Chapter 34: DNA Replication Flashcards
The process in which the DNA within a cell makes an exact copy of itself.
DNA Replication
Eukaryotic cells (self-cycle) replicate in what phase?
S phase
Must be done rapidly with control and accuracy
DNA Replication is considered
Highly coordinated
The E. coli genome has…
4.6 million base pairs
The E. coli is replicated in _____, at a rate of _____!
less than 40 minutes
Rate of 2000 nucleotides per second
Old duplex is conserved and new duplex composed of two completely new strands
Conservative
Sections of the old duplex dispersed somewhat randomly to the two daughter duplexes
Dispersive
Single strand of DNA is _____
Unstable
Base pairs face _____
Inward - poses a challenge for replication
Conservative replication takes a strand of DNA …
opens up the duplex and make a copy and close it back up
Start with original and make an exact copy
Dispersive replication is where you end up with
a hybrid where you could break the backbone of the DNA and religate it together
“Patchwork”
Two daughter duplexes with one each of the old and new strands
Semiconservative
Semiconservative was proposed by
crick and watson start with double stranded, split apart and end up with a copy. Two resulting strands with half of original and after second generation, two new strands
N14 is the _____ of nitrogen
Natural isotrope
Meselson Stahl Experiment
Developed a method to Get a very fine density gradient established in the tube and match it to the DNA
Can separate DNA that has N14 from N15 based on density
What gets labeled in the Meselson Stahl experiment?
Bases get labeled
In the Meselson Stahl experiment, First generation results eliminate _____
Second generation eliminates _____
First - conservative model
Second - dispersive model
New DNA is all grown in the presence of..
N14
By two generations, you end up with…
2 peaks at equal intensity (One peak is 50/50, the other is N14)
By the first generation, we know that the _____ model is not correct.
Conservative
By the second generation, progressively enrich…
Towards fully N14 but not with the discreet jump with two different peaks
_____ is at the heart of DNA replication
DNA Polymerase
Adds nucleotides 1 by 1
Phosphodiester bond formation
What are the 4 important characteristics of DNA Synthesis?
- Deoxynucleoside triphosphates and Mg2+ are required. dATP, dGTP, dCTP and dTTP
- A template strand is used to direct DNA synthesis.
- Chain extension is always 5’ to 3’
- A primer from which the new strand grows must be present
Binding of the _____ induces a structural change, closes over the nucleotide, forming a pocket into which only the correct nucleotide fits.
incoming dNTP
Specificity is determined by two factors:
- the correct hydrogen bonding between the incoming dNTP and the DNA template
- the overall shape of the incoming base
Specificity of replication is dictated by the _____.
Complementarity of bases
DNA Replication requires _____.
Highly processive polymerases
The holoenzyme responsible for the majority of DNA synthesis in E. coli is _____, a multi-protein complex.
DNA polymerase III
DNA polymerase III is highly processive - once it begins catalysis it rarely ____.
Releases the DNA substrate.
DNA’s replication process is…
Semi-conservative
In the Meselson-Stahl experiment, how many bands will conservative DNA show?
2 bands
Separation in the Meselson-Stahl experiment based on…
Density
The Meselson-Stahl experiment was used to determine…
The correct model for replication
The reaction catalyzed by DNA Polymerase produces…
1 extra base and the two phosphate groups drive the reaction forward
The primer will have a _____ and cleaves…
Hydroxyl group and cleaves the two phosphates of incoming base
The induced fit is not efficient, but…
Cannot afford to have errors in DNA, so it is better to have lost a little energy
dNTP removes _____ correct BP and will fall into the _____ section.
1/20
Exonuclease section
The base must match the _____.
Active site
1000x more accurate because of…
Polymerase I exonuclease activity
A holoenzyme does not require….
Any cofactors to activate
“Whole” how it is
DNA Polymerase III almost never…
Stops replicating, process keeps chugging
3 distinct stages of DNA replication:
Initiation, elongation and termination
Initiation
Adding primer
Elongation
Adding bases
Termination
Connecting fragments and ridding of primer/proofreading
DNA replication is carried out by a large complex containing multiple…
Proteins (>20)
The multi-step process of DNA replication:
Initiation
Helix must be unwound
Primer synthesis
Primer removal
One fork, two antiparallel strands
Connecting the pieces
Proofreading and error correction
Replication begins in E. coli at a unique _____ site called the…
245 base pair site called the oriC locus (replication primer)
Helicases are _____ and always require…
Unwinders and always require ATP
Single-stranded binding protein (SSB) bind to _____ to prevent _____.
AT rich sites to prevent reattachment of the helix
The initiation protein allows…
Unwinding of the helix
RNA polymerase (primase) enables…
DNA synthesis to begin
RNA primer adds _____ to serve as primer after DNA is unwound.
10 RNA nucleotides
DNA template ———->RNA Primer ———->Newly synthesized DNA
What fills in the arrows?
- Primase
- DNA polymerase
What prepares the double helix for unwinding?
Topisomerases
When DNA is unwound for replication, _____ occurs due to overwinding in nearby regions of the helix.
Supercoiling
Topoisomerases induce OR eliminate…
Supercoils
Type 1 topoisomerases…
Eliminate supercoiling
Type 2 topoisomerases…
Introduce supercoiling
The “negative” supercoiling starts to…
Unwind (Areas between supercoiling are where its unwound
Bacterial topoisomerase is a…
Therapeutic target
What are two inhibitors of DNA gyrase that are used to treat bacterial infections?
Nalidixic acid and ciprofloxacin
What prevents anthrax poisoning?
Ciprofloxacin
DNA gyrase is what type of topoisomerase?
Type 2 in bacteria
What two things are needed to separate the DNA strands?
Specific helicases and ATP hydrolysis
Helicases require _____ and separates _____ to make the DNA available for DNA polymerase.
ATP and separates double helix
Helicases have how many subunits?
6
Defects in the helicase can result in…
Werner syndrome
Werner syndrome causes…
Premature aging
The site of replication is called the…
Replication fork
The replication fork moves in one direction, so…
Both strands are copied simultaneously
All DNA polymerases synthesize DNA only in the…
5’ –> 3’ direction
Okazaki fragments have…
A primer between each fragment and allow 3’ –> 5’
What begins each okazaki fragment?
RNA Primer
What is the complex containing primase enzyme which synthesizes short pieces of RNA at the replication fork (complementary to the lagging-strand template)?
Primosome
Primosome adds your…
Primer
DNA polymerase uses the RNA primer to start…
The lagging-strand DNA snythesis
A _____ is needed to start each Okazaki fragment.
Primer
The lagging strand is _____ so that it passes through the polymerase active site in _____ direction, allowing synthesis to occur in the _____ direction.
Looped
Active site in 3’ –> 5’
Synthesis in 5’ –> 3’
DNA polymerase I is the _____ polymerase and deals with _____
Proofreading, deals with primer
The joining of Okazaki fragments by _____ and _____
DNA Polymerase I and DNA ligase
Problem: After Okazaki fragment synthesis is complete, there is a _____ between the end of Okazaki fragment and RNA primer.
Gap
Nick translation
DNA Polymerase I extends the Okazaki fragment while its 5–>3 exonuclease activity removes the RNA primer.
Nick translation adds _____ where primer was which fixes gaps.
Base pair
What is required to rejoin the break?
DNA Ligase (only needed for lagging strand)
DNA Ligase solidifies _____ in DNA and causes DNA to become _____ now.
Nicks in DNA
Continuous DNA now
What allows for removal of mismatched bases?
Exonuclease
3’–>5’ exonuclease activity contributes to the remarkably high fidelity of DNA replication - less than ____.
10^-8 errors/BP
Most uncorrected replication errors are subsequently corrected by….
The DNA repair enzymes (NOT proofreading)
Mistake rate is approximately…
1/100,000 bps, but DNA pol III and repair enzymes fix many
Human genome?
3.2 x 10^9 billion
Fruit fly large chromosomes
10x larger than E. coli, more complex (Millions of base pairs)
Human genome BPs?
6 billion
Eukaryotic chromosomes are _____.
Large linear, double-stranded DNA molecules
Prokaryotes have _____ DNA.
Circular
_____ of replication are required with each origin of replication representing a unit or replicon.
Multiple origins
Humans have how many replicons?
30,000 replicons
Proteins called _____ allow only one replication per replicon per round of DNA synthesis.
Licensing factors
DNA pol alpha is equivalent to _____ in prokaryotes
Pol I
Displays primase activity, initiates DNA replication in eukaryotes, generating a DNA molecule approx. 20 dNTP in length
DNA Pol alpha
Don’t incorporate RNA
DNA poly delta: A more processive enzyme, extends the chains. The switch from alpha to delta is called _____
Polymerase switching
Unique structures at the ends of linear chromosomes =
Telomeres
Telomeres are at the _____ of DNA
Free end of linear DNA
Two problems telomeres have:
- Susceptible to damage by nucleases
- Due to nature of DNA synthesis, one strand will shorten upon each round of DNA synthesis
IF telomere is shortened?
BAD = cell death
What replicates telomeres and fixes the overhang/gap?
Telomerase
Telomerase is RNA-dependent _____
DNA pol
Telomerase contains…
An RNA template that it uses to extend the leading strand
In rapidly dividing cells, including cancer cells, telomeres must be maintained by the telomerase to _____, which would lead to cell death.
Prevent shortening
_____ activity is characteristic of cancer cells (replicate uncontrollably).
High-telomerase activity
Which is NOT required for DNA synthesis?
- deoxynucleotide triphosphates
- a primer
- a template strand
- a DNA polymerase
- endonucleases
Endonucleases
The 3’ to 5’ exonuclease activity of DNA polymerase III is critical for:
- the processivity of polymerization
- removal of RNA primers
- the high fidelity of polymerization
- dimerization of the polymerase subunits
- DNA polymerase III does not have a 3’ to 5’ exonuclease activity
The high fidelity of polymerization
Enzymes which use the power of ATP hydrolysis to separate DNA strands are called:
- Restriction endonucleases
- Topoisomerases
- Separases
- Helicases
- Primases
Helicases
You set up a DNA replication reaction in a test tube using enzymes from E. coli. You add all the necessary components EXCEPT that you forget to add DNA ligase. When you then separate the strands to analyze the new DNA, what products would you expect?
- no DNA product at all
- normal full length DNA products only
- a mixture of full length strands, and shorter strands that include both RNA and DNA
- a mixture of full length strands, and shorter strands made of just DNA
- only short strands, which are made of just DNA
A mixture of full length strands, and shorter strands made of just DNA
You try your reaction again, remembering to add DNA ligase. This time, however, you forget to add primase. What products would you expect?
- no DNA product at all
- normal full length DNA products only
- a mixture of full length strands, and shorter strands that include both RNA and DNA
- a mixture of full length strands, and shorter strands made of just DNA
- only short strands, which are made of just DNA
No DNA product at all
Processivity refers to the:
Ability to catalyze many reactions without releasing the substrate
Which statement about DNA replication in eukaryotes in FALSE? Eukaryotic DNA replication:
- involves multiple DNA polymerases
- occurs on linear double-stranded DNA molecules
- utilizes multiple origins of replication
- utilizes DNA primers made by primase
- is highly processive
Utilizes DNA primers made by primase