4: Restriction Endonucleases (MIDTERMS) Flashcards
→ 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
What classification on enzyme does the nuclease fall under?
Hydrolases
The group of enzymes that catalyze bond cleavages by reaction with water.
Hydrolases
What are the 2 kinds of Nucleases?
- Endonucleases
- Exonucleases
Kinds of Nucleases:
Hydrolyze internal bonds within a polynucleotide chain
Endonuclease
T or F
Endonuclease is able to cleave both a staggered (ladder) and blunt pattern
T
Kinds of Nucleases:
Remove nucleotides one at a time from the end of a DNA molecule
Exonuclease
Kinds of Nucleases:
Cutting at the end
Exonuclease
→ Means recognizing a specific/restriction site
→ able to identify certain palindromic sequence
Restriction
→ can only identify a certain sequence which it specifically cuts
→ Made (identified) in early 1950s
→ came from bacteria
Restriction endonucleases
Restriction endonucleases were fist described by who and when?
Werner Arber and Matthew
Meselson in the 1960s
The bacteria studied by Arber and Meselson in 1960’s
Phage lambda
Bacterias which are under constant exposure to foreign DNA (lyzed bacterial & bacteriophages) which result in what?
Transformation or transduction
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
T or F
All strains of bacteria are prone to bacteriophage infection
F (Some strains are IMMUNE)
Process that alters the DNA of bacteria; hence there would be changes in the bacteria which could lead to death.
Bacteriophage infection
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
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)
When virus infects bacteria what are the 2 defense mechanism?
- Restriction enzymes
- Modification (methylation)
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
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.
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)
What defense mechanism occurs to prevent RE from binding to bacterial dNA?
Modification (methylation)
→ Each type of restriction enzyme cuts a dsDNA at
a unique symmetrical sequence of how many nucleotides?
4-8 nucleotides
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
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
T
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
T
Length of base pair cleaved by Type I restriction enzyme?
1000 base pair or more from recognition sequence