(Restriction Enzymes Presentation) - .pdf

Restriction enzymes, also called restriction endonucleases, are specialized proteins that cut DNA at specific sequences. They were first discovered in bacteria, where they serve as a defense mechanism against invading viruses (bacteriophages). By recognizing and cutting foreign DNA at precise sites, bacteria protect themselves from infection.

Each restriction enzyme identifies a particular short DNA sequence (usually 4–8 base pairs long), called a recognition site, and makes a cut either within or near this site. Depending on how they cut, they can produce blunt ends (straight cuts) or sticky ends (overhanging single-stranded DNA). These enzymes have become one of the most important tools in molecular biology and genetic engineering, as they allow scientists to:

Cut DNA at specific points.

Insert, remove, or modify genes.

Create recombinant DNA molecules.

Perform techniques like cloning, mapping, and genetic analysis.

1. TOPIC:TYPES OF RESTRICTION ENZYME
Group-05
KaziRowshonAfroz
2023-1-77-013
NayemHassan
2023-2-77-092
Md. Al Imran Ratul
2023-2-77-067
AfifaZaman
2023-2-77-047
Sadia Rahman
2023-2-77-102

2. RESTRICTION ENZYME TYPE-I
Features:
•Large,multi-subunit
•Restriction+methylation
•Producesrandomfragments
Function:
i.ProtectsbacteriafromphageDNA
ii.CutsforeignDNA
iii.MethylateshostDNA
iv.Recognizessequence,cutsfaraway
v.NeedsATP,Mg²⁺,SAM
Application:
✓Notusedinbiotech(unpredictable)
✓Usedinmechanism&DNA–proteinstudies
Example:EcoKI(E.coli)
Figure:MechanismofEcoKI
Introduction:Molecularscissors→cutDNAatspecificsites
DefenseinbacteriaagainstphageDNA
DiscoveredbyArber,Smith,Nathans→NobelPrize1980

3. TheType-IIrestrictionenzymesarethemostwidelyusedinmolecularbiologyandbiotechnologyduetotheirabilitytocleave
DNAatspecificandpredictablepositionsamongthedifferenttypesofrestrictionenzymes.
Function:
1.ActasmolecularscissorsbycuttingDNAatspecificrecognitionsites.
2.Inbacteria,thispreventsinfectionbyforeignDNA(i.e,bacteriophageDNA).
3.Inbiotechnology,itisusedforDNAcloning,andrecombinantDNA
technology.
Example:EcoRI–aType-IIrestrictionenzyme.
•Recognitionsite:5′-GAATTC-3′
•Cutsite:betweenGandA
•Endformed:Stickyends(5′overhangs).
Application:
I.RecombinantDNAtechnology:cuttingandinsertinggenesintoplasmids.
II.Cloningandgeneexpressionstudies.
III.RestrictionmappingofDNA.
RESTRICTION ENZYME TYPE-II
Figure:MechanismofEcoRIR.E

4. ❑RequireATP,Mg²⁺,S-adenosylmethionine(SAM)foractivity.
❑Recognizeasymmetric,non-palindromicDNAsequences.
❑CutDNA~25–27bpawayfromrecognitionsite.
Function:
▪Partoftherestriction-modificationsystem→protectsbacteriafromforeign
DNA(phages).
▪Cleavesonlywithtwoinverselyorientedrecognitionsites.
▪Producestickyends,notblunt.
Application:
MainlyusedforDNAmapping,protein–DNAinteractionstudies,andATP-
dependentDNAcleavage,ratherthanroutinecloning.
Example:EcoP15I(fromE.coli)–recognizes5′-CAGCAG-3′
RESTRICTION ENZYME TYPE-III
Figure:Mechanismoftype-IIIR.E
Type-IIIrestrictionenzymesaremulti-subunitcomplexesthatrecognizespecificDNAsequencesandcleaveatadefined
distanceawayfromthesiteinanATP-dependentmanner

5. AclassofrestrictionenzymesthatrecognizeandcleavemodifiedDNA,suchasmethylated,hydroxymethylated,orglucosyl-
hydroxymethylatedbases.Theyactasabacterialdefensesystemagainstbacteriophages.
RESTRICTION ENZYME TYPE-IV
Features:
•DetectsandbindstomethylatedormodifiedbasesinDNA.
•CleavesDNAatvariabledistancesfromthemodifiedbase.
•RequiresATP,Mg²⁺ascofactors.
Function:
➢ProtectsbacteriafromphageswithmodifiedDNA
➢Cleavagesiteisnotfixed→cutsatdifferentpositions
➢Notcommonlyusedinbiotechduetounpredictablecleavage
Example:McrBC(fromE.coli)→recognizesmethylatedcytosine.
i.Origin:Foundinmanybacteriaaspartoftherestriction-modificationsystemto
defendagainstviruses.
ii.Application:LimiteduseinbiotechnologycomparedtoType-IIandType-V
enzymes.
Figure:MechanismofMcrBCR.E

6. AclassofrestrictionenzymesthatuseaguideRNA(gRNA)moleculetoidentifyandcleavespecificDNAsequences.Includes
CRISPR-Cassystems.
RESTRICTION ENZYME TYPE-V
Features:
•UsesRNAmolecules(e.g.,crRNA,tracrRNA,orgRNA)torecognize
targetDNA.
•Introducesdouble-strandedbreaksatspecificsites.
•ReliesonCasproteins(e.g.,Cas9,Cas12).
Function:Generatestickyendsorbluntendsdependingontheenzyme.
❑Cas9→Bluntends
❑Cas12a(Cpf1)→Stickyends
Example:CRISPR-Cas9
i.Origin:AdaptedfromtheimmunesystemofStreptococcuspyogenes.
ii.Application:Thefoundationofmoderngenomeediting.
Figure:MechanismofCas12aprotein

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