Lecture 3 Flashcards
How many base pairs are in a human genome?
3x10^9
What is the error rate of DNA replication?
1 mistake in every 10^9 base pairs is seen following DNA replication
How are most errors corrected?
Proofreading and DNA repair
How many nucleotides are changed every cell division?
3
How are errors further corrected?
By post replication repair mechanisms
Why do germ cells need to have a low mutation rate?
To maintain the species
Why do somatic cells need low mutation rates?
To avoid uncontrolled proliferation/cancer
What enzyme synthesizes DNA? How?
DNA polymerase
Brings in an incoming deoxyribonucleoside triphosphate that bonds with the hydroxyl group of the DNA residue on the primer chain. P2O7 falls off and another residue is on the DNA chain
What decides the type of dNTP added to the primer strand?
The template
New chain is assembled in a preexisting template that is complementary to incoming bases
In order to use the template what is required?
The separation of the two parental strands
Needs enough nucleotides - dATP, dGTP, dCTP and dTTP
DNA polymerase requires a primer with a free 3’-OH to begin
How does the primer strand extend?
5’->3’
Replication fork is ___________
Asymmetric
In what order are the strands of DNA replicated?
Both are simultaneously replicated
In what direction does DNA polymerase synthesize DNA?
5’ -> 3’ direction
What is the leading strand?
Strand that is synthesized continuously 5’ ->3’ and its template is in the 3’ -> 5’ direction
What is the lagging strand?
Strand synthesized in segments because its template is in the 5’->3’ direction
How is proofreading done by DNA polymerase?
DNA polymerase makes a mistake out of every 10^9 nucleotides but DNA polymerase tightens its fingers around the active site, before a new nucleotide is added. If the fingers can tighten then the correct base pair is in place
What is exonucleolytic proofreading?
When a incorrect base is added, the base does not pair with the template. DNA polymerase requires a perfectly paired 3’ -OH terminus so the action of the DNA polymerase going backwards 3’-5’ cause it to exonuclease to clip off unpaired residue at 3’primer terminus
Why is DNA replicated 5’->3’?
Because it allows for efficient error correction in a energy conserved way
How is the lagging strand replicated?
Replicated through backstitching process:
- DNA primase synthesized a 10 nt long RNA primer to prime DNA synthesis
- Once DNA polymerase makes fragments of DNA along the lagging strand, RNA primer is erased by RNAseH and replaces it with DNA.
- DNA ligase joins the ends
Why does the DNA primase use RNA and not DNA?
DNA primase can sit down on the DNA without a primer and synthesize RNA, but with this ability there is not much of a proof reading ability. That is possibly why RNA is used so there can be more mistakes but ends up getting erased when RNA is degraded
What must happen before replication takes place?
DNA helicase must unwind DNA
What is the structure of DNA helicase?
6 identical subunits that binds and hydrolyzes ATP
What does the binding and hydrolyzes of ATP in helicase cause?
Conformational change that propels it like a rotary engine along single stranded DNA, passing it through a center hole
What is DNA helicase capable of?
Prying apart the helix at rates of 1000 nucleotide pairs/sec
Since base pairs energetically favor pairing, once separated by helicase, how do the strands stay separated?
Single-stranded DNA binding proteins bind tightly and cooperatively to exposed SS DNA.
They help stabilize unwound DNA and prevent formation of hairpins.
Ultimately, DNA bases remain exposed
How does DNA polymerase stay on DNA?
Sliding clamp keeps DNA polymerase on DNA when moving and releases when double stranded DNA is encountered.
How does the sliding clamp and DNA polymerase assemble?
The assembly requires clamp loader: it hydrolyzes ATP as it loads the clamp onto a primer template junction
What happens to the clamp loader on the leading strand?
Clamp loader does not have to stay around because the sliding clamp remains associated to DNA polymerase for long stretches
What happens to the clamp loader on the lagging strand?
It stays close so it can assemble a new clamp at start of each new Okazaki fragment
What removes almost all errors missed by proofreading?
Mismatch repair proteins
How do mismatch repair proteins know which strand is the new strand and the one to correct?
In E. coli, depends on methylation to distinguish new strand
In human, depends on single strand breaks that are present on lagging strand before Okazaki fragments are ligated.
*how leading strand is fixed is unknown
How do the mismatch proteins function?
MutS binds to mismatch; MutL scans for the nick and triggers degradation of nicked strand
What happens with the mismatch repair gene is mutated?
Cells accumulate mutations at high rate
HNPCC (colon cancer)
What is DNA topoisomerases?
A reversible enzyme that breaks a phosphodiester bond to change superhelicity, thereby relieving supercoiling.
How does supercoiling occur?
As replication fork moves, it creates a winding problem for the parental helix. Every 10bp replicated corresponds to one turn
What is Type I Topoisomerase?
Enzyme that catalyze the relaxation of supercoiled DNA in a thermodynamically favorable process.
Work by creating transient single strand break in DNA which allows the DNA on either side of the nick to rotate freely relative to each other, uses the other phosphodiester bond as a swivel point.
How does resealing occur after Type I topoisomerase releases supercoil tension?
Resealing is rapid and doesn’t require any additional energy since energy is stored in the phosphotyrosine linkage
What is Type II topoisomerase?
Enzyme that makes a transient double stranded break in the DNA.
It is activated at sites on chromosome where two double-stranded helices cross each other.
It uses ATP
It can separate “decatenate” 2 interlocked DNA circles
Can prevent severe tangling problems that would arise
What does type II topoisomerase use ATP for?
- break one double-stranded helix reversibly to create a “gate”
- Causes second strand to pass through
- reseals break and dissociates
Where are replication origins?
A-T rich regions where sequence attracts initiator proteins to pry open DNA
Where is the replication origin in bacteria?
Process of A-T rich regions where sequence attracts initiator proteins to pry open DNA in eukaryotes is similar to bacteria but details and regulation differ.
What is the only point of control for E.coli in replication?
Initiation, so it is highly regulated
When can initiation in E.coli occur?
Proceeds only when sufficient nutrients are present.
Before replication can occur again the bacteria must go through a refractory period.
Why are A-T rich regions typically where replication origin takes place?
Because A-T only has double bonds so it is energetically favored to break two bonds instead of 3 (G-C)
What is a refractory period in E.coli?
Delay until new strand is methylated
How many replication forks are enough for bacterial genomes to be replicated?
2
How long does it take for bacterial genome to be replicated? why?
40 minutes because genomes are small
How long would it take to do an average chromosome with a single ORI (origin of replication)?
Traveling at 50nt/sec, 800 hours
In bacteria, how is DNA helicase + helicase loader attracted?
Initiator proteins bind to specific sites in ORI, forming a complex. And this complex attracts the helicase and loader
How long does the helicase loader remain engaged in bacteria?
Until helicase is properly loaded
Once helicase is loaded on bacteria genome, what proceeds?
Helicase unwinds DNA so primase can make RNA primer on leading strand; remaining proteins assemble to create 2 replication forks with complexes moving in opposite direction with respect to the ORI
When does eukaryotic DNA replication occur?
During DNA synthesis phase (S) which lasts about 8 hours for mammalian cells
Chromosomes are replicated to produce what in eukaryotes?
To produce two complete copies, joined at centromeres until M phase
How is replication activated in eukaryotes?
In clusters (replication units) consisting of 20-80 orgins
Different regions of each eukaryotic chromosome are replicated how?
In a reproducible order during S phase, depending on chromatin structure
when is heterochromatin replicated in eukaryotes? When are regions of genome with less condensed chromatin replicated?
It is late-replicating because timing is related to packing of DNA in chromatin. Less condensed is replicated first
What are the minimum requirements for sequence to be ORI in yeast?
- must have binding site for ORC (origin recognition complex)
- Must have an A-T rich stretch for easy unwinding
- Must have binding site for proteins that help attract ORC
