DNADNA

Structure Structure & Replication& Replication

OverviewOverview

DNA StructureDNA Structure

Structure of DNAStructure of DNA

DNADNA is a is a double-strandeddouble-stranded molecule (two strands) molecule (two strands) The two strands The two strands windwind around each other forming a around each other forming a double helixdouble helix

Each strandEach strand of DNA molecule is a of DNA molecule is a polypolydeoxyribonucleotide:deoxyribonucleotide: (i.e. many (i.e. many monomonodeoxyribouncleotides)deoxyribouncleotides) bbase (A,G,C,T) + ase (A,G,C,T) + ssugar (deoxyribose) + ugar (deoxyribose) + pphosphoric acidhosphoric acid

MonodeoxyribonucleotidesMonodeoxyribonucleotides:: linked by 3`- 5`phosphodiester bonds linked by 3`- 5`phosphodiester bonds (PDE)(PDE)

to form polydeoxyribonucleotideto form polydeoxyribonucleotide In In eukaryotic cellseukaryotic cells (with a nucleus): DNA is associated with various (with a nucleus): DNA is associated with various

types of proteins (nucleoproteins) present in the types of proteins (nucleoproteins) present in the nucleusnucleus

In In prokaryotesprokaryotes, the protein-DNA complex is present in the , the protein-DNA complex is present in the nucleoidnucleoid

33 -` -`55 ` `pphosphohosphoddiieester bonds (PDE)ster bonds (PDE) PDEPDE bonds join the bonds join the 5`-5`-hydroxyl group of the hydroxyl group of the

sugar of one nucleotide to the sugar of one nucleotide to the 3`-3`-hydroxyl hydroxyl group of the sugar of an adjacent nucleotide group of the sugar of an adjacent nucleotide

through a through a pphosphate group –to give a long hosphate group –to give a long unbranched chain of nucleotides.unbranched chain of nucleotides.

The resulted long The resulted long unbranched chainunbranched chain has a has a polaritypolarity, ,

5`-end5`-end the end with free phosphate the end with free phosphate 3`-end3`-end the end with free hydroxyl group the end with free hydroxyl group

both ends are both ends are notnot attached to other attached to other nucleotidesnucleotides

The bases along the backbone are written in The bases along the backbone are written in sequence from sequence from 55`-end of the chain to the `-end of the chain to the 3`-3`-

endend

Phosphodiester bonds can be Phosphodiester bonds can be cleavedcleaved by by 1- chemicals 1- chemicals

2- hydrolysed enzymatically by 2- hydrolysed enzymatically by nucleasesnucleases.. (deoxynucleases: for (deoxynucleases: for DNADNA ribonucleases: for ribonucleases: for

RNA)RNA)

endoendonucleases: cleave the chain at positions nucleases: cleave the chain at positions in thein the

interior of the chaininterior of the chain exoexonucleases: cleave the chain by removing nucleases: cleave the chain by removing individual nucleotides from one individual nucleotides from one

of of the two endsthe two ends

Double helixDouble helix

In the double helix, the two chains In the double helix, the two chains are coiled around a common axis are coiled around a common axis

called the called the axis of symmetryaxis of symmetry

The chains are paired in an The chains are paired in an antiparallel mannerantiparallel manner

the 5`-end of one strand is paired the 5`-end of one strand is paired with the 3`-end of the other strandwith the 3`-end of the other strand

The The sugar-phosphatesugar-phosphate backbone of backbone of each chain is on the outside of the each chain is on the outside of the

molecule (molecule (hydrophilichydrophilic), whereas the ), whereas the basesbases are stacked inside are stacked inside

((hydrophobichydrophobic))

Major grooveMajor groove & & minor grooveminor groove: : provide an access for the binding of provide an access for the binding of

regulatory proteins for their regulatory proteins for their recognition sequences along the recognition sequences along the

DNA chain.DNA chain.

Dactinomycin (actinomycin D)Dactinomycin (actinomycin D)(anticancer drug): (anticancer drug): intercalate into the minor groove of intercalate into the minor groove of

the the DNA double helix ------ interfere withDNA double helix ------ interfere with

replication & transcription.replication & transcription.

Base pairingBase pairing

The The basesbases of one strand of DNA are of one strand of DNA are pairedpaired with the bases of the second with the bases of the second

strand.strand. AAdenine is paired with denine is paired with TThyminehymine GGuanine is paired with uanine is paired with CCytosineytosine

The base pairs are The base pairs are perpendicular to perpendicular to the axisthe axis of the helix of the helix

Accordingly, one polynucleotide chain Accordingly, one polynucleotide chain

of the DNA double helix is always the of the DNA double helix is always the complement of the othercomplement of the other

Chargaff`s RulesChargaff`s Rules: in any sample : in any sample amount of double-stranded DNA, the amount of double-stranded DNA, the

amount of adenine equals the amount amount of adenine equals the amount of thymine & the amount of guanine of thymine & the amount of guanine equals the amount of cytosine… the equals the amount of cytosine… the

total amount of purines equals the total amount of purines equals the total amount of pyrimidines total amount of pyrimidines

The base pairs are held together with The base pairs are held together with hydrogen bondshydrogen bonds

twotwo between between AA & & TT threethree between between GG & & CC

Separation of the two DNA strands Separation of the two DNA strands in the double helixin the double helix

The two strands of DNA separate when hydrogen bonds between the The two strands of DNA separate when hydrogen bonds between the paired bases are disruptedpaired bases are disrupted

Disruption occurs in the laboratory whenDisruption occurs in the laboratory when 1- 1- pHpH of the DNA solution is altered so that the nucleotide base ionize of the DNA solution is altered so that the nucleotide base ionize 2- solution is 2- solution is heatedheated

Melting temperature (TMelting temperature (Tmm)) : is the temperature at which one half of : is the temperature at which one half of the the

helical structure is lost. helical structure is lost. The loss of helical structure is called The loss of helical structure is called denaturationdenaturation Denaturation can be monitored by measuring DNA absorbance at Denaturation can be monitored by measuring DNA absorbance at

260 nm.260 nm. DNA that contains high concentrations of DNA that contains high concentrations of A & TA & T (two hydrogen bonds) (two hydrogen bonds)

denatures at lower temperature than DNA rich in denatures at lower temperature than DNA rich in G & CG & C (three (three hydrogen bonds) hydrogen bonds)

Structural forms of the double Structural forms of the double helixhelix

B-DNAB-DNA:: Right-handed helix Right-handed helix with 10 residues (base pairs) per 360with 10 residues (base pairs) per 360oo turn of the helix turn of the helix The planes of the bases perpendicular to the helical axis.The planes of the bases perpendicular to the helical axis. Chromosomal DNA consists primarily of B-DNAChromosomal DNA consists primarily of B-DNA

A-DNAA-DNA:: Produced by moderately dehydrating the B-DNA form Produced by moderately dehydrating the B-DNA form Right handed helixRight handed helix 11 residues per turn11 residues per turn The planes of the bases are tilted 20The planes of the bases are tilted 20oo away from the perpendicular to the helical away from the perpendicular to the helical axis.axis. Found in DNA-RNA hybrid or RNA-RNA double stranded region.Found in DNA-RNA hybrid or RNA-RNA double stranded region.

Z-DNAZ-DNA left handed helix left handed helix 12 base pairs per turn 12 base pairs per turn Zigzag sugar-phosphate backboneZigzag sugar-phosphate backbone Occur in regions of DNA that have sequence of alternating purines and pyrimidines Occur in regions of DNA that have sequence of alternating purines and pyrimidines

Circular DNA moleculesCircular DNA molecules

EukaryoteEukaryote Each chromosome in the Each chromosome in the nucleusnucleus of the of the eukaryoteeukaryote contains one contains one

long long linearlinear molecule of double-stranded DNA, bound to a complex molecule of double-stranded DNA, bound to a complex mixture of proteins to form mixture of proteins to form chromatinchromatin.-6+.-6+

In addition, eukaryotes have closed In addition, eukaryotes have closed circularcircular DNA molecules in their DNA molecules in their mitochondriamitochondria

ProkaryotesProkaryotes have single, double stranded, have single, double stranded, circularcircular chromosomechromosome Each chromosome is associated with histone-like proteins and RNA Each chromosome is associated with histone-like proteins and RNA that can condense the DNA to form a that can condense the DNA to form a nucleoidnucleoid.. In addition, most species of bacteria contain small, In addition, most species of bacteria contain small, circularcircular

extrachromosomalextrachromosomal DNA molecules called DNA molecules called plasmidsplasmids PlasmidsPlasmids carry genetic information and can undergo replication carry genetic information and can undergo replication

Prokaryotic Prokaryotic DNA synthesisDNA synthesis(REPLICATION)(REPLICATION)

SemiconservativeSemiconservative ReplicationReplication

When the two strands of the DNA When the two strands of the DNA double helix are separated, each can double helix are separated, each can serve as a template for the replication serve as a template for the replication of a new complementary strand.of a new complementary strand.

Each of the individual parental strandsEach of the individual parental strandsremains intact in one of the two new remains intact in one of the two new DuplexesDuplexes

(i.e. (i.e. one of the parental strands is one of the parental strands is conserved in each of the two conserved in each of the two new dublexesnew dublexes))

1-1- Separation of the two complementary DNA Separation of the two complementary DNA strandsstrands

In In prokaryotesprokaryotes, , the two parental strands separate the two parental strands separate in a small in a small single site single site called called origin of replicationorigin of replication. . In In eukaryoteseukaryotes, , replication begins at replication begins at multiple origins of replicationmultiple origins of replication along the DNA helixalong the DNA helix These sites include short sequences These sites include short sequences that are composed almost that are composed almost exclusively of AT base pairs.exclusively of AT base pairs. consensus sequencesconsensus sequences

22 - -Formation of the replication forkFormation of the replication fork

As the two strands unwind and separate, they form As the two strands unwind and separate, they form a region called the a region called the replication forkreplication fork, in which , in which active synthesis occurs.active synthesis occurs.

Replication fork moves along the DNA molecule as Replication fork moves along the DNA molecule as synthesis occurs.synthesis occurs.

Replication of double-stranded DNA is Replication of double-stranded DNA is bidirectionalbidirectional

(i.e. the replication forks moves in both directions (i.e. the replication forks moves in both directions away from the origin)away from the origin)

3`

5`

5`

3`

Replication ForkReplication Fork

Proteins required for DNA strand Proteins required for DNA strand separationseparation

Initiation of DNA replication requires the recognition of Initiation of DNA replication requires the recognition of the the

origin of replication by a origin of replication by a group ofgroup of proteinsproteins that form the that form the

prepriming complexprepriming complex

These proteins are responsible forThese proteins are responsible for::

1-1- Maintaining the separation of the parental strands Maintaining the separation of the parental strands

2-2- Unwinding the double helix ahead of the advancing replication Unwinding the double helix ahead of the advancing replication fork.fork.

Proteins required for DNA strand Proteins required for DNA strand separationseparation

1 -DnaA protein• 20 - 50 monomers of DnaA protein bind to specific nucleotide sequences at the origin

of replication (rich in AT base pairs).• ATP-requiring process causing the double strands to melt (separate) to form localized regions of single-stranded DNA2- Single-stranded DNA-binding proteins (SSB)• SSB bind only to single stranded DNA • keep the two strands of DNA separated in

the area of replication origin.• Protect the DNA from cleavage by

nucleases.3- DNA helicases• Bind to single stranded DNA and then move

into the neighboring double stranded region, to unwind the double helix.

• Require energy provided by ATP

Solving the problem of supercoilsSolving the problem of supercoils

As the two strands of the double helix are separated, As the two strands of the double helix are separated,

positive supercoilspositive supercoils appear which interfere with further appear which interfere with further unwinding of the double helixunwinding of the double helix

DNA topoisomerasesDNA topoisomerases

are responsible for are responsible for

removing these removing these

supercoilssupercoils

DNA TopoisomerasesDNA Topoisomerases

1- 1- Type I DNA TopoisomeraseType I DNA Topoisomerase Cuts Cuts singlesingle DNADNA strandstrand

(by nuclease then ligase)(by nuclease then ligase)

NoNo require for ATP require for ATP

Relax negative supercoils inRelax negative supercoils in

prokaryotesprokaryotes (e.g. in E. coli) (e.g. in E. coli) Relax positive & negative Relax positive & negative

supercoils in supercoils in eukaryoteseukaryotes..

2- 2- Type II TopoisomerasesType II Topoisomerases cuts cuts double double strandsstrands

Relax Relax bothboth positive & negative positive & negative supercoilssupercoils

Required for separation of Required for separation of interlocked molecules of DNA interlocked molecules of DNA following a chromosomalfollowing a chromosomal replication (in prokaryotes & eukaryotes)replication (in prokaryotes & eukaryotes)

Anticancer agents as Anticancer agents as etoposideetoposide target target humasn topoisomerase IIhumasn topoisomerase II

DNA gyraseDNA gyrase A type II topoisomerase A type II topoisomerase found in found in E.coli E.coli Introduce negative supercoils Introduce negative supercoils into relaxed circular DNA into relaxed circular DNA (to facilitates future replication of DNA)(to facilitates future replication of DNA)

Antimicrobial agents, Antimicrobial agents, quinolones (as ciprofloxacin)quinolones (as ciprofloxacin) target DNA gyrasetarget DNA gyrase

33 - -Direction of DNA replicationDirection of DNA replication

DNA polymerase is only able to DNA polymerase is only able to readread the parental the parental nucleotide sequences in the nucleotide sequences in the 3`-5`3`-5` direction direction

It It synthesizessynthesizes the new DNA strands in the the new DNA strands in the 5`-3`5`-3`

directiondirection..

So, beginning with one prenatal double helix, So, beginning with one prenatal double helix, the the two newly synthesized DNAtwo newly synthesized DNA must grow in opposite must grow in opposite

directions:directions:

1- One in 1- One in 5`- 3`5`- 3` direction direction towardstowards the replication fork the replication fork 2- One in 2- One in 5`- 3`5`- 3` direction direction away fromaway from the replication the replication

fork.fork.

3`

5`

5`

3`

REPLICATION FORKs

LAGGING STRAND template

READING DIRECTION

3` 5`

LAGGING STRAND template LEADING STRAND template

LEADING STRAND template

5` 3`

5`3`

5`3`

3`5`

Leading strandLeading strand

- TheThe strand that is being copied strand that is being copied in the direction of the of the

- advancing replication forkadvancing replication fork

Synthesized Synthesized continuouslycontinuously..

Lagging strandLagging strand

- The strand that is being copied in the direction - The strand that is being copied in the direction away away fromfrom the replication fork the replication fork

- Synthesized - Synthesized discontinuouslydiscontinuously with small fragments of with small fragments of DNADNA

(Okazaki fragments)(Okazaki fragments)

- They become eventually - They become eventually joinedjoined to become a single to become a single continuous DNA strandcontinuous DNA strand

44-- RNA Primer SynthesisRNA Primer Synthesis

DNA polymerase can not DNA polymerase can not initiate synthesis of a initiate synthesis of a complementary strand complementary strand of DNA on a totally single of DNA on a totally single stranded templatestranded templateRather, they require an Rather, they require an RNA primerRNA primer RNA primerRNA primer is a short fragment is a short fragment of RNA paired to the single of RNA paired to the single stranded DNA templatestranded DNA template The The RNA primerRNA primer has a has a free –OH group on the 3`-end of the RNAfree –OH group on the 3`-end of the RNAthat serves as an acceptor of that serves as an acceptor of the first nucleotide the first nucleotide

PrimasePrimase It is a specific It is a specific RNA polymeraseRNA polymerase It synthesizes short fragments of RNA (~ 10 nucleotides long), It synthesizes short fragments of RNA (~ 10 nucleotides long), complementarycomplementary & & antiparallelantiparallel to DNA template. to DNA template. RNA primers hybrid with DNA template (RNA primers hybrid with DNA template (UU in RNA pairs with in RNA pairs with AA in DNA) in DNA) In the replication forkIn the replication fork

In the In the leading strandleading strand: only : only one RNA primerone RNA primer is synthesized. is synthesized. In the In the lagging strandlagging strand, , many RNA primersmany RNA primers are constantly synthesized are constantly synthesized

along the along the strandstrand

The building block for RNA primer synthesis is The building block for RNA primer synthesis is NTPs NTPs ((AATP, TP, GGTP, TP, UUTP, TP, CCTP)TP)

PrimosomePrimosome

((PrimasePrimase + + Prepriming complexPrepriming complex))

This complex initiates Okazaki fragment formation by moving This complex initiates Okazaki fragment formation by moving

along the template for the along the template for the lagging strandlagging strand in the 5`-3` direction in the 5`-3` direction

periodically recognizing specific sequences of nucleotides that periodically recognizing specific sequences of nucleotides that

direct it to create an direct it to create an RNA primerRNA primer that is synthesized in the 5`- that is synthesized in the 5`-3` 3`

direction.direction.

Chain ElongationChain Elongation DNA polymerase IIIDNA polymerase III elongates a new DNA strand by adding elongates a new DNA strand by adding

deoxyribonucleotides deoxyribonucleotides dNTPdNTP (d (dAATP, dTP, dGGTP, dTP, dCCTP, TP, TTTP) to the TP) to the 3`-end of the growing chain (one at a time) starting from the 3`-end of the growing chain (one at a time) starting from the RNA primer.RNA primer.

The The sequencesequence of the nucleotides that are added is of the nucleotides that are added is dictateddictated by by the base sequence of the template strand.the base sequence of the template strand.

The new strand grows in the The new strand grows in the 5`- 3`5`- 3` direction, direction, antiparallelantiparallel to to the parental strand.the parental strand.

Energy requiredEnergy required:: Pyrophosphate (Ppi) is released when each new nucleotide is Pyrophosphate (Ppi) is released when each new nucleotide is

added to the growing chainadded to the growing chain Further hydrolysis of Ppi to two PiFurther hydrolysis of Ppi to two Pi Accordingly, a total of two high-energy bonds are used to Accordingly, a total of two high-energy bonds are used to

drive the addition of each dNTPdrive the addition of each dNTP

Chain Elongation StepChain Elongation Step

Location of workLocation of work: : Leading Leading && lagging DNA strands lagging DNA strands

starting from the RNA primer(s).starting from the RNA primer(s).

EnzymeEnzyme : DNA polymerase : DNA polymerase IIIIII

MaterialsMaterials: dNTPs (d: dNTPs (dAATP, dTP, dGGTP, dTP, dCCTP, dTP, dTTTP)TP)

Direction of elongationDirection of elongation: 5`-3` direction: 5`-3` direction

antiparallel to parental strandantiparallel to parental strand

Energy requiredEnergy required: two high-energy bonds : two high-energy bonds

example: example: dATP AMP + Ppi Pi + dATP AMP + Ppi Pi + PiPi

Proofreading of newly synthesized DNAProofreading of newly synthesized DNA

Misreading of the template sequence could result in Misreading of the template sequence could result in mutations.mutations.

To ensure replication correctness of sequence, DNA To ensure replication correctness of sequence, DNA polymerase polymerase IIIIII has a has a proofreading activityproofreading activity ( (3`- 5`3`- 5`

exonuclease activityexonuclease activity))

StepsSteps : :

1- DNA polymerase III 1- DNA polymerase III checkschecks for the correctness of for the correctness of matching matching

of added nucleotide to its complementary base on the of added nucleotide to its complementary base on the template.template.

2- If a wrong nucleotide is added, 3`-5` exonuclease 2- If a wrong nucleotide is added, 3`-5` exonuclease

activity activity editsedits the mistake the mistake

Excision of RNA primers & their replacement Excision of RNA primers & their replacement by DNAby DNA

On the On the lagging strandlagging strand DNA polymerase DNA polymerase IIIIII continues to synthesize DNA until it continues to synthesize DNA until it

isis blocked by an RNA primer.blocked by an RNA primer.

At that stage,At that stage, 1- RNA primer is 1- RNA primer is removed removed (excised) by (excised) by DNA polymerase DNA polymerase II (by 5`-3` exonuclease activity)(by 5`-3` exonuclease activity)

2- Then, the gap is 2- Then, the gap is filledfilled by by DNA polymerase DNA polymerase II (by 5`-3` polymerase activity)(by 5`-3` polymerase activity)

3-3- DNA polymerase DNA polymerase II proofreadsproofreads the filled gap DNA the filled gap DNA (by 3`- 5` exonuclease activity)(by 3`- 5` exonuclease activity)

DNA Polymerase IIIDNA Polymerase IIIDNA Polymerase IDNA Polymerase I5`-3`polymerase activity5`-3`polymerase activity

synthesis of new DNA strandsynthesis of new DNA strand5`-3`polymerase activity5`-3`polymerase activity

synthesis (filling) of gap of synthesis (filling) of gap of removed RNA primer removed RNA primer

Step 2Step 2

3`-5` exonuclease activity3`-5` exonuclease activity

proof reading of new strandproof reading of new strand33-`-`55 ` `exonuclease activityexonuclease activity

proof reading of the DNA filling proof reading of the DNA filling the gap of removed RNA primerthe gap of removed RNA primer

Step 3Step 3

5`-3` exonuclease activity5`-3` exonuclease activity

removal (excision) of RNA removal (excision) of RNA primers. primers. Step 1Step 1

FunctionFunction

Synthesis ---- ProofreadSynthesis ---- ProofreadFunctionFunction

Removal ----synthesis ----- Removal ----synthesis ----- ProofreadProofread

DNA ligaseDNA ligase

The final phosphodiester linkage between the 5`-phosphate The final phosphodiester linkage between the 5`-phosphate group on group on

the DNA chain synthesized by DNA polymerase III and the 3`-OH the DNA chain synthesized by DNA polymerase III and the 3`-OH

group on the chain made by DNA polymerase I is catalyzed by group on the chain made by DNA polymerase I is catalyzed by DNA DNA

Ligase.Ligase.

This process requires energy derived from the cleavage of ATP This process requires energy derived from the cleavage of ATP

to AMP + Ppi to AMP + Ppi

Eukaryotic DNA ReplicationEukaryotic DNA Replication The process of eukaryotic DNA replication closely follows The process of eukaryotic DNA replication closely follows

that of prokaryotic DNA synthesis. that of prokaryotic DNA synthesis.

Main DiffrerencesMain Diffrerences::

1- 1- Multiple origins of replicationMultiple origins of replication in eukaryotes. in eukaryotes.

Single origin of replication in prokaryotes.Single origin of replication in prokaryotes.

2- RNA primers are removed by 2- RNA primers are removed by RNase HRNase H in eukaryotes. in eukaryotes.

Eukaryotic Cell CycleEukaryotic Cell Cycle

Pol Pol multisubunit enzymemultisubunit enzyme 1- 1- Primase activityPrimase activity Synthesizes a short RNA primer Synthesizes a short RNA primer 2- 2- Initiates DNA synthesisInitiates DNA synthesis by the 5`-3` polymerase activity. by the 5`-3` polymerase activity. on leading & the beginning of each Okazaki fragment on on leading & the beginning of each Okazaki fragment on the lagging strand ---- adding of a short piece of DNA.the lagging strand ---- adding of a short piece of DNA.

Pol Pol : 1- : 1- completes DNA synthesiscompletes DNA synthesis on the leading strand & on the leading strand & elongates elongates

each Okazaki fragmenteach Okazaki fragment 2- 2- ProofreadProofread newly synthesized DNA newly synthesized DNA

Pol Pol pol pol : : involved in carrying out involved in carrying out DNA repairDNA repair..

PolPol : : replicates mitochondrial DNAreplicates mitochondrial DNA

Eukaryotic DNA polymerase

Eukaryotic DNA polymeraseEukaryotic DNA polymerase

TelomeraseTelomerase

In In eukaryotic cells eukaryotic cells After removal of the RNA primer from the extreme After removal of the RNA primer from the extreme 5`-end5`-end

of the of the lagging strandlagging strand, there is no way to fill the remaining , there is no way to fill the remaining gap with DNA.gap with DNA.

To To solve this problemsolve this problem & to & to protect the ends of the protect the ends of the chromosomeschromosomes from attack by nucleases from attack by nucleases

noncoding sequences of noncoding sequences of DNADNA complexed with complexed with proteinsproteins are are found found

at these ends, called at these ends, called telomeres.telomeres.

TelomeresTelomeres

DNADNA of telomeres consists of repetitive sequence of T`s & of telomeres consists of repetitive sequence of T`s & G`s, base paired to a complementary chain of A`s and C`sG`s, base paired to a complementary chain of A`s and C`s

TTxx G Gyy: where : where xx and and yy are in the range of are in the range of 1 : 41 : 4

The The TG strandTG strand is is longerlonger than its complement leaving a than its complement leaving a region of single stranded DNA at the 3`-end of the double region of single stranded DNA at the 3`-end of the double helix helix

(few hundred nucleotides long).(few hundred nucleotides long).

This single stranded region folds back on itself, forming a This single stranded region folds back on itself, forming a structure that is stabilized by structure that is stabilized by proteinprotein to protect the ends of to protect the ends of the chromosomesthe chromosomes

In In aging cellsaging cells, the ends of their chromosomes get slightly , the ends of their chromosomes get slightly shorter with each cell division until the telomeres are gone shorter with each cell division until the telomeres are gone and DNA essential for cell function is degraded.and DNA essential for cell function is degraded.

This phenomenon is related to cellular aging & death.This phenomenon is related to cellular aging & death.

3`

5`

TG TG TG

AC

3`

5`AC

TG

T/G repetitive sequences (longer)

A/C repetitive sequences (shorter)

DNA of the telomere

Single stranded TG region folds back on itself Single stranded TG region folds back on itself

3`

5`

TG sequence

AC sequence

5` 3`

telomereformed by DNA & protein protects the ends of the chromosome

In In aging cellsaging cells, the ends of their chromosomes , the ends of their chromosomes get get

slightly shorter with each cell division until the slightly shorter with each cell division until the

telomeres are gone and DNA essential for cell telomeres are gone and DNA essential for cell

function is degraded.function is degraded.

This phenomenon is related to cellular aging This phenomenon is related to cellular aging

& death& death..

So, what is a telomerase ?So, what is a telomerase ?

Cells that Cells that do not agedo not age (as germ-line cells and cancer cells) contain an enzyme (as germ-line cells and cancer cells) contain an enzyme called called

telomerasetelomerase responsible for replacing these lost ends of telomeres. responsible for replacing these lost ends of telomeres.

TelomeraseTelomerase is a special kind of reverse transcriptase that carries its own RNA is a special kind of reverse transcriptase that carries its own RNA moleculemolecule of about 150 nucleotides long. of about 150 nucleotides long.

This RNA contains copies ofThis RNA contains copies of A/C A/C sequence that is complementary to sequence that is complementary to T/GT/G repeat repeat sequence sequence

Steps of telomere elongationSteps of telomere elongation

1-The telomerase RNA serves as template for extending 1-The telomerase RNA serves as template for extending T/GT/G DNA strand (longer DNA strand (longer strand)strand)

2-The 3`-end of the RNA serves as a primer for DNA polymerase to extend the 2-The 3`-end of the RNA serves as a primer for DNA polymerase to extend the

A/CA/C DNA DNA strand (shorter strand)strand (shorter strand)

3-Once the next repeat sequence is complete, telomerase RNA is translocated 3-Once the next repeat sequence is complete, telomerase RNA is translocated to the newly to the newly

synthesized end of the DNA and the process is repeated.synthesized end of the DNA and the process is repeated.

Reverse TranscriptaseReverse Transcriptase A A retrovirus as HIVretrovirus as HIV, carries the genome in the form of single-stranded , carries the genome in the form of single-stranded RNARNA

molecul.molecul.

Following infection of a host cell, the viral enzyme, Following infection of a host cell, the viral enzyme, reverse transcriptasereverse transcriptase, , usesuses the RNA as a template for the synthesis of viral DNA the RNA as a template for the synthesis of viral DNA Then, DNA becomes integrated into host chromosomes.Then, DNA becomes integrated into host chromosomes.

Reverse transcriptase Reverse transcriptase movesmoves along the RNA template in the along the RNA template in the 3`-5`3`-5` direction, direction, synthesizing new DNAsynthesizing new DNA in the in the 5`-35`-3` direction` direction

No proofreadingNo proofreading in reverse transcriptase high mutation rate of such viruses in reverse transcriptase high mutation rate of such viruses

As an As an attempt to prevent HIV infection from progressing to AIDSattempt to prevent HIV infection from progressing to AIDS, patients are , patients are treated by treated by inhibitors of reverse transcriptaseinhibitors of reverse transcriptase (nucleosides or non-nucleosides (nucleosides or non-nucleosides inhib.), inhib.),

in addition to protease inhibitors (which targets another HIV maturation enzyme.in addition to protease inhibitors (which targets another HIV maturation enzyme.

Inhibitors of DNA synthesis by nucleoside Inhibitors of DNA synthesis by nucleoside analogsanalogs

DNA chain growth can be blocked by the incorporation of DNA chain growth can be blocked by the incorporation of certain certain

nucleoside analogs that have been modified in the sugar nucleoside analogs that have been modified in the sugar portion of the portion of the

Nucleoside (as the 3`- end is not present for DNA chain Nucleoside (as the 3`- end is not present for DNA chain elongation)elongation)

NUCLEOSIDE ANALOGSNUCLEOSIDE ANALOGS

1- Removal of (OH) from 3`-carbon of deoxyribose1- Removal of (OH) from 3`-carbon of deoxyribose 2`, 3` dideoxyinosine (didanosine)2`, 3` dideoxyinosine (didanosine)

2- Conversion of deoxyribose to another sugar (arabinose)2- Conversion of deoxyribose to another sugar (arabinose)

Cytosine arabinoside (cytarabine, araC) : cancer therapyCytosine arabinoside (cytarabine, araC) : cancer therapy Adenine arabinoside (vidarabine, araA) : antiviral agentAdenine arabinoside (vidarabine, araA) : antiviral agent

3- Chemically modification of the sugar moiety3- Chemically modification of the sugar moiety

Zidovudine (AZT)Zidovudine (AZT)

These sugars are generally supplied as These sugars are generally supplied as nucleosidesnucleosides which are then converted which are then converted to to

nucleotides by cellular salvage enzymesnucleotides by cellular salvage enzymes

These nucleotides blocks DNA replication by preventing further chain These nucleotides blocks DNA replication by preventing further chain elongationelongation

So, these compounds slow the division of rapidly growing cells (cancer) and So, these compounds slow the division of rapidly growing cells (cancer) and viruses. viruses.

Cytosine arabinoside (cytarabine, Cytosine arabinoside (cytarabine, araC) used as an anticancer araC) used as an anticancer chemotherapychemotherapy

Adenine arabinoside (vidarabine, Adenine arabinoside (vidarabine, araA)araA)

used as an antiviralused as an antiviral

Zidoviduline (AZT)Zidoviduline (AZT)

Organization of the eukaryotic Organization of the eukaryotic DNADNA

Typical human DNA contains 46 chromosomesTypical human DNA contains 46 chromosomes Total DNA is ~ one meter longTotal DNA is ~ one meter long

It is important to effectively packaging such large amount of It is important to effectively packaging such large amount of genetic material into a volume of the size of a cell nucleus for genetic material into a volume of the size of a cell nucleus for effective replication & gene expressioneffective replication & gene expression..

To fulfill so, DNA interacts with large number of proteins To fulfill so, DNA interacts with large number of proteins Eukaryotic DNA is associated with basic proteins called histones.Eukaryotic DNA is associated with basic proteins called histones.

These serve to order the DNA into basic structural units called These serve to order the DNA into basic structural units called nucleossomes (resemble beads on a string)nucleossomes (resemble beads on a string)

Nucleosomes are further arranged into more complex structures Nucleosomes are further arranged into more complex structures

that organize and condense the long DNA molecules into that organize and condense the long DNA molecules into chromosomes chromosomes

Histones and the formation of nucleosomesHistones and the formation of nucleosomes

55 classes of histones classes of histones

H1, H2A, H2B, H3 & H4H1, H2A, H2B, H3 & H4

Histones are positively charged at physiologic pH Histones are positively charged at physiologic pH (high contents of lysine & argenine)(high contents of lysine & argenine)

They can form ionic bonds with negatively charged They can form ionic bonds with negatively charged DNA DNA

(in addition with Mg2(in addition with Mg2++ can can neutralizeneutralize –ve charged DNA –ve charged DNA phosphate groups)phosphate groups)

1- 1- Formation of the nucleosomeFormation of the nucleosome

Two molecules each of Two molecules each of H2AH2A, , H2BH2B, , H3H3 and and H4H4 form the structural form the structural core of core of

the the individual nucleosomeindividual nucleosome (bead). (bead).

Around this core, a segment of DNA double helix is wound Around this core, a segment of DNA double helix is wound twice twice forming a negatively supertwisted helix.forming a negatively supertwisted helix.

Neighboring nucleosomes are joined by Neighboring nucleosomes are joined by linker DNAlinker DNA ~ 50 base ~ 50 base pairs long. pairs long.

H1H1 binds to the binds to the linker DNAlinker DNA chain between the nucleosome beads. chain between the nucleosome beads.

H1 H1 is the most tissue-specific and species-specific of the histone.is the most tissue-specific and species-specific of the histone.

2- 2- Higher levels of organizationHigher levels of organization

Nucleosomes can be packed more tightly to form Nucleosomes can be packed more tightly to form

polyuncleosome (nucleofilament polyuncleosome (nucleofilament oror 30-nm fiber) 30-nm fiber)

The fiber is organized into The fiber is organized into loopsloops that are anchored a that are anchored a nuclear scafold proteinsnuclear scafold proteins. .

Additional levels of organization lead to the final Additional levels of organization lead to the final chromosomalchromosomal structure. structure.

DNA RepairDNA Repair

DNA repair is required in the following casesDNA repair is required in the following cases::

1- 1- MismatchesMismatches: which occur after DNA replication including : which occur after DNA replication including proofreadingproofreading

incorrect base-pairingincorrect base-pairing insertion of one to few extra nucleotidesinsertion of one to few extra nucleotides

2- 2- DNA damageDNA damage due to environmental insults: due to environmental insults: leads to alteration or removal of nucleotide basesleads to alteration or removal of nucleotide bases chemicalchemical: nitrous oxide : nitrous oxide radiationradiation: UV light : UV light :(can fuse 2 pyrimidines adjacent to each other in :(can fuse 2 pyrimidines adjacent to each other in

DNA)DNA)

High-energy radiationHigh-energy radiation: (can cause double-stranded breaks): (can cause double-stranded breaks)

3- 3- Spontaneous alteration or loss of bases from mammalian Spontaneous alteration or loss of bases from mammalian DNADNA (at a rate of thousands per cell per day) (at a rate of thousands per cell per day)

Effect of unrepaired mismatch and DNA damageEffect of unrepaired mismatch and DNA damage::

A permanent A permanent mutationmutation loss of control over the proliferation of the mutated cells loss of control over the proliferation of the mutated cells

CANCERCANCER

General Steps of RepairGeneral Steps of Repair

RecognizingRecognizing the lesion the lesion

ExcisionExcision of the damaged section of the DNA strand of the damaged section of the DNA strand

Fill the gapFill the gap using the sister strand as a template using the sister strand as a template

AA. . Strand-directed mismatch repair Strand-directed mismatch repair systemsystem

in cases of replication errors escaping the proofreading function during DNA in cases of replication errors escaping the proofreading function during DNA synthesis synthesis

1-1- Identification of the mismatched strandIdentification of the mismatched strand

Proteins which identify and remove the mispaired nucleotide(s) must be Proteins which identify and remove the mispaired nucleotide(s) must be able to able to

discriminate between the template strand and the newly synthesized discriminate between the template strand and the newly synthesized strand strand

containing the mistake.containing the mistake.

GATC sequencesGATC sequences occurring every 1000 nucleotides are occurring every 1000 nucleotides are methylatedmethylated on the on the adenine adenine

The methylation is not done immediately after synthesis ---- so, the newly The methylation is not done immediately after synthesis ---- so, the newly

synthesized DNA is synthesized DNA is temporarily hemimethylatedtemporarily hemimethylated

(i.e. the parental strand is methylated but, the newly synthesized strand is (i.e. the parental strand is methylated but, the newly synthesized strand is not) not)

2- 2- Repair of damaged DNARepair of damaged DNA

i- endonuclease i- endonuclease cutscuts the mismatched strand. the mismatched strand.

ii- the mismatched base(S) are ii- the mismatched base(S) are removedremoved..

iii- the gap left by removal of the bases is iii- the gap left by removal of the bases is filledfilled using the sister using the sister strand as a strand as a

template by a 5`-3` DNA polymerase. (template by a 5`-3` DNA polymerase. (DNA polymerase IDNA polymerase I in in E.coliE.coli))

iv- The cut ends of the DNA are iv- The cut ends of the DNA are ligatedligated (spliced) by DNA ligase. (spliced) by DNA ligase.

A defect in mismatch repair in humans may cause A defect in mismatch repair in humans may cause herditary herditary nonpolyposis colon cancer (HNPCC),nonpolyposis colon cancer (HNPCC), an inherited cancer an inherited cancer

A

G

C

G A

T New strand 5`

parental strand 3`

C

GT

AG

C

NOT Methylated

Methylated

G

C

C

G G

T

G

C

G

C

C

G

A

A

T

CUT (endonuclease)

5`

3`

3`5`

3`5`

5`

3`

3`

5`

DNA polymerase 5`- 3`

ligase

C

removal

Gap filling

mismatch is repaired

Mismatch CH3

BB-Repair of damage caused by ultraviolet -Repair of damage caused by ultraviolet lightlight

Exposure of a cell to Exposure of a cell to ultraviolet lightultraviolet light can result in the covalent joining of can result in the covalent joining of two two

adjacent pyrimidines (usually thymines), producing a dimer (adjacent pyrimidines (usually thymines), producing a dimer (thymine thymine dimerdimer))

prevents DNA polymerase from replicating the DNA strand prevents DNA polymerase from replicating the DNA strand beyond beyond

the dimer formation.the dimer formation.-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Thymine dimersThymine dimers has to be excised for the continuity of DNA replication has to be excised for the continuity of DNA replication

by the following system:by the following system:

1- 1- RecognitionRecognition & & excisionexcision of the dimer by of the dimer by UV-specific endonucleaseUV-specific endonuclease.. called: called: uvrABCuvrABC excinuclease excinuclease on both 5` & 3` sides of the dimeron both 5` & 3` sides of the dimer

2- Damaged oligonucleotide is 2- Damaged oligonucleotide is releasedreleased, leaving a , leaving a gapgap in the DNA strand. in the DNA strand.

3- The 3- The gap is filledgap is filled by nucleotides using the sister strand as a template. by nucleotides using the sister strand as a template.

Ultraviolet Radiation & CancerUltraviolet Radiation & Cancer

Pyrimidine dimers can be formed in the skin of humans exposed to Pyrimidine dimers can be formed in the skin of humans exposed to

unfiltered sunlight.unfiltered sunlight.

In In xeroderma pigmentosumxeroderma pigmentosum ( a rare genetic disease), the cells ( a rare genetic disease), the cells cannot repair the damaged DNA extensive accumulation of cannot repair the damaged DNA extensive accumulation of mutationsmutations

SkinSkin CancersCancers

The most common form of this disease is caused by the The most common form of this disease is caused by the absence absence

of of UV-specificUV-specific excinucleases excinucleases..

CC-- Correction of base alterations (base excision Correction of base alterations (base excision repair)repair)

I -I - The bases of DNA can be The bases of DNA can be alteredaltered either either : : 1- 1- spontaneously spontaneously (as the case of (as the case of ccytosine which is spontaneously deaminated slowly ytosine which is spontaneously deaminated slowly

to to uuracil)racil)

2- 2- by the actionby the action of deaminating or alkylating compounds as nitrous of deaminating or alkylating compounds as nitrous oxideoxide

nitrous oxide is formed by the cell from precursor as nitrosamines, nitrous oxide is formed by the cell from precursor as nitrosamines, nitrites & nitrates.nitrites & nitrates. nitrous oxide deaminates nitrous oxide deaminates cytosinecytosine, , adenineadenine & & guanineguanine

II- II- Bases are Bases are lostlost spontaneously spontaneously

~ 10,000 purine bases are lost per cell per day.~ 10,000 purine bases are lost per cell per day.

Base alterations or loss can be corrected by base excision Base alterations or loss can be corrected by base excision repairrepair

Uracil is Uracil is recognizedrecognized by specific by specific glycosylasesglycosylases

removalremoval from the from the deoxyribose-deoxyribose-

phosphate backbone of the phosphate backbone of the strand.strand.

leave a apyrimidine site (AP-leave a apyrimidine site (AP-site)site)

recognized & excision by recognized & excision by specific specific

AP- endonucleaseAP- endonuclease

removal of the empty sugar-removal of the empty sugar-

phosphate residue by phosphate residue by deoxyribose phosphate deoxyribose phosphate

lyase lyase

Gap fillingGap filling by DNA polymerase by DNA polymerase I & ligaseI & ligase

D-D- Repair of double-stranded breaksRepair of double-stranded breaks

High energy radiation or oxidative free High energy radiation or oxidative free radicals can radicals can

cause double stranded breaks in DNA --- cause double stranded breaks in DNA --- lethal to lethal to

cells.cells.

Double-stranded breaks in DNA can occur Double-stranded breaks in DNA can occur naturally naturally

during gene rearrangementduring gene rearrangement

Repair of Double-stranded DNA breaks (cont.)Repair of Double-stranded DNA breaks (cont.)

11- - nonhomologous end-joining repairnonhomologous end-joining repair

The ends of two DNA fragments are brought together by a The ends of two DNA fragments are brought together by a group of proteins that cause their religation.group of proteins that cause their religation.

This does not require that the two DNA sequences have any This does not require that the two DNA sequences have any sequence homologysequence homology

Error prone & mutagenicError prone & mutagenic

Defects in this repair system are associated with pdp to Defects in this repair system are associated with pdp to cancer & immunodeficiency syndrome.cancer & immunodeficiency syndrome.

2-2- Homologous recombination repairHomologous recombination repair

Uses the enzymes that normally perform genetic Uses the enzymes that normally perform genetic recombination between homologous chromosomes during recombination between homologous chromosomes during meiosismeiosis

This system is used by the lower eukaryotes to repair double-This system is used by the lower eukaryotes to repair double-stranded stranded

breaks.breaks.