Molecular Biology of the Cell Fifth Edition

Chapter 5 DNA Replication, Repair,

and Recombination

Copyright © Garland Science 2008

Alberts • Johnson • Lewis • Raff • Roberts • Walter

Germ-line cells and somatic cells carry out fundamentally different functions

DNA Replication

DNA Repair

Homologous Recombination

Mobile Genetic Elements and Viruses

DNA ReplicationReplicate = Copy

DNA Replication

• Base-Pairing Enables DNA Replication

• DNA Synthesis Begins at Replication Origins

• New DNA Synthesis Occurs at Replication Forks

• The Replication Fork Is Asymmetrical

• DNA Polymerase Is Self-correcting

• Short Lengths of RNA Act as Primers for DNA Synthesis

• Proteins at a Replication Fork Cooperate to Form a

Replication Machine

• Telomerase Replicates the Ends of Eucaryotic Chromosomes

Base-paring enables DNA replication

• Must be carried out with speed and accuracy. • 3.2 x 109 (32억개) nucleotides into 24 chromosome• Awe-inspiring! This copy in about 8 hours. • 1,000 books (Essential Cell Biology 3rd Ed. x 1,000 with almost no errors)

DNA replication

• Each DNA strand can serve as a template, or mold, for the synthesis of a new complementary strand (Fig 6-2).

• DNA acts as a template for its own duplication (Fig 6-3)

• Performed by a cluster of proteins that together form a “replication machine”.

• Semiconservative• Each of the daughter DNA = Original (old) strands + New

strand (Fig 6-4).

DNA synthesis begins at replication origins

• The DNA double helix is normally very stable due to the large number of hydrogen bonds between the bases on both strands.

• Only temperature approaching those of boiling water provide enough

thermal energy to separate these strands.

• DNA double helix must first be opened up and the two strands separated to expose unpaired base.

Replication Origin

• The position at which the DNA is first opened.

• ≈100 base pairs (bacteria or yeast).

• Easy to open.• Attract the initiator proteins.

• Particular sequence of nucleotides.

• DNA rich in A-T base pairs are typically found at replication origin.

• A bacteria genome: a single origin of replication.

• The human genome: 10,000 origins (allows a cell to replicate its DNA relatively quickly; At many places at once).

Initiator proteins

• Bind to the DNA and pry the two strands apart, breaking the hydrogen bonds between the bases (Fig 6-5).

• Individually each hydrogen bond is weak (not require a large energy input to be separated).

• Separating a short length of DNA does not require a large energy input

and can occur with the assistance of these proteins at normal temp.

New DNA synthesis occurs at replication forks

Replication forks

• Y-shaped junctions.

• Replication machine is opening up DNA double helix, making a new daughter strand.

• Two replication forks are formed.

• Move away in both directions (“bidirectional”).• Move very rapidly (1,000 nt/s in bacteria; 100 nt/s in humans).

• Synthesizes new DNA in the 5’ to 3’ directions.

• Only one direction.

• dNTP (deoxyribonucleoside triphosphate) is used.

• Hydrolysis of the NTP provides the energy for the reaction.

• Forms phosphodiester bond.

• Stays associated with the DNA for many cycles (cf. RNA polymaerase II).

• So accurate : only about one error in every 107.

DNA polymerase

The replication fork is asymmetrical

• One new DNA strand is being made in a 3’ to 5’ direction.

• Whereas the other new is in the opposite direction (5’ to 3’)

• The replication fork is therefore asymmetrical.

DNA polymerase catalyze the growth of DNA in only one direction.

How is the problem solved?

“backstitching” maneuver!

• Leading strand: continuously.

•Lagging strand: discontinuously

•Okazaki fragments: DNA polymerase works in the 5’ to 3’

direction for each new piece. (Successive Separate Small Pieces)

•All cells, procaryotic or eucaryotic, have leading and lagging strands

(common feature).

DNA polymerase is self-correcting

DNA polymerase is so accurate : only about one error in every 107 (lower than can be explained by AT, GC base-pairing).

1. Careful Monitoring: incoming nucleotide + template.

• 5’-to-3’ polymerization activity (add the next).

2. Proofreading (self-correcting)• At the same time as DNA synthesis

• Error-correcting activity.

• Check whether the previous nucleotide added is correctly.

• 3’-to-5’ exonuclease activity (remove the mispaired).

• The two activities (polymerization + proofreading) are tightly coordinated by different domains within the polymerase.

Why DNA polymerase synthesize DNA only in the 5’-to-3’ direction?

Fig. 6-15 legend

Because DNA polymerase can join (recruited to DNA) a

nucleotide only to a base-paired nucleotide (double strand), it cannot start a new DNA strand.

Then, how does the DNA replication start at replication fork

without the help of DNA polymerase?

Short lengths of RNA act as primers for DNA synthesis

• RNA primer • 10 nt long, provides a base-paired 3’ end.

• A starting point for DNA polymerase.

• Complementary base-pairing with DNA.

• Primase• Joins two nucleotides together w/o the need for a base-paired end.

• Synthesizes a short length of a closely related type of nucleic acid, RNA (Ribonucleic acid, Does not synthesize DNA)

• An example of RNA polymerase.• Not proofread: A high frequency of mistakes. But, automatically

removed and replaced by DNA.

On the lagging strand...

•New primers are needed continually (cf. leading strand - only starting point).

•Three additional enzymesNuclease: breaks apart the RNA primer.

DNA polymerase (or repair polymerase): replaces the RNA with DNA

using the adjacent Okazaki fragment as a primer.DNA ligase: nick sealing.

• In this way, the cell’s replication machinery ensure that all of the DNA is copied faithfully.

Fig. 6-15 legend

Proteins at a replication fork cooperate to form a Replication Machinery

Legend p. 209

• Nuclease, repair polymerase, and ligase.

• Unzipping of DNA double helix (Two proteins cooperate)

• Helicase: uses the energy of ATP hydrolysis to pry apart the double helix as it speeds along the DNA.

• Single-strand binding protein: clings to the single-stranded DNA

exposed by the helicase and transiently prevents it from re-forming base pairs.

• Sliding clamp: keeps the DNA polymerase firmly attached to the DNA.

• Clamp loader: hydrolyzes ATP and locks a clamp around the DNA.

DNA helicase The structure of DNA helicase

sliding clamp

Legend p. 209 Current view of the action of replication machines

Replication Machinery (Multienzyme complex)

• Most of the proteins in DNA replication • Held together in a large mutienzyme complex.• Enable DNA synthesis on both strands in a coodinated manner.

• Sewing machine (재봉틀) composed of protein parts and powered

by nucleoside triphosphate hydrolysis.

• How these components fit together and work as a team

is not entirely understood.

Telomerase replicate the ends of eucaryotic chromosomes

‘end-replication problem’

: there is no place to lay down the RNA primer needed to start the Okazaki fragment!

Bacteria: circular DNA molecules as chromosomes.

Eucaryotes: “Solve this problem in an ingenious way”

• Special repetitive tamdem nucleotide sequences (GGGGTTA) at the ends of chromosomes which are incorporated into

telomeres (about 1,000 times).

Telomerase

• Attracted by the telomeric DNA sequences.

• Having RNA template as a part of the enzyme.

• A large protein-RNA complex.

• Replenishes the nucleotides in 5’-to-3’ direction, which are lost

each time a eucaryotic chromosome is duplicated.

• Contains reverse transcriptase domain (from RNA to DNA).

• The length of telomere sequences is balanced.

• The repetitive DNA sequences then acts as a template.

Telomerase in action

The Function of Telomerase

Allows replication of chromosome ends.

Protection: distinguishes from the double-strand break.

Replicative cell senescence: provide a safeguard against the

uncontrolled cell proliferation.

Human fibroblast (섬유아(芽) 세포) proliferates for 60 cell division.Produce only low levels of telomerases.Telomeres gradually shorten each time they divide.

When inserting an active telomerase gene, telomere length is maintained

and cells continue to proliferate indefinitely.

DNA Repair

Mutations can have severe consequences for a cell or organism

Mutation

A permanent changes in the DNASickle cell anemia

• A genetic (inherited) disease.• A single nucleotide change in hemoglobin (Hb).

• Nucleotide mutation (A to T)

• Change in translation from glutamic acid (Glu, E) to valine (Val, V).

Hemoglobin (Hb)• Iron-containing oxygen-transport

metalloprotein in the red blood cells of all vertebrates.

• Carries oxygen from the respiratory organs (lungs or gills) to the rest of the body (i.e. the tissues) where it releases the oxygen to burn nutrients to provide energy to power the functions of the organism

Sickle cell anemia (continued)

• Two copies of the mutant β-globin gene.

• Incorrect sequence of β-globin amino acid.

• Sickle shape of RBC.

• Sickle-cell Hb is less soluble (fibrous precipitates).

• Sickle cells are more fragile.

• Reduced number of RBC.

• Weakness, dizziness, headaches, pain, and total organ failure).

• patients are more resistant to malaria (since the parasite grows

poorly in RBC from sickle-cell patients).

“The importance of protecting reproductive cells

(germ cells) against mutation”

Sickle cell anemia

Cancer

• Uncontrolled (unchecked) cell proliferation.

• Nucleotide changes in somatic cells.

• 30% of the deaths in Europe and North America.

• Gradual accumulation of changes in the DNA.

• Cancer incidence increases dramatically as a function of age.

How do cells protect their DNA from the accidental damages?

Genetic Stability for Replication

✓ Accuracy ✓ DNA polymerase

✓ Correcting the copying mistake ✓ DNA polymerase

✓ Repairing the accidental damage ✓ Repair system

A DNA mismatch repair system removes replication errors that escape the replication

machine

• Despite the safeguards, errors do occur.

• A backup system (DNA mismatch repair) for errors.

• Corrects the rare mistakes.

• Increasing the overall accuracy of DNA copy to one mistake in

109 nt copied.

DNA mismatch repair system• Recognizes DNA mismatches.

• Removes (excises) one of the two strand of DNA.

• Resynthesizes the missing strand.

• Always excise only the newly synthesized DNA strand: excising the other strand (the old strand) would preserve the mistake instead of correcting it.

How the mismatch repair machinery distinguishes the two DNA strand in eukaryotes?

• Not yet known.

• However, newly replicated DNA strands are preferentially nicked.

• Nicks (single-stranded breaks) could provides the signal that directs the mismatch repair machinery to the appropriate strand.

DNA is continually suffering damage in cells

• depurination

• deamination

• thymine dimer

p. 74

Depurination & Deamination • The most frequent chemical reactions.

• Create serious DNA damage in cells.

• Depurination• Release guanine as well as adenine from DNA (does

not break the phosphodiester backbone).

• Spontaneous reaction: 1012 purines will be lost.

• Deamination • Converts cytosine to Uracil.• Spontaneous reaction.

UV radiation in sunlight

• Thymine dimer

• Two adjacent thymine bases attached to one another.

• Stall the DNA replication machinery at the site of the damage.

• Xeroderma pigmentosum (색소건피증): cannot repair thymine dimers, because they have inherited a defective gene for one of proteins involved in the repair process (skin cancer).

“All of these types of damage, if unrepaired, would have disastrous consequences for an organism”

Mutations or nucleotide deletions

mutationsnucleotide deletions

The stability of genes depends on DNA repair 1. Excision

2. Resynthesis 3. Ligation

“The thousands of random chemical changes occur in the DNA everyday”

“The existence of two copies of the genetic information”

3 steps Key enzyme process

Excision* Nuclease The damaged DNA is recognized and removed

Resynthsis Repair DNA polymerase

Binds to the 3’-hydroxyl end of the cut and fills in the gap

Ligation DNA ligase The nick in the helix is sealed.

* A series of different enzymes, each specialized for removing different types of DNA damage.

The importance of repair process

• The large investment that cells make in DNA repair enzymes.

• Yeast cells contain more than different proteins.

• DNA repair pathways are even more complex in humans.

50Double-strand breaks can be repaired

rapidly but imperfectly

• Double-strand breaks • A particularly dangerous type of DNA damage

• Leave no intact template strand.

• Ionizing radiation, mishaps at replication fork, strong oxidizing agents, and metabolites produced in the cells.

• Non-homologous end-joining• Two broken ends are simply rejoined.• Nucleotides are usually lost.• Quick and dirty mechanism.

A record of the fidelity of DNA replication and repair is preserved in genome sequences

• Changes in DNA accumulate remarkably slowly in the course of evolution.

• Natural selection: those that have harmful consequences are

usually eliminated.• Humans vs. chimpanzees (98% identical DNA).

• Thanks to the faithfulness of DNA replication and repair, 100 million years

have scarcely changed its essential content (Fig. 6-28)• The gene that determines maleness.• Humans vs. whales

Homologous Recombination ����������� ������������������  

•Homologous recombination• The exchange of genetic information between a pair of

homologous chromosomal DNA (Repair process).

• Genetic variation is also important to allow organisms to

evolve (Genetic diversity during meiosis)

Homologous recombination requires extensive regions of sequence similarity

• It promotes the exchange of DNA sequences between chromosomes.

• It takes place only between DNA duplexes.

• The match need not be perfect for homologous recombination to

succeed, but it must be very close.

Homologous recombination can flawlessly repair DNA double-strand breaks

• It is often initiated when a double-strand break occurs shortly after a stretch of DNA has been replicated.

• At that time, the duplicated helices are still in close proximity to one another.

Homologous recombination exchange genetic information during meiosis

* Although the two original DNA molecules must have similar sequences, they do not have to be identical; thus a crossover can create DNA molecules of novel nucleotide sequences.

“chromosomal crossovers”• Two homologous chromosomes come together, undergo a genetic change.

• The site of exchange can occur anywhere in the

homologous sequences.

• No nucleotide sequences are altered.• So precise events.

• Generates new combinations of DNA.

• Sexually or asexually reproducing organisms.

A specialized enzyme deliberately slices

• A special enzyme cuts both strands of the double helix, creating a complete break.

• The 5’ end are then chewed back by a DNA-digesting enzyme, creating protruding single-stranded 3’ ends.

• Each of these single strands then searches for a homologous DNA helix, leading to the formation of a “joint molecule”.

A. Homologous recombination begins with a double strand break in a chromosome.

Cross-strand exchange

06_30_Holliday_junct.jpg 06_31_cross-strand EM.jpg

• The crucial intermediate : ‘Cross-Strand Exchange’ or ‘Holiday junction’

• To regenerate two separate DNA molecules, the two crossing strands must be cut.

• Without rotation (or isomerization): no homologous recombination

• After rotation: crossing strands.

Results: The two DNA molecules have crossed over, and two molecules of novel DNA sequence have been produced.

B. The rotation of a Holiday Junction allows recombination to occur

✓ Allows an organism to repair DNA that is damaged on both strands of the double helix.

✓ Can fix other genetic accidents that occur during nearly every round of DNA replication.

✓ Essential for the accurate chromosome segregation during meiosis (ch. 20).

1.C. Advantages of homologous recombination

Text p.215

Mobile Genetic Elements and Viruses

Text p. 221

• ‘mobile genetic elements’• Jumping DNA.• Short DNA sequences.• Changes that alter the order of genes or even add new

information (cf. Homologous recombination, conservative).

• Insert into virtually any sequence within the genome.

• Most lack the ability to leave the cell where they reside.

• Their movement is restricted to a single cell and its descendants.

• cf. virus (escape from one cell and infect another).

Mobile genetic elements encode the components they need to movement

• Site-specific recombination allows DNA exchange between DNA

helices that are dissimilar in sequence.

• Does not require DNA sequence similarity.

• Encode a specialized recombination enzyme that mediates its movement.

• Also called ‘Transposons’.

Bacterial transposons

enzyme that catalyzes the movement

Sequences recognized only by its own transposase

enzyme that inactivate indicated antibiotics

Bacterial mobile genetic elements (Transposons)

• Transposase: an enzyme that carries out some of the DNA breaking and joining reaction.

• Contains ‘Antibiotics resistant genes’ : ampicillin (ampR) and

tetracycline (tetR).

DNA-only transposons movements

• Bacteria transposons move by two mechanisms

• Cut+Paste (nonreplicative transposition)

• Copy+Paste (Replicative transposition)

The human genome contains two major families of transposable sequences

• Nearly half of the human genome is made up of various mobile genetic elements.

• Most have moved not as DNA but via an RNA intermediate. (Retrotransposons unique to eucaryotes)

1. Donor DNA to RNA (RNA polymerase)

2. RNA to DNA (reverse transcriptase)

3. Insertion of DNA copy to target genes.

* The reverse transcriptase is encoded by the L1 element itself.

3 steps

L1 elements

• A highly repeated sequence.• LINE (long interspersed nuclear element)

• cf. SINE (short interspersed nuclear element)

• About 20% (LINE), 15% (SINE) of the total human genome.

• Most copies are immobile (accumulation of mutations).

• Translocation of L1 can sometimes result in human disease:

• Hemophilia (血友病): is caused by insertion of an L1 element into Factor VIII gene (essential for proper blood clotting).

Alu sequence• Present in about 1 million copies in our genome.

• Do not encode their own reverse transcriptase.

L1 and Alu sequence• Have proliferated in primates relatively recently in evolutionary

time.

• Must have had major effects on the expression of many of our

genes

L1 and Alu elements have multiplied to high copy numbers relatively recently in evolutionary time.

Legend for Fig. 6-35

Viruses

광견병

유행선 이하선염

홍역

천연두

황열병

대상포진

단핵증

후천성면역결핍증

독감

(소아)마비

감기

A형 간염

발진, 구순

간염

Viruses are fully mobile genetic elements that can escape from cells

• First noticed as disease-causing agent.

• Essentially genes enclosed by a protective coat.

• Utilize the cell’s machinery to express their genes as proteins and to replicate their chromosomes.

• Often lethal to the cells; the infected cell breaks open (lyses) and thereby releases the progeny viruses.

• Examples: cold sore (입가의 ��) by herpes simplex virus, blisters (물집) by the chicken pox virus.

• Attack mammalian, plant, or even bacterial cells.

• Viral genome can be made of DNA or RNA.

• Viruses can reproduce themselves ‘only inside a living cell’, where they are able to hijack the cell’s own biochemical machinery.

• Viral genomes typically encode the viral coat proteins as well as proteins that co-opt (舊, attract) host enzymes to replicate their genome.

• Viral coats come in different shapes and sizes.

• Viral genome: three ~ several hundred genes.

Retrovirus reverse the normal flow of genetic information

• Found only in eucaryotic cells.

• Resembles retrotransposon.

• DNA is synthesized using RNA as a template by ‘reverse

transcriptase’ encoded by retroviral genome.

• The life cycle of retrovirus includes reverse transcription + integration into the host genome.

• Retrovirus

•HIV (human immunodeficiency virus, causing AIDS) genome can persist in the latent state as a DNA provirus.

• The retroviral DNA is inserted, or integrated into a randomly selected site in the

host genome by a ‘integrase’ enzyme

• This ability of the virus to hide within host cells complicates any attempt to treat the infection with antiviral drugs.

• AIDS ‘viral reverse transcriptase’ is one of the prime target of drug development.