Enzymes Flashcards
What would be at the 5 and 3 end of a DNA molecule that has been exposed to a
nuclease?
5’: PO4
3’: -OH
Explain what it means when nucleases can be damage / structure / sequence specific.
- Damage: Scan for damage -repair damaged DNA (DNA pol. add new bases to site)
- Structure: (recognise) intermediates generated by DNA repair mechanisms
- Sequence: recognise sequence of DNA (& break P-O bond on or near it)
Sequence specific nucleases are also referred to as?
Restriction endonucleases
Differentiate between endonuclease and exonuclease enzymes
- Exonuclease: Cuts DNA from free ends; cuts ssDNA
- Endonuclease: cut in b/w (middle) DNA; cuts dsDNA
Which essential cofactor do nucleases require to work?
Mg2+
Differentiate between blunt ends and sticky ends.
- Blunt: no overhanging bases @ ends (P-O bonds broken)
- Sticky: overhanging bases @ ends (have non-paired nucleotides) (P-O bond & H-bond broken)
what is a 5’ or 3’ overhang is when referring to sticky ends.
5’ ————— 3’ (5’ no pair)
(5’ eg) 3’ ————- 5’
(3’ eg) 5’ ————–3’
3’ —————-5’ (3’ no pair)
What kind of enzyme is DNAase I? What is it used for?
Endonuclease. Remove DNA from solution (e.g. DNA footprinting analysis)
How does DNAseI behave with different cofactors?
- Mg2+: Cuts independently = random fragments (P-O bond cut randomly along strands of ds DNA)
- Mn2+: Cleaves at dsDNA leaving 1-2 nucleotide overhangs
Explain how ligases work, and how they are used during DNA replication.
form P-O bond b/w 5’ P & 3’ OH of 2 strands (blunt/sticky).»_space; join Okazaki fragments tog. in DNA replication
When would an exonuclease be used?
When you want to cut overhangs -> blunt ends
What circumstance would you want to use an endonuclease?
For Host Restriction Modification Systems- use restriction endonuclease to cut thru DNA @ specific sequence
Which catalytic activity of DNA pol I is not present in the Klenow fragment? And what’s the other 2 catalytic activity
5’ to 3’ exonuclease bc it acts as an invasion pol.- displace downstream bases as it fills dNTP in ss section in DNA.
- 5’ to 3’ polymerase = proofread
- 3’ to d’ exonuclease = cut/fill 3’ overhangs
Which polymerase does not require a template strand? What’s it used for & its requirements?
Terminal Deoxynucleotidyl Transferase.
> make 3’ overhangs (on ssDNA/dsDNA 3< bp)
- Mg2+ & dNTP
Explain how the bacterial restriction modification system protects the cell against viral DNA?
Restriction endonucl./enzyme (RE) defends cell from invading DNA. But Bact. DNA & phage DNA is similar recognition capacity, so to distinguish itself from viral DNA, methylase (M) adds methyl group to that capacity. If recog. site for RE is methylated =can’t cut DNA w/ methyl = “self’ DNA proteted. While invading DNA is cleaved bc not methylated
Describe the different DNA polymerases E. coli uses for different cellular processes
- Pol I, II, III: Proofreads
- Pol I: 5’ to 3’ exonuclease
Which Type of RE enzyme will reliably cut at a specific sequence? Why is this important?
Type II cuts w/in recognition site: recog. seq. asymetrical (palindrome). Important bc we know where the the DNA will cleave
What is a palindromic sequence?
When base sequence is read the same from 5’ to 3’ on both strands on dsDNA
Role of exonuclease III?
acts on 3’ overhangs on dsDNA on 3’ to 5’ direction
Where is T4 DNA ligase obtained from? And what is its cofactor
- T4 bacteriophage
- ATP
List the 3 catalytic activities of DNA polymerase I
- 5’ to 3’ polymerase
- 5’ to 3’ exonuclease
- 3’ to 5’ exonuclease
What is Nuclease S1 & describe how specificity is affected by [ ]
exonuclease isolated from Aspergillus.
- Lo [ ]= cuts both ss & ds DNA
- Hi [ ]= cuts only dsDNA, ds RNA or DNA:RNA
