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In which phase of the cell cycle is dna replicated?
1.1.G1G1
2.2.SS
3.3.G2G2
4.4.M PhaseM Phase
DNA Replication Overview
Overview:1.DNA strands separate.2.Complimentary base
pairing (A-T and G-C).3.THREE STEPS
1.Initiation2.Elongation3.termination
4.Tons of enzymes involoved.
An entire “team” of An entire “team” of enzymesenzymes and and proteinsproteins are are
responsible for responsible for each stage of each stage of replication.replication.
Step 1: Initiation
• Origins of Replication (ori) – specific sequence of nucleotides along DNA where replication begins–Replication proceeds in BOTH directions of the replication bubble
–Replication fork: Y-shaped area of bubble where DNA elongates
Enzymes• Helicase• SSB’s• Topiosomerase
TOPOISMOERASTOPOISMOERASEE
DNA directionality Review
Identify the 3’ carbon on the nucleotide.
DD
CC BB
AA
EE
0%
20%
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20%
0%
1. A
2. B
3. C
4. D
5. E
What would the complementary DNA strand be for the following
sequence?5’-CGTATG-3’5’-CGTATG-3’
0%0%0%0%
5’GCATAC- 3’ 5’GCAUAC- 3’
3’GCATAC- 5’ 3’GCAUAC- 5’
1.1. 5’GCATAC-3’5’GCATAC-3’
2.2. 5’GCAUAC-3’5’GCAUAC-3’
3.3. 3’GCATAC-5’3’GCATAC-5’
4.4. 3’GCAUAC-5’3’GCAUAC-5’
Stage 2: Elongation• ADD new NT’s• DNA Polymerase III –
adds new NT’s to the growing end of the new DNA strand – Only added to 3’ side
Priming of DNA for synthesis
• Primase Primase enzyme: attaches enzyme: attaches to parent strand and to parent strand and makes/lays down primers…makes/lays down primers…
• PRIMERSPRIMERS = short segment of RNA nucleotides used to begin the replication
• Primer formation MUST PRECEDE DNA replication
It takes energy…
• Nucleotide added as Triphosphate…to provide energy
Each equipped with energy!Each equipped with energy!
Polymerase
Addition of Addition of nucleotidesnucleotides
Question
This cleaving is…This cleaving is…
Hydrolysis or Dehydration Hydrolysis or Dehydration SynthesisSynthesis
Endergonic vs ExergonicEndergonic vs Exergonic
Leading vs lagging strand
New strands are synthesized differently
• Leading StrandLeading Strand:• 3’-5’ template/parent
– 5’-3’ new strand/daughter
– Continuous DNA synthesis towards Y of replication fork
• Lagging StrandLagging Strand:– 5’-3’ template/parent– 5’-3’ in fragments– Discontinuous synthesis
away from opening replication fork
Replication of the LEADING strand
1.1. PrimasePrimase adds an RNA primer
2. DNADNA Polymerase Polymerase IIIIII adds NT’s in the daughter 5’ to 3’ direction
3. Elongation is continuouscontinuous towards the opening replication fork
Replication of the LAGGING strand
• Same as leading except…Same as leading except…
• Replication is discontinuousdiscontinuous, through multiple segments (Okazaki Okazaki fragmentsfragments)
• Proceeds away from the direction of opening of the replication fork
• DNA ligaseDNA ligase bonds Okazaki fragments
All according to FORK
Stage 3: Termination
• When Replication Fork encounters neighboring ORI
Real Time Video
DNA polymerases
• DNA polymerase III– 1000 bases/second!– main DNA builder
• DNA polymerase I– 20 bases/second– editing, repair & primer removal
Arthur Kornberg1959
Roger Kornberg2006
Editing & proofreading DNA
• 1000 bases/second = lots of typos!
• DNA polymerase I (Nuclease Activity)– proofreads & corrects
typos
– repairs mismatched bases
– removes abnormal bases
– reduces error rate from 1 in 10,000 to 1 in 100 million bases
1
2
3
4
What does it really look like?
telomeres
• Repeated base pairs
• TTAGGG repeated 100-1000x to make strands equal
• Non-coding
• Postpones DNA erosion at end
The scientist(s) whose experiment confirmed semiconservative
replication.
Her
shey
& C
hase
Mes
elson
& S
tahl
Ave
ry, McC
arty
, ...
Wat
son
& Cr
ick
Griffi
th
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1.1. Hershey & ChaseHershey & Chase
2.2. Meselson & StahlMeselson & Stahl
3.3. Avery, McCarty, & Avery, McCarty, & MacLeodMacLeod
4.4. Watson & CrickWatson & Crick
5.5. GriffithGriffith
The enzyme whose function is to separate the two strands of DNA
prior to replication.
0%
96%
4%
0%
0% 1.1. DNA ligaseDNA ligase
2.2. PrimasePrimase
3.3. TopoisomeraseTopoisomerase
4.4. HelicaseHelicase
5.5. DNA PolymeraseDNA Polymerase
The enzyme whose function copy the template DNA strand one
nucleotide at a time.
78%
0%
0%
13%
9% 1.1. DNA ligaseDNA ligase
2.2. PrimasePrimase
3.3. TopoisomeraseTopoisomerase
4.4. HelicaseHelicase
5.5. DNA DNA PolymerasePolymerase
The enzyme whose function is to form phosphodiester linkages between okazaki fragments.
0%
0%
9%
4%
87% 1.1. DNA ligaseDNA ligase
2.2. PrimasePrimase
3.3. TopoisomeraseTopoisomerase
4.4. HelicaseHelicase
5.5. DNA PolymeraseDNA Polymerase
The enzyme whose function is to correct the “overwinding” that occurs ahead of
the replication fork.
0%
0%
100%
0%
0% 1.1. DNA ligaseDNA ligase
2.2. PrimasePrimase
3.3. TopoisomeraseTopoisomerase
4.4. HelicaseHelicase
5.5. DNA PolymeraseDNA Polymerase
