1 DNA Replication and Transcription Biosynthesis of DNA and RNA Replication of DNA Action of DNA Polymerases DNA Damage and Repair Synthesis of RNA Post-transcriptional

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DNA Replication and Transcription

Biosynthesis of DNA and RNA

DNA Replication and Transcription

Biosynthesis of DNA and RNA

Replication of DNA

Action of DNA Polymerases

DNA Damage and Repair

Synthesis of RNA

Post-transcriptional Modifications of RNA

Base Sequences in DNA

Replication of DNA

Action of DNA Polymerases

DNA Damage and Repair

Synthesis of RNA

Post-transcriptional Modifications of RNA

Base Sequences in DNA

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Replication of DNAReplication of DNA

During replication, each original strand of DNA is used as a template.

DNA replication is semiconservative.DNA replication is semiconservative.Each new DNA duplex is composed of an original strand and a new one.

It has been demonstrated that this semiconservative process is universal for all cells.

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DNA replicationDNA replication

Parent DNA

Firstgeneration

Secondgeneration

semiconservative conservative

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Replication of DNAReplication of DNA

Replication begins at a disecrete point on theDNA molecule and proceeds bidirectionally.

DNA replicationDNA replication

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Replication processReplication process

In the late 1950, John Cairns observed structures during the replication of DNA in E. coli cells - replication forksreplication forks.

In circular DNA, replication is observed as occurring at a discrete point and proceeding in both directions.

In linear DNA, replication is initiated as several sites which grow in both directions - replication bubblesreplication bubbles.

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Circular DNA replicationCircular DNA replication

replicationforks

terminus

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Linear DNALinear DNA

Replicationbubbles

Blue originalRed daughter

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Action of DNA polymerasesAction of DNA polymerases

DNA polymerase IDNA polymerase IThe first enzyme discovered that would

catalyze the synthesis of DNA.

dNTP + (dNMP)n (dNMP)n+1 + PPi

dNTP deoxyribonucleoside triphosphates(dATP, dGTP, dCTP, dTTP)

(dNMP) preformed DNA with n or n+1 mononucleotides

PPi pyrophosphate

Mg2+

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DNA polymerase IDNA polymerase I

• Mg2+ complexes the nucleotide.

• Energy is supplied from the release of pyrophosphate.

• DNA acts as a template.

• The DNA must have a primer segment with a free 3’-hydroxyl group - for attachment of the new nucleotide.

Elongation occurs at the 3’ end and proceeds in the 5’ -> 3’ direction.

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DNA polymerases II and IIIDNA polymerases II and III

• Additional polymerases have been discovered in E. coli.

• These enzymes have many of the same reaction requirements of DNA polymerase I.

• It is now believed the DNA polymerase III is the main enzyme used for DNA replication.

• The other forms most likely serve proofreading or repair functions.

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DNA polymerasesDNA polymerases

PolymeraseCharacteristic I II III

Molecular weight 103,000 88,000 900,000

Polypeptide subunits 1 4 10

Polymerization rate 16 - 20 7 250-1000

(nucleotides/sec)

Activity 3’ -> 5’ exonuclease Yes Yes Yes

5’ -> 3’ exonuclease Yes No No

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Okazaki fragmentsOkazaki fragments

• All know DNA polymerases catalyze chain elongation in the 5’ 3’ direction.

• The two template strands are oriented in an antiparallel fashion.

• Only one strand can be processed in a continuous fashion - 3’ 5’ parent strand.

• The complementary strand is synthesized in the 5’ 3’ direction as discontinuous fragments - Okazaki fragments.

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Okazaki fragmentsOkazaki fragments

3’

5’

3’

5’

Leading strand

Lagging strand (Okazaki fragments)

The fragments are still added in the 5’ 3’ direction.They are covalently linked in later steps.

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DNA ReplicationDNA Replication

Prokaryotic DNA replication - Prokaryotic DNA replication - E. coliE. coliThis multistep process involves several proteins.

Protein Function

Helicase Begins unwinding of DNA helix

DNA gyrase Assists unwinding

SSB proteins Stabilizes single DNA strands

Primase Synthesis of RNA primer

DNA polymerase III Elongation of chain by DNA synthesis

DNA polymerase I Removal of RNA primer and fill in

gap with DNA

DNA ligase Closes last phosphoester gap

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Prokaryotic ReplicationProkaryotic Replication

Step oneStep one• Helicase recognizes and binds to the origin for

replication.• It catalyzes the separation of the two DNA strands.• DNA gyrase assists in unwinding and the

replication fork is formed.

DNA gyrase

Helicase

ATP

ADP

5’

3’

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Step twoStep two• Exposed single strands of DNA must then be

stabilized and protected from cleavage of the phosphodiester bonds.

• SSB proteins perform this function.• Complementary strands are now available as

templates.SSB protein

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Step threeStep three• Primase initiates synthesis by producing a

short strand of RNA (4-10 nucleotides.)• This is only required once for the leading

strand. Separate initiation is required for all Okazaki fragments.

• DNA polymerase III can then process the 3’-hydroxyl group.

primer RNA

primase

DNA polymerase III

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StStep fourep four• After ‘priming’, DNA polymerase III can then

process the 3’-hydroxy group.• The leading strand continues in the direction

of the advancing replication fork.• The lagging strand fragments stop when they

reach another fragment.

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Step fiveStep five• The RNA primers are removed by the 5’->3’

nuclease action of DNA polymerase I.• Remaining gaps are filled by DNA polymerase

I.

DNApolymerase I

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Step sixStep six• DNA ligase is used to complete the final

phosphoester bond.• Termination of replication occurs when the two

replication forks meet on the circular DNA.

DNA ligase

ATPADP

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Eukaryotic replicationEukaryotic replication

There is much we do not understand about this more complicated process.

Important differenceImportant difference

TelomeresTelomeres - specialized DNA ends that consist of hundreds of repeating hexanucleotide sequences

The sequence for humans is AGGGTTAGGGTT

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Eukaryotic replicationEukaryotic replication

TelomeraseTelomerase

• The enzyme that catalyzes the synthesis of DNA ends.

• Unusual enzyme (ribozyme) that contains an RNA molecule that serves as a template.

• The RNA guides the addition of the correct nucleotides.

• Varying activity levels of telomerase may serve to regulate cell division and aging.

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DNA Damage and RepairDNA Damage and Repair

MutationMutation

• Sudden, random alteration of original DNA code that changes the genotype.

• It may be as simple as one wrong nucleotide.

• It can be harmful/deadly or be a positive change. (evolution - rare)

Can be caused by chemical or environmental factors - mutagensmutagens.

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DNA Damage and RepairDNA Damage and Repair

There are ~4000 know human genetic diseases. Many result from mutation of a single gene.

Sickle cell anemia.Sickle cell anemia. Mutation of gene that makes part of hemoglobin.

Male pattern baldness.Male pattern baldness. Characteristic thinning of hair in males. Linked to a pair of genes.

Albinism.Albinism. Lack of ability to produce tyrosinase which catalyzes the conversion of tyrosine to DOPA.

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MutationsMutations

Spontaneous mutationsSpontaneous mutationsChanges that occur during normal genetic

and metabolic function.

Two typesTwo types• Mistakes in the incorporation of

deoxyribonucleotides during DNA replication.

• Base modifications caused by hydrolytic reactions.

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Spontaneous MutationsSpontaneous Mutations

Final mistakes during E. coli replication are very rare.

• 1 error in every 1010 base pairs.

• Actual error rate for base incorporation may be much higher ( 1 in every 104-105).

• Repair mechanisms will correct most mismatched bases.

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Replication errors are of three types:Replication errors are of three types:

• Point mutation - substitution of one base pair for another.

• Insertion of one or more extra base pairs.

• Deletion of one or more base pairs.

Substitution is the most common type of spontaneous mutation.

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E. coli cells have systems to detect and repair mismatched bases.

The general mechanism proceeds in four steps.The general mechanism proceeds in four steps.

• Endonuclease-catalyzed cleavage of phosphoester bond holding the incorrect base.

• Removal of mismatched base by an exonuclease.

• Incorporation of the correct base by DNA polymerase I or III.

• Closure of the final gap by DNA ligase.

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PhotodimerizationPhotodimerization

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thyminedimer

UV lightExposure to UV lightcan cause adjacentthymines to covalently link.

This results in adistortion of the DNAmolecule and breaks the hydrogenbonding with theadenine.

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Thymine dimer repair in E. ColiThymine dimer repair in E. ColiTo repair the damage, a photoreactivating enzyme binds to thymine dimer.

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Visible light activatesthe enzyme

which breaksthe dimer,

restoringoriginal structure.

The enzyme is then releasedfrom the repaired DNA.

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Thymine repair in humansThymine repair in humans

DNA repair is more complex than in E. Coli requiring at least 5 enzymes.

Human repair mechanism must:• cleave the sugar phosphate backbone• remove bad section• rebuild a new section

Xeroderma PigmentosumXeroderma Pigmentosum Genetic disorder where repair mechanism does not work.

Can result in multiple skin cancers by age 20.

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Induced mutationInduced mutation

A number of environmental factors can induce a mutation - mutagens.

RadiationRadiationIonizing - X-rays, rays, cosmic raysNonionizing - UV light

Intercalating agentsIntercalating agentsFlat, hydrophobic chemicals that are typically aromatic. They can insert between stacked base pairs resulting in insertion or deletion of bases.

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Intercalating agentsIntercalating agents

intercalatingagent

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Induced mutationInduced mutation

Chemical mutagensChemical mutagens

• Reactant with bases - formaldehyde

• Base analogs - 2-aminopurine, 5-bromouracil

• Acridine dyes - proflavin

• Alkylating agents - mustard gases

• Others - carcinogens

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Synthesis of RNASynthesis of RNA

Some basic termsSome basic terms• TemplateTemplate - the strand of DNA used for the

synthesis of RNA. It is read in the 3’-> 5’ direction.

• Coding strandCoding strand - the ‘other’ DNA strand.

• TranscriptTranscript - the RNA molecule. It is synthesized in the 5’ -> 3’ direction.

The first base in a gene is numbered +1. Additional bases are numbered sequentially. ‘Upstream’ bases are assigned negative numbers. There is no zero value.

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DNA-directed RNA synthesisDNA-directed RNA synthesis

Prokaryotic cells rely on DNA-directed polymerase (RNA polymeraseRNA polymerase)to catalyze all steps in the transcription of RNA.

The process occurs in three stages:

InitiationInitiation

ElongationElongation

TerminationTermination

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RNA synthesisRNA synthesis

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In the first step, RNA polymerase bindsto a promoterpromoter sequenceon the DNA chain.

This insures that transcription occurs in the correct direction.

The initial reaction is toseparate the twoDNA strands.

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initiationsequence

terminationsequence

‘Special’ basesequences inthe DNA strandindicate where RNA synthesis starts and stops.

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The elongation process continues until an entire gene is transcribed. This is catalyzed by RNA polymerase

NTP + (NMP)n (NMP)n+1 + PPi

DNA template

NTP ribonucleoside triphosphate,ATP, GTP, CTP, UTP

(NMP) preformed RNA with n or n+1mononucleotides

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Once the terminationsequence isreached, thenew RNA moleculeand the RNA synthaseare released.

The DNA recoils.

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The transcription process differs for eukaryotic cells

• Three classes of RNA polymerases for the transcription process (I, II, II)

• I transcribes large ribosomal RNA genes.

• II is for protein-encoding genes.

• III is used during transcription of tRNA and 5S rRNA

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RNA-directed RNA synthesisRNA-directed RNA synthesis

An alternate mode of RNA synthesis is found in RNA viruses.

They induce formation of RNA replicase in the host cell and use RNA as a template.

Direction of synthesis is 5’ -> 3’.Direction of synthesis is 5’ -> 3’.

Same basic mechanism.Same basic mechanism.

RNA transcript is complementary to the RNA transcript is complementary to the RNA RNA template.template.

There are no editing, proofreading or There are no editing, proofreading or repair repair activities.activities.

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Post-transcriptionalmodifications of RNA

Post-transcriptionalmodifications of RNA

Primary transcriptsPrimary transcriptsNewly synthesized RNA molecules. They are typically inactive.

Several types of post-processing may be conducted to produce a mature form of RNA that is active.

The processing varies based on the type of RNA.

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tRNA and rRNA processingtRNA and rRNA processing

Four types of processesFour types of processes

• Trimming of the ends by phosphoester bond cleavage

• Splicing to remove introns

• Addition of terminal sequences

• Hetrocyclic base removal

Prokaryotic cells do not demonstrate intron removal.

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tRNA post-processingtRNA post-processing

ACC

- OH3’

5’5’

3’

pre-tRNA tRNA

Several stepssome catalyzed

by ribonuclease P

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mRNA processingmRNA processing

While prokaryotic mRNA requires little or no alteration, eukaryotic mRNA must be modified in the nucleus before use.

There are three processes that occur

• Capping

• Poly A addition

• Splicing of coding sequences

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CappingCapping

Modification of the 5’ end.Modification of the 5’ end.

• Hydrolytic removal of a phosphate from the triphosphate functional group.

• Guanosine triphosphate (GTP) is used to attach a GMP, resulting in a 5’ -> 5’ triphosphate covalent linkage.

• The end guanine residue is then methylated at N7

Additional capping may include methylation at ribose hydroxyl groups.

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Poly A additionPoly A addition

Modification of the 3’ end.Modification of the 3’ end.Most mature mRNA have a 3’ tail of from 20

to 250 nucleotides.

• Initially an endonuclease catalyzes the removal of a few 3’ base residues.

• Addition of adenine residues is catalyzed by polyadenyl polymerase.

This tail is thought to stabilize mRNA by increasing resistance to cellular nucleases.

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Intron removalIntron removal

Exon Intron Exon ExonIntronGene

Primary transcript

MatureRNA

reseal transcript

transcription

removal of introns

Splicing

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Base sequences in DNABase sequences in DNA

Efforts are underway to characterize the entire human genome. This requires methods for simple and inexpensive sequencing of DNA.

Two methods meet this need.Two methods meet this need.

• Maxam-Gilbert chemical cleavage

• Sanger chain-termination sequencing

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The US human genome projectThe US human genome project

Methods for DNA sequencing are greatly assisting this project.

It is a joint program of the Department of Energy and the National Institutes of Health.

This project is a part of a larger international effort to characterize the genomes of humans and several model organisms.

X chromosomeX chromosomep

q

22.322.222.1

21.321.2

21.111.411.3

11.2311.22

11.21 11.111.111.2

12

13

21.121.2

21.3

22.122.2 22.3

2324

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coagulation factor IX, hemophilia B

blue-monochromatic color blindnesscoagulation factor VIIIc, hemophilia Ahomosexuality, male

cleft palate

growth control factor, X-linkedXg blood group

ocular albinismsensorineural deafness

anemia, sideroblastic, with spinocerebellar ataxia

lymphoproliferative syndrome

Simpson dysmorphia syndrome

Y chromosomeY chromosome

q

p11.311.211.111.1

11.2111.2211.23

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acetylserotonin methyltransferasetestis-specific protein, Y-linkedXg blood group

ribosomal protein S4, Y-linkedtestis determing factorzinc finger protein, Y-linked

stature, Y-linked

Documents

1 DNA Replication and Transcription Biosynthesis of DNA and RNA Replication of DNA Action of DNA Polymerases DNA Damage and Repair Synthesis of RNA Post-transcriptional