Module 2-2 Flashcards
What does the semi-conservative replication suggest?
1 old strand and 1 new strand in each double stranded DNA molecule
What does the conservative replication model suggest?
New and old strand re-anneal after replication
What would the result be in dispersive replication?
one new and one old strand on each double stranded DNA molecule
What was the key to the Meselson-Stahl experiments?
differential labeling of the DNA strands
What element did Meselson-Stahl rely on?
that heavy Nitrogen 15 could be incorporated into DNA and label old strands 15N and new 14N
What were the findings of the Meselson-Stahl experimet?
After 3 generations of replication of E. coli labeled with 15N initially, a strand of 14N was seen as well as a smaller 15N/14N band, supporting semi-conservative nature
What was Eukaryotic replication studied using?
autoradiography using 3H-thymidine in the bean Vicia faba
How were the Eukaryotic replication studies done?
1.An unlabeled chromosome was replicated with 3H-thymidine and resulted in 2 labeled sister chromatids
2. After these replicated with unlabeled thymidine, found one labeled and one unlabeled chromatid as well as reciprocal regions of both chromatids labeled
SUPPORTED SEMI-CONSERVATIVE replication
What is the Origin of Replication?
Where replication of DNA starts
What is the Replication Fork?
where DNA strands are unwound
What is the Replicon?
a length of DNA replicated at one replication fork
What was found when studying E. coli replication using radioisotopes and autoradiography?
that there was one origin of replication (approx 245bp) and that the whole chromosome was replicated in one replicon (Theta replication)
*Supported bi-directional replication
What were the Kornberg experiments looking for?
enzyme that was able to replicate DNA in an in vitro system
What was the enzyme Kornberg found?
DNA polymerase I (DNA Pol I)
What was required in Kornberg’s experiments?
dNTP’s (A, T, C, G) and a template DNA (with partial compliment)
How does DNA Pol I synthesize the reactions?
Joins 5’ P to 3’ OH and releases 2 P
What were the problems of DNA Pol I in vitro?
- Slower rate of synthesis than in vivo
- Worked better on single stranded template than double stranded
- DNA Pol I also degraded DNA (exonuclease activity)
What is Biologically active DNA?
DNA that’s capable of supporting metabolic activities and directs reproduction of the organism from which it was originally duplicated
What was Kornberg’s reasoning?
IF DNA Pol I could make DNA that was biologically active, THEN it must be the major catalyzing force of DNA synthesis in cell
Describe Kornberg’s experiment with the øX174 phage.
- strand was replicated with BU 32P labeled strand with DNA polymerase I and DNA ligase
- and - strand were formed as double stranded DNA (RF duplex)
- DNase nicks one strand and are denatured to get seperate - strand and nicked + strand
- These undergo density gradient centrifugation and 32P/heavy BU-containing - strand is isolated
- This is replicated with a normal non-BU or labeled strand with DNA polymerase and DNA ligase
- and - strand formed when synthetic + strand is synthesized as new RF
- This is reacted with DNase and heated to give a + strand and nicked - strand
- These undergo density gradient centrifugation and gives unlabeled light synthetic + strands which are added to E. coli protoplasts and form new phages (it is infectious)
DEMONSTRATES BIOLOGICAL ACTIVITY
What were the conclusions from Kornberg’s experiment with the øX174 phage?
DNA Pol I demonstrated biological activity of DNA that it replicated
*DNA replication was accurate as any alteration in the 5386 bases would have probably rendered it non-viable
What were other DNA polymerases and their similarities?
DNA polymerase II and III
- all elongate DNA from a primer, 5’-3’ polymerization
- all have 3’-5’ exonuclease activity
- ONLY DNA POL I has 5’-3’ exonuclease activity
How many subunits is DNA Pol III holoenzyme dimer made of?
10 (BIG STRUCTURE)
What do the a and E subunits do?
a does 5’-3’ polymerization
E does 3’-5’ exonuclease
What is a replisome?
DNA polymerase III holoenzyme and other proteins at replication fork
What causes the replication bubble formation in E. coli?
binding by DnaA and ATP hydrolysis
What unwinds DNA in E. coli?
DnaB and DnaC
*NEED ATP AS ENERGY TO BREAK H BONDS
How is the tension relieved in DNA unwinding?
DNA gyrase enzyme (a DNA topoisomerase)
*makes single or double stranded breaks and the strands are released (NEED ATP)
What is the primer to allow replication of DNA template?
Short segment of RNA (5-15 nucs) complementary to DNA
*done by RNA polymerase (RNA primase) that allows DNA pol III to proceed
How are both strands of DNA synthesized?
DNA POL III ONLY SYNTHESIZES 5’-3’
- One strand is continuously synthesized on the leading strand
- Lagging strand is synthesized in segments (okazaki fragments)
How is the lagging strand synthesized?
RNA primers are added to lagging strand and DNA polymerization begins from these fragments and proceed until next RNA primer is encountered
How long are okazaki fragments in E. coli?
1000 - 2000bp
What removes the initial RNA primers?
DNA Polymerase I (5’-3’ exonuclease activity)
What fills the gap from the removal of the RNA primers?
DNA Polymerase I
What joins the ends of the gaps from the filling of the primer region?
DNA ligase which catalyzes phosphodiester bonds to seal the gap
How is the replication proofread to insure accuracy?
All polymerases have 3’–>5’ exonuclease activity
*detect and excise mismatches
What is the overall replication model?
- Helicase/primase at replication fork
- single stranded binding proteins stabilize open helix
- Gyrase removes tension
- Beta subunit clamps bind DNA
- Lagging and leading strand are synthesized simultaneously
- RNA primers removed (DNA pol I), gaps filled (DNA Pol I), okasaki fragments joined (DNA ligase)
How does the rate of Eukaryotic DNA replication compare to that of prokaryotic?
Eukaryotic replication is slower
What model was used to study eukaryotic DNA replication?
yeast
What were the multiple origins of replication called?
Autonomously replicating sequences (ARS)
What step of Cell cycle are ARS bound in?
G1 by specific proteins
What proteins must be disassociated and re-associated during replication?
Histone proteins
What are Pol a and g?
key eukaryotic DNA polymerases
- synthesis begins with pol a (low processivity)
- then switches to pol g to continue replication (100x rate increase)
What size are okazaki fragments in eukaryotes?
100-150 nucs
What problem does the antiparallel nature of DNA lead to?
problems at the end of chromosomes (TELOMERES)
*The final gap is unable to be filled, which shortens the ends of the chromosome during each round of replication
What replicates the telomeres?
the enzyme Telomerase that contains RNA and proteins which serve as guide and template and basepairing overlap is followed by reverse transcription of the overlap which extends the lagging strand
What is the sequence of human telomeres?
DNA 5’-TTAGGG-3’
RNA 3’-AAUCCC-5’
not active in most somatic cells
What accomplishes DNA recombination?
A series of enzymatic processes
- Endonuclease nicking
- Ligation
- Branch migration (determines length of transfer)
- Duplex separation forming Holliday structure
- Endonuclease nicking
- Ligation
How is Gene conversion performed?
DNA recombination causes mismatches if wild type and mutant type are in recombined region
- mismatch can be excises on either strand
- subsequent repair can lead to conversion to mutant or wild type
