Broad Course Objectives for DNA Replication Students will be able to: • describe the historic experiment that demonstrated DNA replication follows a semi-conservative model. • describe the process of DNA replication in prokaryotes at the biochemical level • explain how proofreading and repair is accomplished during DNA synthesis
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Broad Course Objectives for DNA Replication Students will be able to: describe the historic experiment that demonstrated DNA replication follows a semi-conservative.
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Broad Course Objectives for DNA Replication
Students will be able to:• describe the historic experiment that demonstrated
DNA replication follows a semi-conservative model.• describe the process of DNA replication in
prokaryotes at the biochemical level• explain how proofreading and repair is accomplished
during DNA synthesis
Outline/study guide—DNA Replication
• At what point in the cell cycle does DNA replication occur?
• When two DNA molecules (or chromosomes) are made from one, where do the parental strands end up, vs. the newly synthesized strands? (i.e. semiconservative replication)
• Why can DNA only be synthesized in the 5’ 3’ direction?
• What are the enzymes and proteins involved in DNA synthesis? What is the function of each and at what point do they act?
• At what point does RNA function in DNA replication?• What determines the lagging strand vs. the leading strand? How does
this change on the “other” side of the replication origin?• How are the Okazaki fragments joined into one continuous DNA strand?
• How does the DNA replication machinery correct errors made during replication?
• Are human chromosomes linear or circular? Bacteria?• Why do linear chromosomes (but not circular chromosomes) have a
problem with telomeres becoming shorter and shorter with each round of replication? How do some cells get around this?
48 year old woman with Werner Syndrome
Progeria
T A
G
C
A
G
A T
T A
T
G
GA
A
C
C
CT
T
G
C G
T A
T A
C
G
A T
T A
C G
T
A T
C G
C
C G
A T
C G
CA
CGG
C
Incomingnucleotides
Original(template)strand
Original(template)strand
Newlysynthesizeddaughter strand
Replicationfork
(a) The mechanism of DNA replication (b) The products of replication
After one generation, DNA is “half-heavy”(consistent with both semi-conservative and dispersive models)
After ~ two generations: DNA is “light” and “half-heavy”(Consistent with which model?)
Why does DNA (and RNA) only “grow” in the 3’ direction?
New strand Template strand
5’ end 3’ end
Sugar A TBase
C
G
G
C
A
C
T
PP
P
OH
P P
5’ end 3’ end
5’ end 5’ end
A T
C
G
G
C
A
C
T
3’ end
Nucleosidetriphosphate
Pyrophosphate
2 P
OH
Phosphate
Fig from Cambell and Reece, 7th ed(Like Brooker, fig 13-15)
Brooker, fig 13.10
Origin of replication
Replicationforks
Direction ofreplication fork
1st Okazakifragment
First and second Okazakifragments have beenconnected to each other.
1st Okazaki fragmentof the lagging strand
2nd Okazakifragment
3rdOkazakifragment
Primer
Primer
The leading strand elongates,and a second Okazaki fragmentis made.
The leading strand continues to elongate. A third Okazaki fragment is made, and the firstand second are connected together.
Primers initiate DNA synthesis.Synthesis of the leading strand occurs inthe same direction as movement of thereplication fork. 1st Okazakifragment of lagging strand ismade in opposite direction.
DnaA proteins bind to DnaA boxes and toeach other. Additional proteins that causethe DNA to bend also bind (not shown).This causes the region to wrap aroundthe DnaA proteins and separates theAT-rich region.
DNA helicase
DNA helicase (DnaB protein) binds to theorigin. DnaC protein (not shown) assiststhis process.
DNA helicase separates the DNA in bothdirections, creating 2 replication forks.
From Cold Spring Harbor Symposia of Quantitative Biology, 28, p. 43 (1963). Copyright holder is Cold Spring Habour Laboratory Press.
Replicationfork
Bacteria only have one origin on their (circular) chromosome
Replication rate
• Eukaryotic DNA replication– Typical human chromosome length: 100 million bp– Time to replicate a chromosome: minutes to hours– Hundreds of origins per chromosome– Replicon = ~20,000 to 300,000 bp long– 500-5000 bp / minute at each replication fork
(slower than bacterial replication; that much harder to “unwind” the DNA for replication).
• Bacterial (prokaryotic) replication: – Single circular chromosome (~4.6 million base pairs
[bp])– Single origin of replication single replicon
(“Replication Bubble”)
Requirements of DNA Replication in a complex organism
• Very low error rate:– One human cell: 6 billion bp of DNA. A
copying error rate of 1 error/million nt 6000 errors with every cell division
• Very fast copy rate– E. coli –1000 nt per minute 3 days to
replicate (real life: 20 minutes per cell cycle; 1000 nt per second)
Brooker, fig 13.21
DNA polymerase cannot linkthese two nucleotides togetherwithout a primer.
No place for a primer
3′
5′
Linear chromosomes (eukaryotic) cannot easily replicate the ends of chromosomes
Chromosome gets shorter at the telomeres with each replication if overhang is left.
In humans and most complex organisms, telomerase is only used in continuously dividing stem cells (e.g. spermatogonia stem cells) most cells get shorter telomeres over time (age). What happened to Dolly, the cloned sheep? (she was generated from a skin cell with shorter telomeres, and she aged early)
Linear chromosomes (eukaryotic) must fill in gap left by RNA primer
Telomere
Telomerase
Eukaryoticchromosome
Repeat unit
3′
3
5′
T T A G G G T T A
A A T C C C A A TC C C A A U C C C
G G G A G G GT T A T TG G G
T T A G G G T T A
CC C A A U C C C
G G G T T A T T G
GG
T T AG G G A G G G
C C C A A U C C C
TT
C C C A A T A A A AT C C C U A AC UC CC C C
T T A G G G T T A G G G T T A T T GT T AG G G A G G G G G
T T A G G G T T A G G G T T A T T GT T AG G G A G G G G G GT T A G G
A A T C C C A A T
A A T C C C A A T
A A T C C C A A T
RNA
RNA primer
Telomerase synthesizesa 6-nucleotide repeat.
Telomerase moves 6nucleotides to the right andbegins to make another repeat.
The complementarystrand is made by primase,DNA polymerase, and ligase.