ENDONUCLEASESENDONUCLEASES

Enzyme which produced single double stranded cut within DNA Enzyme which produced single double stranded cut within DNA molecule (that is they do not digest DNA molecules from their molecule (that is they do not digest DNA molecules from their

end)end)

Why need the modern biotechnology(manipulate Why need the modern biotechnology(manipulate the DNA)the DNA)

Uses of the molecular biotechnologyUses of the molecular biotechnology• Technical development in the PCRTechnical development in the PCR• In the several manufacturing processIn the several manufacturing process

Different Enzyme use in the manipulation, different Different Enzyme use in the manipulation, different purpose purpose

• Protease(detergents)Protease(detergents)• Glucose oxidase(glucose to gluconic acid, industrial chemical, Glucose oxidase(glucose to gluconic acid, industrial chemical,

diagonastic blood sugar)diagonastic blood sugar)• Renin(cheese)Renin(cheese)• papain(papaya plant, animal feed)papain(papaya plant, animal feed)• Glucose isomerase(glucose to fractose)Glucose isomerase(glucose to fractose)• papsin(animal protease, in manufacturingpapsin(animal protease, in manufacturingIndustryIndustry

• urokinase(anti-trombiontic agent)urokinase(anti-trombiontic agent)• asperaginase(cancer chemotherapy)asperaginase(cancer chemotherapy)• Tyrosine hydroxylase(parkansons disease)Tyrosine hydroxylase(parkansons disease)

Enzymes use to manipulate the DNA, recombinantEnzymes use to manipulate the DNA, recombinant Restriction endonucleases\knives\scissors\scalpalRestriction endonucleases\knives\scissors\scalpalType of restriction enzymeType of restriction enzymeType1Type1• Not useful for gene manipulationNot useful for gene manipulation• Interact with unmodified recognition sequenceInteract with unmodified recognition sequence• Traveling distance 1000-5000npTraveling distance 1000-5000np• Cleavage only one strand of DNA at random siteCleavage only one strand of DNA at random site• Create a gap of 75n in lengthCreate a gap of 75n in length• Cleavage site is non-specificCleavage site is non-specific

Type11Type11• Useful for gene manipulationUseful for gene manipulation• Recognized a particular DNA sequenceRecognized a particular DNA sequence• Mg ion for recognition for restrictionMg ion for recognition for restriction

Type111Type111• They require for ATP, mg ionThey require for ATP, mg ion• They have intermediate properties between type1 and type11They have intermediate properties between type1 and type11

NamingNaming ofof restrictionrestriction enzymeenzymeEco r1( eco spp name, genus name general name,1 one enzyme Eco r1( eco spp name, genus name general name,1 one enzyme

have more then one restriction enzyme)have more then one restriction enzyme)

Target sit e of restriction endonucleaseTarget sit e of restriction endonucleaseNature of the cut endNature of the cut end• Blunt end style Blunt end style (a) 5-----------gg cc----------------5(a) 5-----------gg cc----------------5 3-----------cc gg---------------33-----------cc gg---------------3Cut across both strand of DNACut across both strand of DNAJoin both strand without introducing materialJoin both strand without introducing material

• Sticky end styleSticky end style(b) 5----------GAA TTC--------------3(b) 5----------GAA TTC--------------3

3----------CTT AAG-------------53----------CTT AAG-------------5

(b2) 5----------G AATTA------------3(b2) 5----------G AATTA------------3

3-----------CTTAA G-----------53-----------CTTAA G-----------5

Cut on the single strandCut on the single strand

Isoschizomers Isoschizomers (restriction enzyme)(restriction enzyme)isolate from different organism but recognized identical base isolate from different organism but recognized identical base

sequence e.g. Asp 718(Achromobactor spp) and sequence e.g. Asp 718(Achromobactor spp) and Kpn1(Klebiella pneumoniae) Kpn1(Klebiella pneumoniae)

g/gtacccg/gtaccc

ccatg\gccatg\g

Ligases\suturesLigases\suturesInvolved in the repairing of the DNAInvolved in the repairing of the DNA

Sealing and reunion of the DNASealing and reunion of the DNA

Take part in the DNA replication and recombination Take part in the DNA replication and recombination

Production of the hybrid DNA( genetic engineering)Production of the hybrid DNA( genetic engineering)

Seal the nick chainSeal the nick chain

Activity of ligasesActivity of ligases

1.Sticky end ligation1.Sticky end ligation

5 pGATCC--------G pGATCC-------G 35 pGATCC--------G pGATCC-------G 3

3 G-------CCTAGp G------CCTAGp 53 G-------CCTAGp G------CCTAGp 5

DNA ligase + ATPDNA ligase + ATP

5 pGATCC---------GGATCC5 pGATCC---------GGATCC-----------G 3-----------G 3

3 G---------CCTAGG3 G---------CCTAGG-----------CCTAGp 5-----------CCTAGp 5

Pairing of fragment is the transientPairing of fragment is the transientWeak hydrogen bodingWeak hydrogen bodingFragment is stabilized by formation of covalent bond b\t 5 Fragment is stabilized by formation of covalent bond b\t 5

phosphoryl one strand and 3 hydroxyl on the other strandphosphoryl one strand and 3 hydroxyl on the other strand

2.Blunt ended ligations2.Blunt ended ligations• blunt DNA can be ligated blunt DNA can be ligated • There is no base paring hold fragment together temporarilyThere is no base paring hold fragment together temporarily• Blunt end is the useful for joining together DNA fragment which Blunt end is the useful for joining together DNA fragment which

are not produced by same restriction enzymeare not produced by same restriction enzymeCleavage with hind 111 cleavage with EcoriCleavage with hind 111 cleavage with Ecori ---------A 3 5 pAATTC-------------------A 3 5 pAATTC---------- ---------TTCGAp 5 3 G-------------------TTCGAp 5 3 G---------- s1 nuclease (which digest single strand)s1 nuclease (which digest single strand)---------A 3 5 pC--------------------A 3 5 pC------------------- TP5 3 G------------------- TP5 3 G----------- DNADNA ligase (high concentration) +ATP ligase (high concentration) +ATP --------------AC-------------------------AC----------- ---------------TG--------------------------TG-----------

S1 nucleasesS1 nucleases Analyze DNA-RNA hybrid structure to map transcriptAnalyze DNA-RNA hybrid structure to map transcript

It is use for DNA mappingIt is use for DNA mapping

Hairpin lop structure formed during synthesis of cDNA digested by Hairpin lop structure formed during synthesis of cDNA digested by this enzymethis enzyme

DNA polymerase 1,holoenzymeDNA polymerase 1,holoenzyme1. Bifunctional activity (5 exonucleatic activity in addition to DNA 1. Bifunctional activity (5 exonucleatic activity in addition to DNA

synthesis)synthesis)• 5 exonuclease activity degrade the DNA strand which 5 exonuclease activity degrade the DNA strand which

complementary to the template strand and thus forming a complementary to the template strand and thus forming a nicknick

• DNA synthesis began at 3end of the nick and produce new DNA synthesis began at 3end of the nick and produce new strand of the DNA complementary to the template strand of the DNA complementary to the template

2. Prepare labeled DNA of high specific activity 2. Prepare labeled DNA of high specific activity

3. Catalyze de novo DNA synthesis3. Catalyze de novo DNA synthesis

DNA polymerase 1,klenow fragmentDNA polymerase 1,klenow fragment• DNA polymerase 1+protease-------two protein fragmentDNA polymerase 1+protease-------two protein fragment

Large fragment is called klenow fragmentLarge fragment is called klenow fragment• Dose not exhibit 5 exonuclease activity Dose not exhibit 5 exonuclease activity • Use to synthesized DNA when there is no need of removing Use to synthesized DNA when there is no need of removing

DNA strand which is complementary to template strandDNA strand which is complementary to template strand• For production of second strand of cDNAsFor production of second strand of cDNAs

T4 DNA polymeraseT4 DNA polymerase• Lake 5exonuclease activityLake 5exonuclease activity• active in 3 exonuclease activityactive in 3 exonuclease activity

(It act on both 3 end of double stranded DNA resulting in 5 single (It act on both 3 end of double stranded DNA resulting in 5 single stranded extension dna complementry to these single strand stranded extension dna complementry to these single strand are synthesis with deoxynucleoside triphosphase enzyme) are synthesis with deoxynucleoside triphosphase enzyme)

• Useful in generating 5 single strand endUseful in generating 5 single strand end

Tag DNA polymerase (thermus aquaticus)Tag DNA polymerase (thermus aquaticus)• It lacks 5 to 3 and 3 to5 exonucleatic activityIt lacks 5 to 3 and 3 to5 exonucleatic activity• It is ideal for both automated and manual DNAIt is ideal for both automated and manual DNA

sequencing (because fast, highly progressive littlesequencing (because fast, highly progressive little

or no 3-exonucleatic activityor no 3-exonucleatic activity• It is use in the PCR and can withstand high temperature and It is use in the PCR and can withstand high temperature and

DNA sequencingDNA sequencing

Ribonuclease (RNAase H)Ribonuclease (RNAase H)• Is an endonuclease that degrade RNA portion of the RNA-DNA Is an endonuclease that degrade RNA portion of the RNA-DNA

hybridhybrid• Key enzyme in the cDNA cloning procedureKey enzyme in the cDNA cloning procedure• Use to detect the DNA-RNA hybridUse to detect the DNA-RNA hybrid• Use to remove the poly A tail from mRNAUse to remove the poly A tail from mRNA

Reverse transcriptaseReverse transcriptase• RNA dependent DNA polymeraseRNA dependent DNA polymerase• Enzyme require DNA primer complementary to RNAEnzyme require DNA primer complementary to RNA

template template • Mg and Mn for initiation of transcriptionMg and Mn for initiation of transcription• In vitro synthesis of complementary DNA from mRNAIn vitro synthesis of complementary DNA from mRNA• It is also contain DNA dependent DNA polymeraseIt is also contain DNA dependent DNA polymerase

Activity responsible for second strand formation in cDNAActivity responsible for second strand formation in cDNA• Reverse transcription mediate the conversion of genetic Reverse transcription mediate the conversion of genetic

information mRNA molecule to d strand DNA moleculeinformation mRNA molecule to d strand DNA molecule

Poly (A) polymerasePoly (A) polymerase• In vitro gene manipulationIn vitro gene manipulation

• Catalyze the addition AMP unit to the 3 end of RNACatalyze the addition AMP unit to the 3 end of RNA

Deoxyribonuclease 1Deoxyribonuclease 1 DNase 1 is an end nuclease which digest either single or DNase 1 is an end nuclease which digest either single or

double stranded DNA.double stranded DNA. Addition of mgAddition of mg+2+2 ensure random cleavage ensure random cleavage

mn+2 gives cleavage nearly at the same place on both mn+2 gives cleavage nearly at the same place on both strandstrand

enzyme are enable the scientist to design and engineer the enzyme are enable the scientist to design and engineer the gene and thus produce desire specificationgene and thus produce desire specification