4: Restriction Endonucleases (MIDTERMS) Flashcards by Ma. Clariza Luna

→ Degrade DNA molecules by breaking the phosphodiester bonds that link one nucleotide to the next in a DNA strand

→ May be specific for DNA or RNA

Nucleases

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What classification on enzyme does the nuclease fall under?

Hydrolases

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The group of enzymes that catalyze bond cleavages by reaction with water.

Hydrolases

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What are the 2 kinds of Nucleases?

Endonucleases
Exonucleases

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Kinds of Nucleases:

Hydrolyze internal bonds within a polynucleotide chain

Endonuclease

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T or F

Endonuclease is able to cleave both a staggered (ladder) and blunt pattern

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Kinds of Nucleases:

Remove nucleotides one at a time from the end of a DNA molecule

Exonuclease

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Kinds of Nucleases:

Cutting at the end

Exonuclease

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→ Means recognizing a specific/restriction site

→ able to identify certain palindromic sequence

Restriction

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→ can only identify a certain sequence which it specifically cuts

→ Made (identified) in early 1950s

→ came from bacteria

Restriction endonucleases

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Restriction endonucleases were fist described by who and when?

Werner Arber and Matthew
Meselson in the 1960s

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The bacteria studied by Arber and Meselson in 1960’s

Phage lambda

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Bacterias which are under constant exposure to foreign DNA (lyzed bacterial & bacteriophages) which result in what?

Transformation or transduction

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Process where where the host bacteria will manifest new characteristics which in a way are detrimental to the bacterial species.

Transformation or transduction of bacteria

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T or F

All strains of bacteria are prone to bacteriophage infection

F (Some strains are IMMUNE)

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Process that alters the DNA of bacteria; hence there would be changes in the bacteria which could lead to death.

Bacteriophage infection

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This enzyme is a “host defense mechanism” of the bacterium, produced to cut the foreign DNA into pieces resulting in the destruction of such.

Restriction endonuclease

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T or F

Bacterium produces an enzyme that supports the replication and direct synthesis of new phage particles

F (produces enzyme that DEGRADES PHAGE DNA before it has time to replicate and direct synthesis of new phage particles)

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When virus infects bacteria what are the 2 defense mechanism?

Restriction enzymes
Modification (methylation)

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Use this card to familiarize yourself for the steps of restriction modification system

When DNA of bacteriophage enter the bacteria, the pre-manufactured restriction enzymes will bind to the phage DNA at certain identified parts.
These enzymes will then cut the phage DNA resulting to fragmentation, thereby making the phage DNA ineffective.
Self-DNA is protected by methylation of the guanine and cytosine thereby blocking the enzyme from binding to that site to prevent self-cutting of DNA

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In restriction modification system, What happens when DNA of bacteriophage (virus) enetr bacteria?

pre-manufactured restriction enzymes (antibodies) will bind to the phage DNA and cut the phage DNA resulting to fragmentation–making the phage DNA ineffective.

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T or F

Once Restriction enzyme (antibodies) bind to phage DNA, it results to replication of DNA thus making it multiply)

F (results to FRAGMENTATION thus making it INEFFECTIVE)

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What defense mechanism occurs to prevent RE from binding to bacterial dNA?

Modification (methylation)

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→ Each type of restriction enzyme cuts a dsDNA at
a unique symmetrical sequence of how many nucleotides?

4-8 nucleotides

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when acted upon by a restriction enzyme will linearize the molecule

Circular genomic or plasmid DNA

when acted upon by a restriction enzyme will result in the formation DNA fragments

Linear DNA

When a linear DNA is acted upon a RE it will result in formation of DNA fragments for how many restriction sites?

twice the number of unique restriction sites

T or F the rule that linear DNA when acted upon RE produces twice the number of unique restriction sites also applies when two or more enzymes are used in combination during restriction analysis of a given DNA material.

T or F Restriction endonuclease are independent from palindrome

F (RE are DEPENDENT from palindrome)

Sequences recognized by REs read the same from left to right as they do from right to left on the complementary strand.

Palindromic sequence

T or F REs can only identify palindromic sequences and specifically cuts it

In nomenclature of restriction endonuclease what represents the FIRST THREE LETTERS?

Shortened abbreviation of the organism

In nomenclature of restriction endonuclease what represents the FOURTH LETTER

Strain of the bacteria

In nomenclature of restriction endonuclease what represents the ROMAN NUMERAL?

indices if the same organism contains several different REs (The class of the REs produced)

Identify what type of restriction enzyme: → Co factors: Mg2+ ions, Sadenosylmethionine (SAM), and ATP → The recognition sequences are quite long with no recognizable features → Methylation reaction is performed by the same enzyme which mediates cleavage → Little value for gene manipulation

Type I Restriction Enzyme

What cofactor/s does Type I Restriction Enzyme have?

Mg2+ ions, Sadenosylmethionine (SAM), and ATP

T or F In Type I Restriction Enzyme recognition sequences are quite long with recognizable features such as symmetry

F (NO RECOGNIZABLE FEATURES)

T or F Type I Restriction Enzyme cleave at DNA at nonspecific sites

Length of base pair cleaved by Type I restriction enzyme?

1000 base pair or more from recognition sequence

T or F In Type I restriction enzyme methylation occurs after it is cleaved

F (BEFORE IT IS CLEAVED/CUT)

T or F Type I restriction enzyme are recommended for clinical premises and mol bio lab since they have value for gene manipulation

F (NOT RECOMMENDED, ONLY FOR RESEARCH)

consists of the identified sequence that the RE will cut

Restriction recognition site

area that will be cut/cleaved

Cleavage site

Identify what type of restriction enzyme: → Favorable to use in molecular biology → Recognition site is 4-6 bp sequence → Site-specific as they hydrolyze specific phosphodiester bonds in both DNA strands → used as the key material in molecular biology and recombinant DNA techniques including genome mapping, RFLP analysis, DNA sequencing, and cloning

Type II Restriction Enzyme

T or F There is separate proteins for restriction and methylation for Type II restriction enzymes as compared to Type I

Restriction and modification are mediated by separate enzymes so it is possible to cleave DNA in the absence of modification a. Type I restriction enzyme b. Type II restriction enzyme c. Type III restriction enzyme

b. Type II restriction enzyme

T or F The restriction activities in Type II restriction enzymes require cofactors like ATP or Sadenosylmethionine

F (do NOT require, only Mg2+)

What are the 2 types of DNA Ends Produced Using Type II RE

1. Sticky ends (5' and 3' ends) 2. Blunt ends

Types of DNA Ends Produced Using Type II RE: Staggered ends on a DNA molecule with short, single stranded overhangs

Sticky ends

Types of DNA Ends Produced Using Type II RE: Have straight cut, down through the DNA, that results in a flat pair of bases on the ends of the DNA

Blunt ends

What are the 2 kinds of sticky ends?

1. Sticky 5’ ends (5’ overhang) 2. Sticky 3’ ends (3’ overhang)

What kind of sticky ends is shown in the photo:

Sticky 5’ ends (5’ overhang)

What kind of sticky ends is shown in the photo:

Sticky 3’ ends (3’ overhang)

T or F If ever a sequence in the DNA has been cleaved, it can be reverted back

T (as long as it finds complementary base pair and ligase)

Identify what type of restriction enzyme: → Possess both restriction and modification activities → Recognizes specific sequences and cleave 25-27 bp outside of or near the recognition sequence, in a 3’ direction → Requires 2 restriction sites in opposite orientation → Requires Mg2+ ions

Type III Restriction Enzyme

Length of basepair that Type II Restriction Enzyme cleaves

4-6 bp

Length of basepair that Type III Restriction Enzyme cleaves and location

25-27 bp (from recognition site)

T or F Type II Restriction Enzyme requires 2 restriction sites in opposite orientation

F (TYPE III)

Identify what type of restriction enzyme: → Cleave outside of their recognition sequences → Require 2 such sequences in opposite orientations within the same DNA molecule →Rarely give complete digests

Type IV Restriction Enzyme

What is the cofactor, cleavage site, and example of Type 1 restriction enzyme

Cofactor: ATP, SAM, MG2+ Cleavage Site: Cleaves at sites AWAY from recognition site Ex: ECO B, ECO R

What is the cofactor, cleavage site, and example of Type 2 restriction enzyme

Cofactor: MG2+ Cleavage Site: Cleave within or at a short distance from recognition site Example: EcoRI, BamHI, Sphl

What is the cofactor, cleavage site, and example of Type 3 restriction enzyme

Cofactor: ATP, MG2+ Cleavage Site: Cleave at sites 25-27 bp from recognition site Example: EcoPI, HinfIII

What is the cofactor, cleavage site, and example of Type 4 restriction enzyme

Cofactor: MG2+ Cleavage Site: Cleave close to or within recognition sequence (pero sabi previsouly outside daw gulo mo henrick) Example: Mrr

What is the Source, Recognition/Cleavage site, and Nature of cut ends of: EcoRI

Source: Escherichia coli RY13 Recognition/ Cleavage Site: 5’ G AATTC 3’ 3’ CTTAA G 5’ Nature of cut ends: Sticky 5’ overhang

What is the Source, Recognition/Cleavage site, and Nature of cut ends of: BAMHl

Source: Bacillus amaloliquifaciens H Recognition/ Cleavage Site: 5’ G GATTC 3’ 3’ CCTAA G 5’ Nature of cut ends: Sticky 5’ overhang

What is the Source, Recognition/Cleavage site, and Nature of cut ends of: HindIII

Source: Haemophilus influenza Rd Recognition/ Cleavage Site: 5’ A AGCTT 3’ 3’ TTCGA A 5’ Nature of cut ends: Sticky 5’ overhang

What is the Source, Recognition/Cleavage site, and Nature of cut ends of: Alul

Source: Arthrobacter luteus Recognition/ Cleavage Site: 5’ AG CT 3’ 3’ TC GA 5’ Nature of cut ends: Blunt end

Which RE's have sticky 5' overhang?

EcoRI, BamHI, HindIII, SalI (BEHS)

Which RE's have blunt end?

AluI, BalI, HaeIII, SmaI (SABAH con yelo)

Which RE's have sticky 3’ overhang?

Apalm, ApII, PstI (PAA)

Restriction Enzymes are affected by what factors?

1. Enzyme substrate cocentration 2. Temperature 3. pH

This provides optimal condition for RE

Restriction Buffer

T or F Buffer contains cofactors as well

Composition of RE buffer: correct ionic strength

NaCl or KCl

Composition of RE buffer: proper pH

Tris HCl

Composition of RE buffer: enzyme cofactor

MgCl2

Composition of RE buffer: alcohol for methylation that cutes sequence to prevent fragmentation of DNA

2-Mercaptoethanol

Composition of RE buffer: Stop digestion by chelating cations

EDTA

T or F Once buffer, RE, and DNA to be cleaved have been mixed, it will then be subjected to electrophoresis

Direction of migration of DNA in gel electrophoresis?

Cathode (negative) → Anode (positive) (- → +)

Factors that affects migration of DNA in gel electrophoresis?

1. charge 2. size

Relation between the size of the molecule and its movement?

inversely proportional (heavier size = slow migration and vice versa)

Causes of RE indigestion?

1. Optimal temperature 2. Incomplete Digestion

Cause of RE indigestion: Optimal Temperature Optimal temperature of Restriction Enzymes?

37°C

Cause of RE indigestion: Optimal Temperature Too hot of a temperature results to what activity?

Denaturation of enzyme

Cause of RE indigestion: Optimal Temperature Too cold of a temperature results to what activity?

Enzymatic activity lowered (slower Too cold resction), may require longer digestion time

Produced because of inoptimal conditions; conditions (temperature, ph, etc.) are not appropriate

Star activity

What are the causes of incomplete digestion?

1. Reaction condition 2. Enzyme requirement 3. Substrate DNA Conditions

Cause of incomplete digestion: Reaction condition examples of reaction condition?

1. Too little enzyme 2. Too much substrate 3. Nonoptimal temp 4. Short incubation time (TTTT)

Cause of incomplete digestion: Enzyme Requirement examples of enzyme requirement

1.Cofactor 2. Recognition sequence requirements 3. Site preference

Cause of incomplete digestion: Substrate DNA Conditions examples of substrate DNA conditions

1. Methylation 2. Restriction site proximity 3. Structure

Applications of Restriction Endonucleases?

1. Recombinant DNA Technology 2. Single Nucleotide Polymorphisms (SNP) 3. Restriction Enzyme DNA Mapping

Applications of Restriction Endonucleases: Assist in the insertion of a gene into a plasmid vector in cloning techniques.

Recombinant DNA Technology

Applications of Restriction Endonucleases: Recombinant DNA Technology a circular DNA found in bacteria that is used in gene cloning

Plasmid

Applications of Restriction Endonucleases: Distinguish gene alleles by specifically recognizing single base changes in DNA known as?

Single Nucleotide Polymorphisms (SNP)

Applications of Restriction Endonucleases: A change of one base pair eliminates a restriction cleavage site

Single Nucleotide Polymorphisms (SNP)

Applications of Restriction Endonucleases: This is a method used to map an unknown segment of DNA by breaking it into pieces and then identifying the locations of the breakpoint.

Restriction Enzyme DNA Mapping

Applications of Restriction Endonucleases: The positions of the sites can be inferred based on the sizes of the resultant DNA fragments

Restriction Enzyme DNA Mapping