DNA replication in Nucleic acid Biochemistry

8/9/2019 DNA replication in Nucleic acid Biochemistry

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NUCLEIC ACIDNUCLEIC ACIDBIOCHEMISTRYBIOCHEMISTRY

BCHS 4306

Lecture 3:DNA Replication/ Repair/Recombination


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FUNDAMENTAL PROPERTIES OF DNA

Encode traits.

Self-Replication.

Mutation.

Transmission of traits.


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The Goal Transmission ofgenetic material

Mitosis Somatic cell division

Meiosis - Gamteogenesis


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The Human Genome is made up of 23ses of !h"omosomes

Male Female


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DNA replication in eukaryotesTightly coordinated with the cell cycle


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MetaphaseHomologous Chromosomes pair at the

metaphase plate AnaphaseCentromeres divide and sister chromatids

split

The Essence of Mitosis:Parent (2n) to Daughter cells (2n)

!n

2n

2n


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Each gamete contains only one copy of a chromosomeHomologous chromosomes pair during meiosis I

#stdivision

2nd division

gametes

The Essence of Meiosis:Parent (2n) to Daughter cells (1n)

2n 2n

2n 2n

n n n n


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Spermatogenesis & Oogenesis


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Fertilization and Development

n + n

(Haploid)

2n

(Diploid)


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The Mechanism for doublingDNA

DNA Replication

Key requirements

Accuracy and speed


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DNA ReplicationDNA Replication

Initiation

Elongation

Formation of replication fork

Termination

Telomere shortening/lengthening


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A. Replication InitiationProblem 1- Where to start?

Origin of Replication Replication begins at the origins of

Eukaryotic DNA has Multiple Origins of Replication


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Problem 2

DNA polymerization occurs only on aprimed template


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DNA Polymerization:Needs 4 critical factors

1. A template strand of DNA to copy

2. DNA polymerase III and I

3. An RNA primer to extend on

4. Free nucleotides (dATP, dCTP, dGTP, dTTP)Added to the 3-OH end of the new chain as it grows

Template


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Nucleotides are the building blocks (units) ofDNA

A Nucleotide = sugar + phosphate + base


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DNA synthesisInvolves polymerization of nucleotide bases

Polymerization of a free nucleotide to the growing strand by formation of a phosphodiester bond between

5-phosphate of the free nucleotide and

3-OH of the growing strand.


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DNA ReplicationInvolves nucleotide polymerization


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B. DNA strand elongationProblem 3:Replication occurs only in the 5 to 3 direction

DNA strands are anti-parallel.

Replication can only proceed in 5 to 3 direction.

Only one strand can carry out continuous synthesis. Leading strand

The opposite strand (Lagging strand) requires a different strategy.

because DNA synthesis cannot occur in the 3 to 5 direction.


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DNA is made of two anti-parallelhelices

5& '&

5&'&


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The Replication ForkThe Replication Fork


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Solution:Two strategies for the two strands


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DNA replication in eukaryotesmultiple origins replicate chromosomes rapidly


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Problem 4

Lagging strand is discontinuous has gapsLagging strand is discontinuous has gaps

Solution 5-3 exonuclease activity of DNASolution 5-3 exonuclease activity of DNApolymerasespolymerases


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Messelson-Stahl Experiment


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DNA Replicationis Semi-Conservative


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Problem 5Problem 5

Maintaining high accuracyMaintaining high accuracy

At high speedAt high speed


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DNA Replication:High Accuracy & High Speed

Veryhigh fidelity(accuracy). Only 1 error in 1010bases.

Achieved by the proof reading function of DNA polymerase I

1. Pol I 3-5 exonuclease activity recognizes mismatches andcorrects them during replication.

2. Pol I 5-3 exonuclease activity destroys RNA primer of Okazakifragments

RNA primase lack proof reading function. Therefore RNA primers on okazaki fragments may have a highdegree of errors.

Pol I degrades RNA primers through 5-3 exonuclease activity

And replaces with DNA.


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DNA ReplicationSolution - 3-5 exonuclease activity of DNA Pol I


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Problem 6

Unwinding the double helix

Maintaining single stranded DNA

Relaxing supercoils

DNA t d l ti


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DNA strand elongationother problems

Unwinding the double helixUnwinding the double helix Helicases= disrupt hydrogen bonds which hold two strandstogether.

Maintaining the single strands and preventing the duplexMaintaining the single strands and preventing the duplex

from reformingfrom reforming Single stranded binding proteins(SSB)

Relaxing the supercoils forms by the unwinding of theRelaxing the supercoils forms by the unwinding of thedouble helix by the helicases.double helix by the helicases. Topoisomerases

Breaks supercoiled DNA ahead of the replication fork via asingle stranded break or double stranded break.

Relaxes the supercoils by rotating DNA.

Rejoins the breaks to form a contiguous strand.


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Topoisomerase-DNA Gyrase:Relaxing supercoils ahead of the fork

Unwinding the doublehelix

ahead of the fork is thefirst step

Carried out byTopoisomerasewhich is a

DNA gyrase


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DNA Replication:DNA Replication:High Accuracy & High SpeedHigh Accuracy & High Speed

High speed.

TheE.coligenome which is 5 Megabases is replicated in40 minutes!

This occurs at a rate of 2000 nucleotides per second.

The replication fork must move at the rate of 1000 basesper second.

Achieved through the replisome

complicated enzyme complex that mediates all aspectsof replication


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Replisome:Replisome:The cellular replication machineryThe cellular replication machinery


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DNA replication in eukaryotesDNA replication in eukaryotesProblem 7: Eukaryotic DNA is packaged into chromatinProblem 7: Eukaryotic DNA is packaged into chromatin

Prokaryotes DNA naked in the cytoplasm

Eukaryotes have DNA packaged into chromatin and therefore theadditional task of

Disassembling the nucleosomes during replication initiation

and reassembling the nucleosome after replication termination.


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DNA replication in eukaryotesAssembly of nucleosomes following replication termination

Chromatin assembly factor (CAF-1) binds histones and targets them

to the replication fork.

CAF-1 and histones bind the clamp protein proliferating cell nuclearantigen (PCNA) which brings the histones onto the chromatin.


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Problem 8Problem 8

Telomere shortening

DNAReplication


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DNA ReplicationProblem: Chromosome ends remain un-replicated resulting in

telomere shortening

The leading strand isuna)le to completereplication.

*t the ends ofchromosomes.

The ver+ ,rst R* primercannot )e replaced.


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DNA Replication in Eukaryotes:Problem: Chromosome ends remain unreplicated

hromosome shortening /ith each c+cle.


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Telomere problemTelomere problem

Telomere shortening is a good thing..

Limits lifetime of the majority of cells in our body (somaticcells)..

Through process of aging or senescense.

However some cells need to be immortal

Stem cells need to have unlimited proliferative potential

Needs telomere lengthening process..


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The Solution : TelomereLengthening

5& TT*GGGTT*GGGTT*GGG.....'&


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Telomerase extends telomeres:Consequence-repeats at chromosome ends


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Telomerase:A special enzyme that extends telomeres

Telomerase has an RNA associated with it.

Acts like the RNA primer and allows the end to be filled in.

Elongates shortened end.

Telomere lengthening


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Telomere lengthening

Figure "-2a


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Defects in telomere replication Inability to lengthen telomeres Werner syndrome - prematureaging

Inappropriate lengthening of telomeres - cancer

ReverseTranscription


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Reverse TranscriptionDNA replication using RNA as a template


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Polymorphism vs. MutationPolymorphism vs. Mutation

Polymorphismsare variations in the DNA sequence of anindividual genome.

Generally the termPolymorphismrefers to Changes in the DNA sequence which has no impact on gene function And therefore are silent in relation to phenotype.

Can be within a gene or outside genes.

Mutationsare polymorphismswhich result from changes in theDNA sequence ofa gene.

When the mutations impacts gene function it manifests as aphenotype(heritable trait).

Mutationis the ultimate source of evolutionary change

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MutationsCover a broad array of different changes in the DNA

May affect

Entire chromosomes

Parts of chromosomes

Gene Regions

1.Affect single base pair regions-SNPs and INdels

2.Alter the number of copies of a small repeated sequence

Di-nucleotide repeats used extensively as markersto track disease.

Tri-nucleotide repeats (Involved in a special class ofdiseases)

More complex repeats

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Point MutationsPoint Mutations

An alteration in the DNA sequence in a single location.

The change can involve a single nucleotide or a small stretch ofadjacent nucleotides.

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S t M ttiS t M tti


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Spontaneous MutationSpontaneous MutationThree MechanismsThree Mechanisms

1. Spontaneous depurination

1. Mammalian cells spontaneously lose 10,000 purines from itsDNA in a 20 hr. cell generation at 37oC.

2. Spontaneous deamination

3. Oxidative damage of bases

1. Caused by superoxide radicals, H2O2, and (.OH) radicals

produced as byproducts of normal aerobic metabolism.

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SpontaneousMutationMechanisms


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Spontaneous Mutation MechanismsDepurination and Deamination

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Consequences of deaminationConsequences of deamination

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Mutation Mechanisms:3. DNA Replication Errors

DNA Replication is usuallyhighly accurate.

Only 1 mistake in every 109base pairs!

2


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DNA Rep#i!aion E""o"sFa## ino h"ee !ae$o"ies

#. 0ase Su)stitutions

2. 0ase 1nsertions and eletions

'. Repeat e3pansions

'


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DNA Replication Errors1. Base Substitutions

DNAReplicationErrors


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DNA Replication Errors2.Insertions / Deletions (indels)


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DNA Replication Errors3. Tri nucleotide repeat expansion

MutationRate


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Mutation RateVariable from gene to gene

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Average = 1 x 10-6mutations / gene / generationRange : 10-7- 10-4mutations / gene / generationMeans that about 1/20 gametes has a newly mutated gene


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Induced MutationsInduced Mutations

Mutagens increase the mutationsrate

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Induced Point Mutations: MechanismsInduced Point Mutations: Mechanisms

1. Base Replacement

2. Base Alteration

3. Base Damage

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Mutagens


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MutagensAre characterized by two properties

Mutational specificity

Transitions (purine-to-purine or pyrimidine-to-pyrimidine_

Transversions (purine-to-pyrimidine or pyrimidine-to-

purine) Insertion/deletions

Preferential location = HOT SPOTS

"(

l


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1. Base Replacement :Results in mispairing of nucleotide bases during DNA replication

Tautomeric shifts

Keto form (normal) to imino form

Keto form (normal) to enol form

Base Analog

5-bromouracil

2-amino purine

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T Shf


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Tautomeric Shifts5-Bromouracil is an analog of Thymidine (T)

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The keto-form of 5-BU basepairs normally with adenine

The ionized-form of 5-BU basepairs with guanine

U.A to C.G transition

TautomericShifts


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Tautomeric Shifts2-amino purine is an analog of adenine (A)

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When protonated 2-APmispairs with cytosine

A.T to G.C transitions

2-AP can pair with Thymidineas normal

A.T to G.C transition


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2. Base Alteration2. Base Alteration

Mutagenic Agents Alkylating agents Intercalating agents

Results in Mispairing of nucleotide bases during DNAreplication

The consequence is a mutation

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AlkylatingagentsmediatedmutationAlkylatingagentsmediatedmutation


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Alkylating agents mediated mutationAlkylating agents mediated mutationTransition G.C to A.T or T.A to C.G

Alkyl Agents

EMS (ethylmethanesulfonate)

NG (nitrosoguanidine)

Add alkyl groups (ethyl group by EMS and methyl

group by NG) to many positions of all 4 bases.

O-6-alkyl-guanine results in mispairing of G with T.

Transition G.C to A.T

O-4-alkyl-thymidine results in mispairing of T with G

Transition T.A to C.G

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I l AIt lti A t


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Intercalating AgentsIntercalating Agents

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Flat planar molecules which mimic base pairs

slip in between stacked nitrogen bases at the core of the doublehelix.

Inducessingle base pair insertions or deletion

3. Base Damage3. Base Damage


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ggG.C to T.A.

Aflotoxin B1 Carcinogen which attaches to guanine at N-7

breaks bond between base and sugar

to create an apurinic site

Causestransversions G.C to T.A.

UV-generated photoproducts

Formscyclobutane pyrimidine photodimers.

CausesC to T transitions mainly.

Transversions, frame-shifts, large duplications and deletions.

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DepurinationinducedbyaflatoxinDepurinationinducedbyaflatoxin


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Depurination induced by aflatoxinDepurination induced by aflatoxinG.C to T.A

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Aflatoxin B1(AFB1) is a powerful carcinogen.

Attaches to the N7 position of Guanine (G).

Results in the breakage of the bond between AFB1bound G and the sugar.

Liberates base.

Results in an apurinic site.

Often results in the addition of an adenine in the apurinic site.

Resulting in aG.C to T.A transversion

IonizingRadiationIonizingRadiation


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Ionizing RadiationIonizing Radiation

Can penetrate through skin and affectgermline (UV cannot)

Can ionize or break DNA directly

Can ionize other chemicals that canreact with DNA

Is there a threshold?

$(

h d h


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UV Light Produces Thymine DimersUV Light Produces Thymine Dimers

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An increasing number of mutationsAn increasing number of mutationsassociated with human disease areassociated with human disease are

knownknown

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Mutations


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MutationsLocation, Location, Location!

SickleCellAnemiaapointmutationintheSickleCellAnemiaapointmutationinthe


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Sickle Cell Anemia a point mutation in theSickle Cell Anemia a point mutation in thecoding regioncoding region

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Normal Allele for

Hemoglobin A

Mutant Allele for

Sickle Cell(Hemoglobin S)

Glu6Val

Mutations at the splice junctions can alsoMutations at the splice junctions can also


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lead to disease:lead to disease:

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Neurofibromatosis

HuntigntonsDisorderTripletrepeatHuntigntonsDisorderTripletrepeat


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Huntigntons Disorder - Triplet repeatHuntigntons Disorder - Triplet repeatexpansionexpansion

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FragileXsyndrome


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Fragile X syndromeCGG expansion in the 5 region

DNA replication in Nucleic acid Biochemistry

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