today you will… explain the process of dna replication. identify the specific roles of the enzymes...

Enter question text...

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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

Meselson & Stahl

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

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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’

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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

Replication Video

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

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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.

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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.

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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.

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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.

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0% 1.1. DNA ligaseDNA ligase

2.2. PrimasePrimase

3.3. TopoisomeraseTopoisomerase

4.4. HelicaseHelicase

5.5. DNA PolymeraseDNA Polymerase