These cuts produce what scientists callThese cuts produce what scientists call“blunt ends”“blunt ends”

5’ TGACGGGTTCGAGG CCAG 3’3’ ACTGCCCAAGGTCC GGTC 5’

““blunt ends” and “sticky ends”blunt ends” and “sticky ends”Remember how Remember how HaeHaeIII produced a III produced a

“blunt end”? “blunt end”? EcoEcoRI, for instance, makes a staggered RI, for instance, makes a staggered

cut and produces a “sticky end”cut and produces a “sticky end”

5’ GAATTC 3’3’ CTTAAG 5’5’ GAATTC 3’3’ CTTAAG 5’

5’ G AATTC 3’3’ CTTAA G 5’

blunt endblunt end

sticky endsticky end

Some more examples of restriction sites of Some more examples of restriction sites of restriction enzymes with their cut sites:restriction enzymes with their cut sites:

HindHindIII: 5’ AAGCTT 3’III: 5’ AAGCTT 3’ 3’ TTCGAA 5’3’ TTCGAA 5’

BamBamHI: 5’ GGATCC 3’HI: 5’ GGATCC 3’ 3’ CCTAGG 5’3’ CCTAGG 5’

AluAluI: 5’ AGCT 3’I: 5’ AGCT 3’ 3’ TCGA 5’3’ TCGA 5’

Restriction enzymes recognize and Restriction enzymes recognize and make a cut within specific palindromic make a cut within specific palindromic sequences, known as sequences, known as restriction sitesrestriction sites, , in the genetic code. This is usually a in the genetic code. This is usually a

4- or 6 base pair sequence.4- or 6 base pair sequence.

Example?

DNA LIGASESDNA LIGASES

DNA Ligases close nicks in the DNA Ligases close nicks in the phosphodiester backbone of DNA.phosphodiester backbone of DNA.

Biologically, DNA Ligases are essential Biologically, DNA Ligases are essential for the joining of Okazaki fragments for the joining of Okazaki fragments during replication, and for compliting during replication, and for compliting short-patch DNA synthesis occuring short-patch DNA synthesis occuring in DNA repair process.in DNA repair process.

DNA POLYMERASEDNA POLYMERASE A DNA polymerase is an enzyme that A DNA polymerase is an enzyme that

catalyzes the polymerization of catalyzes the polymerization of deoxyribonucleotides into a DNA strand.deoxyribonucleotides into a DNA strand.

DNA polymerases are best known for their DNA polymerases are best known for their role in DNA replication in which the role in DNA replication in which the polymerase" reads” an intact DNA strand polymerase" reads” an intact DNA strand as a template and uses it to synthesize the as a template and uses it to synthesize the new strand.the process copies a pieces of new strand.the process copies a pieces of DNA.DNA.

DNA polymerases use a mg++ for DNA polymerases use a mg++ for catalytic activitiy.catalytic activitiy.

Type . Pol I, pol II, pol IIIType . Pol I, pol II, pol III

KLENOW FRAGMENTKLENOW FRAGMENT The Klenow fragment is alarge protein The Klenow fragment is alarge protein

fragment produced when DNA polymerase fragment produced when DNA polymerase I from E.coli. Is enzymatically cleaved by I from E.coli. Is enzymatically cleaved by the protease subtilisin.the protease subtilisin.

First reported in 1970, it retains the 5’ to3’ First reported in 1970, it retains the 5’ to3’ polymerase activity and the 3’ to5’ polymerase activity and the 3’ to5’ exonuclease activity for removal of exonuclease activity for removal of precoding nucleotides and proofreading, precoding nucleotides and proofreading, but loses its 5’ to 3’ exonuclease activity.but loses its 5’ to 3’ exonuclease activity.

EXONUCLEASEEXONUCLEASE Exonucleases are enzymes that work by Exonucleases are enzymes that work by

cleaving nucleotides one at a time from cleaving nucleotides one at a time from the end of a polynucleotide chain.the end of a polynucleotide chain.

A hydrolyzing reaction that breaks A hydrolyzing reaction that breaks phosphodiester bonds at either the 3’ or phosphodiester bonds at either the 3’ or the 5’ends occurs.the 5’ends occurs.

Eukaryotes and Prokaryotes have 3 types Eukaryotes and Prokaryotes have 3 types of exonucleases involved in the normal of exonucleases involved in the normal turnover of mRNA :3, to 5’ exonuclease, turnover of mRNA :3, to 5’ exonuclease, which is a dependent decapping protein,5’ which is a dependent decapping protein,5’ to 3’ exonuclease, an independent protein, to 3’ exonuclease, an independent protein, and poly(A)- specific 3’ to 5’ exonuclease.and poly(A)- specific 3’ to 5’ exonuclease.

TERMINAL DEOXYNUCLEOTIDYL TERMINAL DEOXYNUCLEOTIDYL TRANSFERASETRANSFERASE

Terminal Deoxynucleotidyl Transferase, Terminal Deoxynucleotidyl Transferase, also known as TdT and terminal also known as TdT and terminal transferase.transferase.

TdT catalyses the addition of nucleotides TdT catalyses the addition of nucleotides to the 3’ terminus of a DNA molecule.to the 3’ terminus of a DNA molecule.

The preferred substrate of this enzyme is a The preferred substrate of this enzyme is a 3’ overlapping, but it can also add 3’ overlapping, but it can also add nucleotides to blunt or recessed 3’ ends.nucleotides to blunt or recessed 3’ ends.

Cobalt is a necessary cofactor, however Cobalt is a necessary cofactor, however the enzymes catalizes reaction upon Mg the enzymes catalizes reaction upon Mg and Mn administration in vitro.and Mn administration in vitro.

ALKALINE PHOSPHATASEALKALINE PHOSPHATASE Alkaline phosphatase(ALP), is a Alkaline phosphatase(ALP), is a

hydrolase enzyme responsible for hydrolase enzyme responsible for removing phosphate groups from removing phosphate groups from many types of molecules, including many types of molecules, including nucleotides, proteins and alkaloids.nucleotides, proteins and alkaloids.

The process of removing the The process of removing the phosphate group is called phosphate group is called Dephosphorylation.Dephosphorylation.

POLYNUCLEOTIDE KINASEPOLYNUCLEOTIDE KINASE Polynucleotide kinase (PNK) is an Polynucleotide kinase (PNK) is an

enzyme that catalyzes the transfer of enzyme that catalyzes the transfer of a phosphate from ATP to the 5’ end a phosphate from ATP to the 5’ end of either DNA or RNA.of either DNA or RNA.

The enzymatic activity of PNK is The enzymatic activity of PNK is utilized in two types of reactions:utilized in two types of reactions:

Forward reaction.Forward reaction. Exchange reaction.Exchange reaction.

FORWARD REACTIONFORWARD REACTION PNK transfers the gamma phosphate PNK transfers the gamma phosphate

from ATP to the 5’ end of a from ATP to the 5’ end of a polynucleotide (DNA or RNA). The polynucleotide (DNA or RNA). The target nucleotides is lacking a 5’ target nucleotides is lacking a 5’ phosphate either because it has phosphate either because it has been dephosphorylated or has been been dephosphorylated or has been synthesized chemically synthesized chemically

EXCHANGE REACTIONEXCHANGE REACTION Target DNA or RNA that has a 5’ Target DNA or RNA that has a 5’

phosphate is incubated with an phosphate is incubated with an excess of ADP-in this setting, PNK will excess of ADP-in this setting, PNK will first transfer the phosphate from the first transfer the phosphate from the nucleic acid on to an ADP , forming nucleic acid on to an ADP , forming ATP and leaving a dephosphorylated ATP and leaving a dephosphorylated target. PNK will then perform a target. PNK will then perform a forward reaction and transfer a forward reaction and transfer a phosphate from ATP on to the target phosphate from ATP on to the target nucleic acid.nucleic acid.

REVERSE TRANSCRIPTASEREVERSE TRANSCRIPTASE This enzyme by using the template This enzyme by using the template

of RNA ,synthesize the new strand of of RNA ,synthesize the new strand of DNA .DNA .