©2001 Timothy G. Standish Isaiah 40:28 28Hast thou not known? hast thou not heard, that the...

Isaiah 40:28

28 Hast thou not known? hast thou not heard, that the everlasting God, the LORD, the Creator of the ends of the earth, fainteth not, neither is weary? there is no searching of his understanding.

ReplicationReplicationTimothy G. Standish, Ph. D.

The Information Catch 22The Information Catch 22With only poor copying fidelity, a primitive

system could carry little genetic information without L [the mutation rate] becoming unbearably large, and how a primitive system could then improve its fidelity and also evolve into a sexual system with crossover beggars the imagination."

Hoyle F., "Mathematics of Evolution", [1987], Acorn Enterprises: Memphis TN, 1999, p20

Tools of ReplicationTools of ReplicationEnzymes are the tools of replication:DNA Polymerase - Matches the correct

nucleotides then joins adjacent nucleotides to each other

Primase - Provides an RNA primer to start polymerization

Ligase - Joins adjacent DNA strands together (fixes “nicks”)

More Tools of ReplicationMore Tools of ReplicationHelicase - Unwinds the DNA and melts itSingle Strand Binding Proteins - Keep

the DNA single stranded after it has been melted by helicase

Gyrase - A topisomerase that Relieves torsional strain in the DNA molecule

Telomerase - Finishes off the ends of DNA strands

Leading StrandLeading Strand

Laging StrandLaging Strand

5’3’

Extension - The Replication ForkExtension - The Replication Fork5’

5’5’3’

5’3’3’

Single strand binding proteins - Prevent DNA from re-anealing

DNA Polymerase

Okazaki fragment

RNA Primers

Primase - Makes RNA primers

5’3’

Gyrase - Relieves torsional strain

Helicase - Melts DNA

Extension - Okazaki FragmentsExtension - Okazaki Fragments

The nick is removed when DNA ligase joins (ligates) the DNA fragments.

3’ 5’5’ 3’

RNA PrimerOkazaki Fragment

RNA and DNA Fragments

DNA Polymerase has 5’ to 3’ exonuclease activity. When it sees an RNA/DNA hybrid, it chops out the RNA and some DNA in the 5’ to 3’ direction.

DNA Polymerase falls off leaving a nick.

DNAPol.

3’ 5’5’ 3’

RNA Primer

DNAPol.

3’ 5’5’ 3’

RNA PrimerLigase

The Role of DNA GyraseThe Role of DNA Gyrase

Helicase

Supercoiled DNA

Gyrase

E. coliE. coli DNA Polymerases DNA Polymerases E. coli has three identified DNA polymerases each of

which has significantly different physical characteristics and roles in the cell

Replication polymerization

10 subunits 600,000 Daltons

II IIIIPolymerase

Major function400 ? 15Molecules/cell

Yes Yes Yes5’- 3’ Polymerization

Yes Yes Yes3’-5’ Exonuclease

Klenow fragment (76,000 Daltons), prepared by mild proteolysis, lacks 5’ to 3’ exonuclease activity and is

used in sequencing

Repair of damaged

Yes No No5’-3’ Exonulcease

Proofreading/ Removal of

RNA primers109,000 Daltons

Telomere

TelomeraseTelomeraseAt the end of linear chromosomes the lagging strand can’t be completed as the last primer is removed and no 3’ hydroxyl group is available for DNA polymerase to extend from

3’5’5’3’

Degradation of RNA primer at the 5’ end

3’5’

5’3’

3’5’5’3’

Next replication

AACCCCAAC

TelomeraseTelomerase

TelomeraseTelomeraseTelomerase is a ribo-protein complex that adds nucleotides to the end of chromosomes thus restoring their length

GGGTTG5’GACCGAGCCTCTTGGGTTG3’CTGGCTCGG

AACCCCAAC

5’GACCGAGCCTCTTGGGTTG3’CTGGCTCGG

GGGTTGGGGTTG

AACCCCAAC

5’GACCGAGCCTCTTGGGTTG3’CTGGCTCGG

GGGTTG GGGTTGGGGTTG

TelomeraseTelomeraseThe TTGGGG repeating telomere sequence can form a hairpin due to unusual GG base pairing

5’GACCGAGCCTCTTGGGTTGGGGTTGGGGTTGGGGTTG3’CTGGCTCGG

Guanine

NGuanine

5’GACCGAGCCTCTTGGGTTGGGGTTGGGG3’GTTGGGG3’CTGGCTCGG

TTGGGGTTGDNA

5’GACCGAGCCTCTTGGGTTGGGGTTGGGGAGAACCCAACCCGTTGGGG3’CTGGCTCGG

DNAPol.

Endo-nuclease

5’GACCGAGCCTCTTGGGTTGGG3’CTGGCTCGG

Endo-nuclease

AGAACCCAACCCGTTGGGGT

GTTGGGG

MutationMutationWhen Mistakes Are MadeWhen Mistakes Are Made

5’ 3’

DNAPol.

5’ 3’

DNAPol.

3’ to 5’ Exonuclease activity

MutationMutationExcision RepairExcision Repair

5’ 3’

3’ 5’

Endo-Nuclease

5’ 3’

3’ 5’

5’ 3’

3’ 5’

5’ 3’

Endo-Nuclease

NicksDNAPol.

5’ 3’

3’ 5’

5’ 3’

3’ 5’

DNAPol.

Endo-Nuclease

5’ 3’

3’ 5’

5’ 3’

3’ 5’

5’ 3’

DNAPol.

Ligase

Endo-Nuclease

Ligase

DNA Replication:DNA Replication:How We KnowHow We Know

There are three ways in which DNA could be replicated:

NewOld

OldConservative - Old double stranded DNA serves as a template for two new strands which then join together, giving two old strands together and two new strands together

OldSemi-conservative - Old strands serve as templates for new strands resulting in double stranded DNA made of both old and new strands

Dispersive - In which sections of the old strands are dispersed in the new strands

ewOld +

The Meselson-Stahl The Meselson-Stahl ExperimentExperiment

The Meselson-Stahl experiment demonstrated that replication is semiconservative

This experiment took advantage of the fact that nucleotide bases contain nitrogen

Thus DNA contains nitrogen

HO ONH2

The most common form of Nitrogen is N14 with 7 protons and 7 neutrons

N15 is called “heavy nitrogen” as it has 8 neutrons thus increasing its mass by 1 atomic mass unit

After 20 min. (1 replication) transfer DNA to centrifuge tube and centrifuge

Disper

sive m

predict

Conservativ

model pre

diction

Semi-c

onservativ

model pre

diction

The Meselson-Stahl The Meselson-Stahl ExperimentExperiment

Prediction after 2 or more replications

Bacteria grown in N15 media for several replications

Transfer to normal N14 media

XThe conservative and dispersive models make predictions that do not come true thus, buy deduction, the semi-conservative model must be true.

The Current Eukaryotic The Current Eukaryotic Recombination ModelRecombination Model

Meiosis Prophase I

Homologous chromosomes

Double strand break

Exo-nuclease

DNAPolymerase

Holliday StructureHolliday Structure

Cutting The Holliday StructureCutting The Holliday Structure

©2001 Timothy G. Standish Isaiah 40:28 28Hast thou not known? hast thou not heard, that the...

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