Module 3 Flashcards
DNA
Deoxyribonucleic Acid
DNA ____ and ____ genetic information
Stores, transmits
What happened to a mouse when injected with virulent / non virulent bacteria. What would happen if heat was added to each bacteria? What does this conclude?
Virulent - mouse dies of pneumonia
Nonvirulent - mouse remains healthy
Heads added (darkens and kills bacteria)
Killed virulent - mouse remains healthy
Killed virulent and live nonvirulent - mouse dies of pneumonia
- concludes that a molecule was transmitting virulence (Frederick Griffith 1928)
How do we know DNA transmits virulence?
- Made a virulent bacteria extract with DNA, RNA, and protein that was extracted from heat-killed virulent cells, and purified to make a solution
- Got 4 solutions of nonvirulent bacteria and added the following to each container:
- Solution only
- Solution + RNase
- Solution + protease
- Solution + DNase
- Containers 1, 2, and 3 became a solution of virulent and nonvirulent bacteria, container 4 remained a nonvirulent bacteria solution
- This indicated that DNA was removed by enzyme DNase, and therefore the cause of virus transmission
Where is genetic info stored?
In genes
What are nucleic acids made of?
Nucleotides
What is a nucleotide?
- building blocks of nucleic acids (DNA)
- made up of a base (A, C, T, or G), a pentose sugar, and at least 1 phosphate group
What makes each nucleotide distinct?
The bases
Characteristics of bases
- Purines (double-ring)
- Adenine or Guanine - Pyrimidines (single-ring)
- Thymine or Cytosine
Nucleoside vs. Nucleotide
Nucleoside: base + sugar
Nucleotide: base + sugar + phosphate (gives (-) charge)
how are nucleotides linked together?
through the phosphate group on the 5’ carbon of nucleotide 2 attacking the 3’ hydroxyl (OH) of nucleotide 1 = phosphodiester bond
phosphodiester bond: C-O-P-O-C
- result is nucleotide 1 has 2 (-) charges on the phosphate, nucleotide 2 has 1 on the left oxygen of phosphate group
DNA has _____. The phosphodiester bonding results in a free ___ phosphate group at the top and a free ___ hydroxyl (OH) group at the bottom.
polarity, 5’, 3’
What is the structure of DNA?
double helix
what 3 conclusions allowed the double helix of DNA to be proved?
- complementary base pairing probably between A + T and C + G (not proved until 2nd finding)
- OH and NH (hydrogen bonds link bases)
- 1 OH and 1 NH between A and T (2 bonds)
- 2 OH and 1 inner NH between G and C (3 bonds) - Chargaff’s Rule - regardless of cell type:
%A = %T and %G = %C - X-Ray Crystallography of DNA - DNA is a helical, repetitive structure
Watson-Crick model
10 base pairs per complete turn (3.4 nm)
diameter of DNA: 2 nm
1. sugar phosphate backbone in rightward direction
2. major groove - large turn
3. minor groove - small turn
base stacking
non-polar, flat sides of bases face inwards and stack tightly
- sugar phosphate backbone is polar
- DNA strands are antiparallel
steps to go from DNA helix -> chromosomes
- DNA helix 2nm diameter
- DNA wrapped twice around histone protein (+) charge, makes nucleosome beads
- Nucleosome beads linked create chromatin fiber (30nm diameter)
- Chromatin fiber is coiled (300nm diameter)
- Coils even more (700nm diameter)
- Condensed chromatid (1400nm diameter)
2 theories of DNA replication
- Conservative - first replication makes 2 parental strands, 2 new ones
- 2nd replication makes 1 parental, 3 new ones - Semi-conservative - first replication makes 2 half parental half new strands
- 2nd replication makes 2 new strands, 2 half parental half new ones
Is DNA conservative or semi-conservative? How was this tested?
DNA is semi-conservative
- radioactive N isotopes used to label DNA in bacteria
1. Parental strands made with 15N label = heavy DNA
2. Daughter strands made with 14N label = light DNA
3. Measured density of DNA after each replication
Finding was also confirmed in eukaryotes using fluorescent nucleotides after 2 rounds of replication
- daughter strand had fluorescence, parental had none (daughter appeared black on screen, parent was lighter colour)
- density of DNA
- density of DNA after 1 replication
- density of DNA after 2 replication
1) 1.722 gm/cm^3
2) 1.715
3) 1.708 daughter, 1.715 parent
Does A-T bond have 2 NH bonds?
No, it has 1 OH and 1 NH because NH is a + bond, do not want them to repel
Steps for DNA synthesis / replication
- Helicase unwinds DNA 5’ -> 3’ (breaks H-bonds)
- forms replication fork
- replication always occurs from 5’ -> 3’ - RNA Primase binds to make RNA primer’s on both strands
- primer allows DNA polymerase to bind and replicate - DNA polmyerase makes complimentary DNA from RNA primer
- adds nucleotides/reads in 5’ -> 3’ - A different DNA Polymerase removes RNA primers to connect Okazaki Fragments on lagging strand
- DNA Ligase attaches all fragments in lagging strand (primers removed and replaced w/ DNA)
What are the stabilizers in DNA replication?
- Topoisomerase II - stabilizes unwound DNA during the process (behind helicase)
- relieves stress of unwounding - Single-strand binding protein (ssBP) - stabilizies DNA strands being replicated
- keep DNA apart
Trombone Loop
lagging strand forms this so both strands are elongated together
- allows the 2 RNA polymerases to stay in contact without strand interference
What enzyme is used when proofreading for misplaced/incorrectly added nucleotides?
DNA Polymerase - turns on exonucleic function by removing incorrect nucleotide, then adds in the correct nucleotide
Origins of replication in:
a) prokaryotes
b) eukaryotes
a) prokaryotes: one origin of replication
- replication starts at origin and moves around circular chromosome in both directions
b) eukaryotes: multiple origins of replication (ORI)
- 2 replication forks in one replication bubble move in opposite directions
- 2 replication bubbles meet to make one large bubble
telomere
- caps at the end of each chromosome
- chromosomes contain 1000s of these repeating sequences (5’ -> 3’)
- function is to prevent chromosome shortening during cell division
direction of leading and lagging strand
- leading strand points TOWARDS replication fork (where Helicase is)
- lagging strand points AWAY from replication fork
True or False: The parental strand and newly synthesized DNA strand are the same length on the leading and lagging strand
False: due to lack of priming, DNA polymerase cannot fill the gap at the very end of the chromosome for the lagging strand
True: for the leading strand
expected vs. observed result of chromosomes after cell division
expected: each time a cell divides, the chromosomes shorten until it bursts and becomes damaged
observed: the chromosomes are protected, length stays the same due to telomeres
how are telomeres incorporated into DNA strands within chromosomes?
- Telomerase protein has an RNA template built into it to synthesize the telomere repeat sequence
- DNA polymerase can fill in the shortened part of the linear DNA by complementary base pairing the new strand with the telomere repeat strand
Telomerase protein activity in:
1. Adult cells
2. Germ and Stem cells
- Adult cells: almost no Telomerase activity
- Germ and Stem cells: has Telomerase activity
consequences of low vs. high Telomerase activity in Germ/Stem cells
Low activity: linked to aging and rare inherited diseases
High activity: associated with cancer
Up to _____ telmorere repeats can be replicated to divide
100
A lack of telomerase activity limits what?
the number of times a cell can divide
PCR and its requirements
Polymerase Chain Reaction: amplification of 1 copy of DNA
Requirements:
- template DNA (to amplify this)
- DNA polymerase
- nucleotides
- 2 Oligonucleotide primers (fwd + reverse), complimentary to sequences of gene of interest (these replace RNA primers) -> the two primers point inwards to each other
Steps for a PCR
- Denaturation of double strand DNA
- using high heat (95 degrees) to break H-bonds -> under b.p. - Annealing of primers to complimentary sequences (60-70 degrees)
- using Hydrogen bonds
- must have excess primers - Extension of the DNA from the primer w/ Taq polymerase
- results in final amplified DNA
-> process repeated 20-30 cycles
Every cycle = 2^n copies
(n = number of cycles)
why does PCR need excess primers created?
to ensure that template DNA doesn’t reanneal
Taq Polymerase
a type of DNA polymerase from Thermus aquaticus (lives in hot springs)
what plays the role of Helicase in PCR?
heat
how does electrophoresis help visualized PCR products?
- agarose gel has pores to place them in
- small fragments move faster
- large fragments move slower
-> DNA is therefore separated by size - electrophoretic buffer well contains ions
- pores block large fragments
which enzyme synthesizes the RNA primer needed to initiate DNA synthesis?
Primase
deoxynucleotide vs. dideoxynucleotide
- deoxynucleotide has a hydroxyl (OH) group on the 3’ carbon, allowing this end to be elongated
- dideoxynucleotide lacks the 3’ hydroxyl group (just H at this end), thus cannot be elongated b/c no OH to attack incoming nucleotide triphosphate
use of dideoxynucleotides
the different fluoreescent labels on the end of each dideoxynucleotide (daughter strand) helps find unknown sequence of parent
microarray
a chip with different DNA sequences with known locations (single strands)
- helps identify disease mutations
cDNA probe
one denatured strand of DNA cut into smaller fragments
- use 2 samples (normal and tumor)
- label w/ fluorescent dyes and combine them
- hybridized probe to microarray and scan for divergences in gene sequences
GWAS
Genome wide association study: helps identify specific disease mutations
- CHIPS can be used to genotype 500,000 - 5 million SNPs
SNP
single nucleotide polymorphism
- a point mutation
DNA damage examples
- ssDNA breaks
- cross-link T bases (DNA adducts)
- missing base
- bulky side group attached to a base
- dsDNA breaks
list all DNA repair mechanisms
- Mismatch repair
- Base-excision repair
- Nucleotide-excision repair
- dsDNA break repair
first 3 are transcription-coupled/global genome repair, 4th is homologous recombination/non-homogolous end-joining
what are the DNA damaging agents?
- Base mismatch: DNA replication stress
- ssDNA breaks: oxygen radicals, ionizing radiation, chemotherapeutics
- DNA adducts: polyaromatic hydrocarbons, UV light
- dsDNA breaks: ionizing radiation, chemotherapeutics
mismatch repair
- MutS protein recognizes mismatched bases, initiates repair process
- MutL and MutH proteins are recruited, MutH breaks backbone some distance away (cleaves DNA)
- exonuclease enzyme removes nucleotides between the 2 proteins
- DNA polymerase fills in missing nucleotides, and DNA ligase joins the backbones
Base excision repair
repairs DNA when a base is damaged (e.g. cytosine loses a nitrogen group)
1. cytosine easily loses amino group, forming a base called uracil (after DNA replication)
2. uracil cannot base pair w/ guanine
3. DNA Uracil glycosylase cleaves uracil base from deoxyribose sugar
4. AP endonuclease cleaves DNA backbone to remove sugar
5. DNA polymerase fills gap and DNA ligase seals it
AP endonuclease
cleaves DNA backbone for base excision repair to remove sugar
DNA Uracil glycosylase
enzyme that cleaves uracil base from sugar for base excision repair
MutL, MutH, MutS
MutL - associates with MutS
MutH - cleaves DNA
MutS - recognizes mistmatched bases in DNA, initiates repair process
Nucleotide excision repair
(similar to mismatch repair)
- UV radiation can make 2 T’s bind to each other incorrectly
- enzymes cleave DNA at sites of damage
- DNA polymerase fills gap, DNA ligase seals DNA
CRISPR
clustered regularly interspaced short palindromic repeats
-> CRISPR-Cas uses short guide RNA (20 nucleotides) to bind to specific DNA sequences
-> Cas9 protein cuts/edits anything that resembles guide RNA (works like a pair of molecular scissors)
2 ways DNA editing used for disease
- allow cell itself to repair cut in DNA, leads to gene’s f’n being turned off
- researchers can insert, repair, or edit a gene by designing small DNA template, changing genome code
-> this maintains gene f’n and replaces a mutation
Benefits of DNA/Genome editing?
CRISPR-Cas9 gene editing helps fight sickle-cell disease in 2 ways:
- template-style editing of hemoglobin
- using guide RNA, Cas9 enzyme repairs faulty Beta-globin gene (subunit of hemoglobin) - remove a protein to prevent hemoglobin production
- Cas9 promotes production of fetal haemoglobin by breaking gene that encodes a repressor
Cons of DNA/Genome editing
- Off-target effects: there can be many sites in genome that look like guide RNA
- On-target effects: after Cas9 cuts DNA, repair mechanisms of cell are unpredictable (can be repaired perfectly -> religation, or some letters inserted/deleted)
- Mosaicism:
(1) if a developing embryo contains a few cells with risky mutations, a biopsy (to see if DNA editing needed) picks up that mutated cell and may lead to unnecessary manipulations
(2) CRISPR-Cas9 treatment may leave too many cells uncorrected to treat the disease (some mutated cells remain)
___ gene = ___ protein
1,1
Central dogma
DNA (in nucleus) -> RNA (in nucleus) -> Protein (in cytoplasm)
DNA vs. RNA
DNA RNA
Sugar: deoxyribose (H on 2’) ribose (OH on 2’)
Bases: A,T,C,G A,U,C,G
5’ end: monophosphate triphosphate
Size: very large smaller (1 gene)
Strands: double single (less stable)
Thymine vs. Uracil
T: base is CH3
U: base is H
RNA
ribonucleic acid
3 stages of DNA transcription
- Initiation: promotor sequence spans few 100bps for general transcription factors to bind
-> transcription starts 25bp downstream of promotor
-> transcription activator proteins bind enhancor sequence
-> general transcription factors recruit RNA Polymerase II, and transcription activator proteins recruit mediator complex to bind to RNA polymerase II + general transcr. factors - Elongation: RNA made from 3’ -> 5’ strand of DNA (RNA transcript grows 5’ -> 3’)
-> no replication fork b/c transcript only being made in 1 direction
-> done on transcription bubble on template strand - Termination: terminator sequence allows Polymerase to fall off DNA template (thus RNA transcription is complete)
coding vs. noncoding template strand
coding - nontemplate strand
noncoding - template strand
Regulation
occurs before initiation of DNA transcription:
- transcription starts at promotor sequence (TATATA sequence b/c easy to open apart) -> dictates what proteins are expressed in cell
- transcription ends at terminator sequence
housekeeping genes
needed all the time and are transcribed consistently
transcription factor in prokaryotes vs. eukaryotes
prokaryotes: sigma factor
eukaryotes: general transcription factor
enhancor sequences
require activation proteins to bind, it is upstream of gene of interest
general transcription factors
6 proteins that initiate transcription when binding to promotor region, recruit RNA Polymerase II
upstream + examples
anything BEFORE specific gene to be transcribed (promotors, enhancers)