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Bchrm 413 Final Exam > 4 Enzymes > Flashcards

4 Enzymes Flashcards

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1
Q

Where do the enzyme that we use to manipulate rna and dna come from

A

Nature have evolved the enzymes to manipulate dna and we use them for own our purposes

Come from:
- cellular replication, recombination, and repair enzymes
- phage or viral enzymes
- mobile DNA/ selfish dna elements

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2
Q

What classes of dna modifying enzymes are there

A

Nucleases
- cut polynucleotides (break binds)

Ligases:
- connect two nucleotides (form new bonds)

kinases:
- add phosphates (form new bonds)

Phosphatases:
- remove phosphates (break binds)

Polymerases:
- combine monomers into a chain (form and break bonds)

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3
Q

How do nucleases generally function

A

Add extra oh to backbone phosphate to help it break away

Can cut two ways to make phosphate end up on either the 5’ or 3’ end

Normally:
Phosphate ends up on 5’ end of the sugar

Less common:
Phosphate gets cut so not on 5’ end, meaning 5’ has oh and 3’ end has phosphate

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4
Q

What are endo and exonucleases

A

Endo: only cut the middle of the dna strand

Exo: only cut at the ends of dna stands

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5
Q

What does is means for nuclease of cut double strand or single stranded

A

Double stranded: cleaves both dna strands

Single: cuts of nucleotides from one strands

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6
Q

Deoxyribuonucleases:

What dnase make the 5’ and 3’ phosphates

Why do we want 5’ phosphate cuts

A

DNase I makes 5’ phosphate

DNase II makes 3’

Want 5’ phosphate because we want to still be able to use the dna , and it need the 5’ phos for other reactions and enzymes

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7
Q

How do ribo nucleases function:

A

They cut rna and make a 2’,3’ cyclic monophosphate

Don’t want to do anything with rna after it’s cut so doesn’t matter that phosphate is on 3’ end

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8
Q

What are the examples of nucleases

A

MNase

Mung bean nucleases

Exonuclease I/III

DNase I/II

type II RE

RNase A/H

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9
Q

What it’s the template, binds broken, products, uses in lab of MNase

A

Micrococcal nuclease is isolated from staphylococcus aureus

Template:
- cuts ss and ds DNA and RNA

Bonds broken
- single stranded cut
- non specific endo/exonuclease

Products
- leave a 3’ phosphate
- makes random short oligos and eventually leaves single nucleotides

Uses in lab
- can’t cut where there is chromatin
- used to find areas that have nucleosomes because can’t cut there (since it’s chromatin structure
- so can do genome sequencing to show where genes are active in the genome because they would cut there

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10
Q

Most dna modifying enzymes need ____

A

Cations to work

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11
Q

What it’s the template, binds broken, products, uses in lab of mung bean nuclease

A

Isolated from mung beans

Template:
- cuts ss DNA and RNA

Bonds broken
- 3-5 exo
- 5-3 exo
So exo in both directions

Products
- leave a 5’ phosphate
- cant keep cutting when reaching Ds region so leaves dS regions intact

Uses in lab
- removes 5’ and 3’ overhangs to make blunt ends (since leaves ds regions intact)

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12
Q

What it’s the template, binds broken, products, uses in lab of exonuclease I

A

Isolated from E.coli

Only works if there are 6 bp ahead of it so can’t fully bind to overhangs since they are 4 BP

Template:
- cuts ss DNA only

Bonds broken
- 3-5 exo
So exo in one direction

Products
- leave a 5’ phosphate

Uses in lab
- removes primers from PCR reactions

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13
Q

What it’s the template, binds broken, products, uses in lab of exonuclease III

A

Isolated from E.colo

Template:
- cuts dsDNA only

Bonds broken
- 3-5 exo
- single stranded cut

Products
- leave a 5’ phosphate
- creates ssDNA

Uses in lab
- used to make ssDNA from dsDNA which is needed for dideoxy sequencing and site directed mutagenesis

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14
Q

What it’s the template, binds broken, products, uses in lab of DNase I/II

A

Found in vertebrate

Template:
- cuts ss or ds DNA

Bonds broken
-nonspecific endonuclease
- single stranded cut

Products
- leave a 5’ phosphate (DNASE I)
- leave a 3’ phosphate (DNASE II)
- creates random short oligo that eventually leave single nucleotides

Uses in lab
- used to remove gDNA from TNA extractions
- can determine site of open chromatins like mnASE because it can’t digest dna bound to chromatin (open chromatin are DNASE I hypersensitive sites (DHS)

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15
Q

What it’s the template, binds broken, products, uses in lab of restriction endo nucleases

A

Found prokaryotes as a host defense mechanism against viral (foreign) DNA

Template:
- cuts ds DNA only

Bonds broken
-specific endonucleases
-cut at specific nucleotide sequences

Products
- leave a 5’ phosphate

Uses in lab
-cloning
- plasmid mapping (digest dna with RE and see if correct band size shows up)

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16
Q

What are the types of RE

A

Mainly type II we talk about

Type I: recognize a two part sequence but don’t make a predictable cleavage pattern

Type II: recognize specific sequences (usually palindromic) and cut at defined positions within their recognition sequence

Type III: recognize two sequences in opposite directions and cleave outside of their recongnition sequence

Type IV: recognize modified (usually methylated) DNA

Type V: use guide RNA to target specific sequences (ex. Cas9)

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17
Q

What are type II RE

A

Recognize and cut at very specific sequences that that are 4-8 BP long (never smaller than 4)

Usually palindromic sequences that they recognize

Cuts make blunt or sticky ends (with at least 2bp overhangs), some have compatible sticky ends with other enzymes

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18
Q

Whag is the naming convention of RE

A

Genus soecies and strain of the bacteria is was isolated from
Ex EcoRI

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19
Q

What is lambda dna

Explain how many BP 4 BP 6bp and 8bp cutters would cut

A

DsDNA from a lambda phage that infects e.coli

4bp: the is a cut site every 256 (4^4)

6 BP: 4096 (4^6)

8 bp: one site every 65,536 (4^8) bp

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20
Q

Examples of RE

A

EcoRI: GAATCC
- from e.coli
- leaves a 5’ overhang of 4 bp (sticky ends) on both strands

EcoRV:
- from e.coli
- leaves a blunt end

SacII
- from streptomyces achromogenes
- leaves a 3’ overhang of 2 bp

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21
Q

What do you have to be careful of when using RE

Give examples

A

The RE may not work well near the ends of the DNA molecules (ex. At the ends of a PCR product)

Doesn’t matter for plasmid tho bc circular

mportant for linear sequences and circular ones of doing restriction forgets (if both cut sites too close not enough BP between them for other RE to cut)

Having 6bp after the RE site is good rule of thumb

EcoRI cuts well when 5bp extra at end, can still cut is one but not well

SACII cuts well even if only one bp at end

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22
Q

What is star activity with RE

A

When under non standard conditions, some RE lose their specificity

This is caused by :

High glycerol concentration:
- all RE are stored in 50% glycerol , so the RE enzyme used shouldn’t exceed 10% of the reaction volume so that there’s not extra glycerol

Non optimal buffer:
- need to make sure buffer is compatible with both enzymes which is why double digests are sometimes hard

Long reaction time:
- leaving the reaction overnight is bad

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23
Q

What is the problem with RE and methylated DNA

A

Two methylatrasfases in ecoli, dam and dcm methylate specific dna sequences

Bad bc if a RE recognition site is methylated the RE can’t cut

So we need to use a dam-/dcm- strain of ecoli with our plasmid

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24
Q

So what all messes up RE usage

A

End of DNA

Star activity

Methylated DNA

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25
What are RNases Where are they found
Since the reaction they do is easy, RNases are small and resilient Found in human skin and bodily fluids, also autoclaving gets rid of all dna but not all rna (which is why we need to be careful with rna contamination) There RNase A and H
26
What is the template, bonds broken, products, and uses n lab of RNASE A
Isolated from the pancreas Template: - cuts ss and ds RNA Bonds broken - endonuclease that cuts after a pyrimidine - single stranded cut Products - leave a 2,3’ cyclic-phosphate - makes short oligos that end in a C/U (because that where it cuts) Uses in lab - removes RNA from genomic or plasmid DNA
27
What is the template, bonds broken, products, and uses n lab of RNASE H
Found in all organisms because it removes RNA primers from Okazaki fragments Template: - cuts RNA in an RNA/DNA duplex (meaning stand of dna and on top is strand of RNA) Bonds broken - nonspecific endonuclease - single stranded cut Products - leave a 5’ phosphate - makes short oligos of RNA Uses in lab: - destroying RNA template after cDNA sysnthesis - cut targeted RNA sequences using complimentary DNA
28
What is the template, bonds broken/formed, products, and uses n lab of DNA ligases
Template: - two split dsDNA pieces or a nicked dsDNA (one bond on one strand is broken - the peices need to be complimentary and have a 5’ phosphate and 3’ OH Bonds broken/formed -phosphodiester bond formed Products -one dsDNA molecules Uses in lab - cloning - attaching adapters for high-throughput sequencing
29
What ligate is used in the lab and Whag does it do What is specificity and how does it ligate
T4 DNA ligase Purified from the e.coli phage T4 Has broad substrate specificity (meaning it works with all types of cut templates) Ligase uses ATP only for energy, not to incorporate the adenine base
30
What are examples of a kinase and a phosphatase Why are their products important
T4 polynucleotide kinase Alkaline phosphatase The ligase rxn needs a 5’ phosphates, if not there ligation can’t happen so kinases important to add that
31
Generally how does DNA 5’(de)phosphorylation work How do the kinase work
Have 5’ polhosphare, the alkaline phosphatase removes that and turns it into 5’ OH If it none of the ends have phosphates, the kinase come in to readd them
32
What is the template, bonds broken/formed, products, and uses in lab of T4 polynucleotide kinase (PNK)
Purified from ecoli phage T4 Template: -ss and ds DNA and RNA with a 5’-OH (to add the phos) Bonds broken/formed -transfers the gamma phosphate of ATP to the 5’ OH (so need ATP for the phosphate and not just for energy like the ligase) Products -5’ phosphate Uses in lab - cloning: oligonucleotides are usually supplied with 5’ Oh - radioactive labelling of DNA: gamma-32P ATP
33
What is the template, bonds broken/formed, products, and uses in lab of alkaline phosphatases
Types: Calf-intestinal alkaline phosphatase (CIP) Shrimp alkaline phosphatase (SAP) Template: -ss and ds DNA and RNA phosphate end (to take it off) Bonds broken/formed - removes 5’ and 3’ phosphates Products -5’ OH and 3’ OH Uses in lab -for clonging it prevents religation of linearized plasmid dna after double digest (so that is only ligates with insert 5’ phos)
34
What is the template, bonds broken/formed, products, and uses in lab of polymerases
Template: -ss DNA and RNA template - a region of ds (primer), so it needs the template ss but needs a region ds to start adding DNA Bonds broken/formed -continuous formation of phosphodiester bonds Products -ss DNA and RNA that may or may not stay bound to the template/ primer (ex. For rna pol the rna doesn’t stay bound to the template, it floats off) Uses in lab - cloning: oligonucleotides are usually supplied with 5’ Oh - radioactive labelling of DNA: gamma-32P ATP
35
What are the examples of polymerases
DNA pol RNA pol Reverse transcriptase RdRP TdT
36
Explain polymerization and how the reaction is carried out What does it need
Polymerization always In 5’-3’ direction with ss rna or ss dna as template Needs : - a triphosphate on the base and uses the alpha phosphate to stay in the strand - a 5’ phos on the nucleotide and 3’ oh on the template - Nucleotides need to match the template The anhydride bond (between the alpha and beta phos ) is broken and that give energy for the reaction Beta gamma phos leave , alpha incolrated into dna (so radio labels alpha phos if want to )
37
Whag types of polymerases are: DNA pol RNA pol Reverse transcriptase RdRP TdT
Template / product DNA pol: dna dependent dna pol RNA pol: DNA dependent rna pol Reverse transcriptase : rna dependent dna pol RdRP: rna dependent rna pol
38
39
What is a template independent polymerase
Terminal transferase (can add dna to rna or dna templates)
40
What are the important polymerase attributes
Processivity Fidelity Specificity Thermostability
41
Describe polymerase processivity
The number of nucleotides processed in a single binding event before it falls off So how long the pol stays bound to the template
42
Describe polymerase fidelity
How accurate the pol is - So how many errors (incorrect base pair it makes), the error rates (chance of error per base) - some pol have 3-5 exonuclease activity (proofreading activity that act as a backspace if incorrect base pair added)
43
What is the error rate for taq pol
3 errors every 10,000 bp (3 x10^-4) This is a lot
44
What pol attributes are important for PCR
Specificity and thermostability
45
DNA dependent dna pol: What is the diffence in how dna pol I and Klenow frangment/t4 dna pol fill nicks
So region in middle of sequence missing DNA pol I: added new base to that gap and removed and readds bases ahead of it Klenow fragment/T4: only fold in the nick, doesn’t remove and readd existing nucleotides ahead of it
46
DNA dependent dna pol product are always
DsDNA The new strand is always attached to the other strand of dna
47
Describe dna pol I domains function and used in lab Where does it come from
Source is E. coli Three domains: - 5-3 dna pol (dna dep dna pol) - 3-5 exonuclease (for proofreading) - 5-3 exonuclease (to remove primer of the lagging strand and also replace existing nucleotides) Function: - digests the strand ahead of it as it polymerizes - primer removal or the lagging strand in dna replication Uses in lab: - used in nick translation to make labeled oligonucleotide probes - used in fluorescent in situ hybridization (FISH) - used in sourthern blot - used in radio active labelling (alpha 32p DNTP)
48
Whag diff in the Klenow fragment and the dna pol I
The Klenow fragment is a fragment of the dna pol I Keeps the 5-3 dna pol activity and 3-5 exonuclease activity BUT loses the 5-3 exonuclease activity (so cant replace existing nucleotides)
49
What is the Klenow fragment now used for
Turn sticky ends to blunt ends: - by fill 5’ overhangs - by removing 3’ overhangs Used to fill gaps Used to label ends of DNA
50
What is the T4 dna POL
Isolated from the T4 bacteriophage Has same domai name function as the Klenow frangemnt But has higher processivity and stronger 3-5 exonuclease activity than Klenow fragment
51
What are the dna dependent dna pol
DNA pol I Klenow fragment T4 dna pol
52
What are rna polymerase (dna depended rna pol) isolated from
Isolated from phages (T7, T3, SP6) - they each have their corresponding promoters in the expression plasmids (ex T7 promoter)
53
What is rna polymerase (dna depended rna pol)
5-3 rna pol that uses a DNA template (so does transcription) Makes ssRNA product Does not require a primer to start polymerizing The promoter must be dsDNA Uses NTP not dNTP
54
How is transcription termination happen in expression plasmids What is the alternative
Usually there’s a transcription termination signal in the plasmid to stop transcription But sometimes it’s not there so you linearize the plasmid to make it so transcription run off happen where the pol fall of at end of linear plasmid
55
What is RNA pol used to make
MRNA for translation RNA for studies on RNA structure Labelled RNA for probes (FISH, northerns) sgRNA FOR CRISPR
56
What is reverse transcriptase
RNA dependent dna pol From retroviruses 5-3’ pol, use dNTP Makes a dsDNA/RNA hybrid from a ssRNA product Requires a primer (DNA or RNA) but the new strand made is DNA (so dNTP) - So dna added on top of RNA using DNA or RNA primer Used in: - c DNA synthesis (mRNA to cDNA) - quantitative PCR to measure RNA levels in the sample by turning rna to dna
57
What do you do once reverse transcriptase reaction is done
Don’t want rna anymore , just want the cDNA So use RNase H (to digest RNA in the rna/dna hybrid) after reaction complete
58
Don’t need to know about RdRP
Okay Slode 50
59
What is tdt and its reaction it catalyzes, uses In lab How does it function
Terminal deoxynucleotidyl transferase (TdT), template independent dna pol Found in vertebrate immune system Reaction: Can only add dna (dntp) to the 3’ ends of any dna in 5-3 direction Template: - ss and ds DNA (blunt or overhangs), so any dna - 5-3 dna pol Function: - if given all four dNTP, it randomly add that to the end - if given a single type (DCTP) only add string of C to the end Uses in lab: - TA cloning - labelling DNA and rapid Ampkification of cDNA ends (RACE)
60
How can function of TDT help in finding dna sequence
- if given a single type (DCTP) only add string of C to the end So then you can do that to the dna and make a primer binding to all those c ‘S (made a primer binding site)

Bchrm 413 Final Exam (13 decks)

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