DNA replication Flashcards
Define helicase
The enzyme that unzips the DNA for replication
Define SSBP protein (AJT photo)
A protein that binds to the single-stranded DNA to avoid hydrogen bonds between the two diff strands
- prevents closing
(REVIEW) why is a DNA strand 5’ to 3’ and 3’ to 5’ (aka why are they anti-parallel?) [add another photo]
For the 5’ to 3’:
- The 5th carbon (phosphate atom) bonds to the third carbon of another
For the 3’ to 5’:
- no “horizontal flip” (my words) — MUST rotate
- to make the nitrogenous bases face each other
- make the hydrogen bonds
- The third carbon (the phosphate atom bonds to the 5th carbon
Describe the process of DNA replication
- Helicase unwinds
- SSBP attaches to nucleotides to avoid closing
- primer attached to nucleotides to let the DNA Polymerase know where to start
- DNA polymerase continuous adding with leading strand (old: 3’ to 5’, new: 5’ to 3’)
- Lagging opposite direction (old: 5’ to 3’. new: 3’ to 5’)
- DNA polymerase moves in intervals bc of it
- okazaki fragments added to DNA replication
- DNA ligase (poly I) “glue” the okazaki frags together by adding a nucleotide in between
- then two semi-conservative daughter DNA is formed
Describe DNA polymerase III [2]
An enzyme that adds nucleotides to the two old DNA strands during replication
- moves in 5’ to 3’ direction
- therefore, leading strand OK with direction
- however, NOT ok with lagging strand
Define RNA primer
a short genetic sequence of RNA that tells the DNA polymerase where to start
Define primase (?)
the enzymes that synthesize RNA primers (from free nucleotides?)
What is the direction of the semi-conservative model (aka old strand and new strand)
For leading:
- old strand: 3’ to 5’
- new strand: 5’ to 3’
For lagging:
- old: 5’ to 3’
- new: 3’ to 5’
Define DNA replication
DNA sequence is copied during S-Phase of cell cycle
Define DNA gyrase
- enzyme that stabilizes DNA structure as it unwinds
- why?: the compression of the wound/ not yet unwinded parts of the DNA unwinding
Define DNA polymerase III
- enzyme
- adds nucleotides in the 5’ to 3’ direction
- (both lagging and leading)
define DNA ligase
joins okazaki fragments on lagging strand
- seals the nicks
Describe the PCR
- synthetic (artificial) method of lengthening specific seuqences of DNA
- useful when theres small amount of DNA available (e.g. crime scenes)
- Taq DNA polymerase enzyme is used for PR
Describe Taq DNA polymerase
- used for PCR
- from thermus acquaticus, heat resistant bacterium
- therefore, resists denaturation at high temperatures
- copies up 1000 nucleotides at a minute
Describe the PCR process
1.) Denaturation
- DNA sample heated to 95*C
- to break hydrogen bonds and separate to 2 strands
2.) Annealing
- DNA sample cooled
- primers attach to the opposite ends of target seq
3.) Elongation (70C - 75C)
- heat-tolerant/ Taq DNA polymerase copies strands
What determines if a DNA strand is 5’ to 3’ or 3’ to 5’
which carbon in the pentose sugar is exposed
What is the role of the RNA primer?
- To let the DNA polymerase know where to start
- By adding a short nucleotide sequence wherein other nucleotides can bond to (?)
In which direction and where can the DNA helicase unwind?
Anywhere and both directions as long as its an A-T rich region
Why is the 3’ carbon the ONLY available carbon for bonding in DNA?
Because it has one bond left
- available for phopshodiester bond
Give the alternative name for PCR
Thermocycle
Define “vitro”
outside
- e.g.: PCR = vitro DNA replication
What is the temperature in the denaturation process of PCR?
90-95*C
Give the temperature for annealing phase of PCR
50-60*C
Give the temperature for the elongation phase of PCR
- 70-75*C
- (for the Taq Polymerase)
Give the purpose of the “Sanger Sequence”
- To know the full genetic sequence
What makes Sanger Sequence different from PCR?
the use of ddNTP
Define ddNTP
dideoxyribonucleic tri-phosphate
- deoxyribose sugar without an Oxygen atom at 3’
- attached to 3 phos
Give the different types of ddNTP
1.) ddATP
2.) ddTTP
3.) ddCTP
4.) ddGTP
What does ddNTP do?
it stops the (further) genetic sequencing
How does ddNTP terminate genetic sequencing?
It removes the oxygen carbon of 3’
- removes the bonding ability due to removal of oxygen’s additional bond
What will happen to the DNA sample because of ddNTP?
Instead of 1 long DNA strand, we get multiple of diff lengths
Describe the process of Sanger Sequencing
1.) DNA sample
- DNA strand split into 4 test tubes
- 1 ddNTP each
- attaches and stops the sequencing at random pts
2.) Gel electrophoresis
- separation of DNA by size
- in wells
- electric current: law of attraction, poles, mvmt.
- most neg: most long -> most positive: least long
3.) Analysis
- read from the bottom up
- determine the sequence
How do the single strand binding proteins prevent repannealing
The pull away the backbones of the strands which breaks the hydrogen bonds
What does the exonuclease do?
The exonuclease removes the RNA primers because of their different structure AND replaces them with DNA
What is the function of DNA ligase
To seal the nicks
Define dNTP
deoxyribonucleoside
Distinguish nucleotide and dNTP
dNTPs:
- from the cytoplasm
- used by DNA polymerase III to make new strands
- Tri-phosphate
- loses two phosphate because of hydrogen and phosphodiester bond
- becomes nucleotide
Nucleotide:
- One phosphate only
Why can’t lytic cycle become lysogenic?
Because of lysis (cell burst)
Discuss the evidence provided by viral structure and function that suggests they evolved from living organisms
- complexity in structure of some viruses would suggest bacteria ancestor
- e.g. maybe from parasitic bacteria
- retroviruses and some eukaryotic cells w/ reverse transcriptase
- genetic material moved out of cells
- obligate parasites, couldn’t exist without cells
- viruses have similar genetic material as cells (DNA and RNA)
Outline the stages in the production of mRNA by transcription
- DNA is unwound by RNA POLYMERASE,, breaks the hydrogen bonds between the DNA nucleotides
- new nucleotides complementary bound to the anti-sense strand by RNA polymerase, transcribing the DNA in a 5’ to 3’ direction.
-* A to U, C to G* - newly synthesized mRNA detaches from anti-sense/template strand strand and unwinds
Give the alternative name for exonuclease
DNA polymerase I
How do dNTPS become Nucleotides?
They bond to others nucleotides and lose 2 phosphate atoms for the hydrogen bond and the phosphodiester bond)
Give the temperatures for PCR (BOOK)
1.) Heating: 90-95C
2.) Cooling: 54C
3.) Elongation: 72*C optimum for TAQ DNA polymerase
Outline the process of DNA profiling [4]
- DNA is obtained from material/individual (e.g. crime scene, hair, blood, mouth, swab, etc.)
- *tandem repeats are copied by PCR ~13 times min.
- gel electrophoresis separates by lengths
- DNA patterns always the same and is unique to individual
- [insert application here]
Explain Chargaff Experiment (brief)
- Main Findings in Support of: Complementary base pairing + ** DNA IS THE GENETIC MATERIAL**
- BEFORE: tetranucleotide — equal nitro base concentration.
- DURING EXP: Sampled the nucleotide concentration of different orgs + viruses
- AFTER: A-T and C-G very similar concentrations, DNA has variation
