Genetic Information Transfer

Genetic Information Transfer

DNA RNA protein

transcription translation replication

reverse transcription

Central dogma

• Replication: synthesis of daughter DNA from parental DNA

• Transcription: synthesis of RNA using DNA as the template

• Translation: protein synthesis using mRNA molecules as the template

• Reverse transcription: synthesis of DNA using RNA as the template

Lecture 2

DNA Replication

（ DNA Biosynthesis）

Section 1

General Concepts of DNA Replication

Double helix structure of DNA

“It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.” Watson & Crick

Characteristics of replication

Semi-conservative replication

Bidirectional replication

Semi-continuous replication

§1.1 Semi-Conservative Replication ——Meselson and Stahl (1958)

Semiconservative replication

•Definition: Half of the parental DNA molecule is conserved in each new double helix, paired with a newly synthesized complementary strand.

•Significance: The genetic information is ensured to be transferred from one generation to the next generation with a high fidelity.

A TTGC

TAACG

A TTGC

TAACG

A TTGC

TAACG

Parent molecule

Daughter molecule

§1.2 Bidirectional Replication

• Replication starts from unwinding the dsDNA at a particular point (called origin), followed by the synthesis on each strand.

• The parental dsDNA and two newly formed dsDNA form a Y-shape structure called replication fork.

Origin

• Examination of T7 DNA replication using electron microscopy

3'

5'

5'

3'

5'

3'

5'3'

direction of replication

Bidirectional replication

• Once the dsDNA is opened at the origin, two replication forks are formed spontaneously.

• These two replication forks move in opposite directions as the synthesis continue.

Replication of prokaryotes

•The replication process starts from the origin, and proceeds in two opposite directions. It is named replication.

Replication of eukaryotes

• Chromosomes of eukaryotes have multiple origins.

• The DNA strands are antiparallel. At a replication fork, both strands of parental DNA serve as templates for the synthesis of new DNA;• All known DNA polymerases synthesize DNA in the 5’ →3’ direction but not in 3’ →5’ direction.

?

§1.3 Semi-continuous Replication

• This dilemma was resolved by Reiji Okazaki ( in the 1960s), who found that a significant proportion of newly synthesized DNA exists as small fragments;

• These units of about a thousand nucleotides are called Okazaki fragments;

– They are 1000 – 2000nt long for prokaryotes and 100-150nt long for eukaryotes.

Reiji Okazaki and his wife Tsuneko Okazaki

解链方向

• The leading strand :the strand synthesized continuously;

• The lagging strand :the strand formed from Okazaki fragments;

• The semi-continuous replication: Continuous synthesis of the leading strand and discontinuous synthesis of the lagging strand represent a unique feature of DNA replication. It is referred to as the semi-continuous replication.

Semi-continuous replication

Section 2

Enzymology

of DNA Replication

Large team of enzymes coordinates replication

Let’s meetthe team…

Template: double stranded DNASubstrate: dNTPPrimer: short RNA fragment with a

free 3´-OH endEnzyme: DNA-dependent DNA poly

merase (DDDP),other enzymes

Protein factor

DNA replication system

Daughter strand synthesis

• Chemical formulation:

• The nature of DNA replication is a series of 3´,5´phosphodiester bond formation catalyzed by a group of enzymes.

Phosphodiester bond formation

(dNMP)n + dNTP → (dNMP)n+1 + PPi

Where’s theENERGY

for the bonding!

energy

We comewith our own

energy!

Enzymes and protein factors

protein Mr # function

Dna A protein 50,000 1 recognize origin

Dna B protein 300,000 6 open dsDNA

Dna C protein 29,000 1 assist Dna B binding

DNA pol Elongate the DNA strands

Dna G protein 60,000 1 synthesize RNA primer

SSB 75,600 4 single-strand binding

DNA topoisomerase 400,000 4 release supercoil constraint

• The first DNA- dependent DNA polymerase (short for DNA-pol I) was discovered in 1958 by Arthur Kornberg who received Nobel Prize in physiology or medicine in 1959.

§2.1 DNA Polymerase

DNA-pol of prokaryotes

•Arthur Kornberg (left) with his son, Roger, after Roger received the 2006 Nobel Prize in Chemistry.

Kornberg liked to refer to his scientific career as a "love affair with enzymes."

• Later, DNA-pol II and DNA-pol III were identified in experiments using mutated E.coli cell line.

• DNA-pol I possess the following biological activity. 1. 53 polymerizing2. The 3` to 5` exonuclease activity3. The 5` to 3` exonuclease activity

Why does a DNA polymerase also need two exonuclease activities?

• DNA-pol I has the function to correct the mismatched nucleotides.

• It identifies the mismatched nucleotide, removes it using the 3´- 5´ exonuclease activity, add a correct base, and continues the replication.

Proofreading and correction

3´→5´ exonuclease activity excise mismatched

nuleotides

5´→3´ exonuclease activitycut primer or excise mutated segment

C T T C A G G A

G A A G T C C G G C G

5' 3'

3' 5'

Exonuclease functions

DNA-pol of E. coli

Klenow fragment

• Klenow fragment: large fragment (604 AA) of DNA pol I, having DNA polymerization and 3´→5´exonuclease activities, and is widely used in molecular biology.

DNA-pol II

• Temporary functional when DNA-pol I and DNA-pol III are not functional.

• Still capable for doing synthesis on the damaged template

• Participating in DNA repairing

DNA-pol III

• A heterodimer enzyme composed of ten different subunits

• Having the highest polymerization activity (105 nt/min)

• The true enzyme responsible for the elongation process

DNA Polymerase III- does the bulk of copying DNA in Replication

β2 subunit:

sliding clamp

§2.2 Primase

• Also called DnaG• Primase (a specific RNA

polymerase) : synthesize primers using free NTPs as the substrate and the ssDNA as the template.

• Primers: short RNA fragments (5-50 nucleotides).

§2.3 Helicase

• Also referred to as DnaB.• It opens the double strand DNA with c

onsuming ATP. (Zip opener) • The opening process with the assistan

ce of DnaA and DnaC

Dna B

Dna C

解链方向

§2.4 SSB protein(single strand DNA binding protein)

• maintains the DNA template in the single strand form in order to • prevent the dsDNA formation; • protect the ssDNA degradation by nucle

ases.

§2.5 Topoisomerase

• Opening the dsDNA will create supercoil ahead of replication forks, the supercoil constraint needs to be released by topoisomerases (type I and II).

• It cuts a phosphoeste

r bond on one DNA strand, rotates the broken DNA freely around the other strand to relax the constraint, and reseals the cut.

Topoisomerase I

• It is named gyrase in prokaryotes.

• It cuts phosphoester bonds on both strands of dsDNA, releases the supercoil constraint, and reforms the phosphoester bonds.

Topoisomerase II

• Antibiotics: ciprofloxacin, novobiocin and nalidixic acid, inhibit the bacterial gyrase.

• Anticancer agents: adriamycin, etoposide, and doxorubicin, inhibit human topoisomerase.

• Connect two adjacent ssDNA strands by joining the 3´-OH of one DNA strand to the 5´-P of another DNA strand.

• Sealing the nick in the process of replication, repairing, recombination, and splicing.

§2.6 DNA Ligase

P

O

O-

O-

O

HO 5’P

O

O-

O-

O3’

3’ 5’

DNA LigaseATP（ NAD+）

AMP5’ 3’

5’ 3’

Section 3

DNA Replication Process

• Initiation: recognize the starting point, separate dsDNA, primer synthesis, …

• Elongation: add dNTPs to the existing strand, form phosphoester bonds, correct the mismatch bases, extending the DNA strand, …

• Termination: stop the replication

Sequential actions

• The replication starts at a particular point called origin.

§3.1 Replication of prokaryotes

a. Initiation

• Genome of E. coli

DNA sequences at the Bacterial origin of Replication DNA sequences at the Bacterial origin of Replication

• The structure of the origin is 248 bp long and AT-rich.

• DnaA recognizes origin.

• DnaB(helicase) and DnaC join the DNA-DnaA complex, open the local AT-rich region, and move on the template downstream further to separate enough space.

• SSB protein binds the complex to stabilize ssDNA.

Formation of replication fork

• Primase joins and starts the synthesis of RNA primers.

• Primasome: protein complex responsible for creating RNA primers on ssDNA during DNA replication.

• Topoisomerase binds to the dsDNA region just before the replication forks to release the supercoil constraint.

Primer synthesis

Dna ADna B Dna C

DNA topomerase

5'3'

3'

5'

primase

3

5

3

5

primer3' HO

5'primas

e

• The short RNA fragments provide free 3´-OH groups for DNA elongation.

• dNTPs are continuously connected to the primer or the nascent DNA chain by DNA-pol III.

• The nature of the chain elongation is the series formation of the phosphodiester bonds.

b. Elongation

• Primers on Okazaki fragments are digested by RNase.

• The gaps are filled by DNA-pol I in the 5´→3´direction.

• The nick between the 5´end of one fragment and the 3´end of the next fragment is sealed by DNA ligase.

Lagging strand synthesis

3'

5'

5'

3'

RNAase

POH

3'

5'

5'

3'

DNA polymerase

P

3'

5'

5'

3'

dNTP

DNA ligase

3'

5'

5'

3'

ATP

RNase

DNA-pol I

• The synthesis direction of the leading strand is the same as that of the replication fork.

• The synthesis direction of the latest Okazaki fragment is also the same as that of the replication fork.

flash

• The replication of E. coli is bidirectional from one origin, and the two replication forks must meet at one point called ter at 32.

• All the primers will be removed, and all the fragments will be connected by DNA-pol I and ligase.

c. Termination

ori

ter

82

32

movie

Replication of prokaryotes

•The replication process starts from the origin, and proceeds in two opposite directions. It is named replication.

§ Replication Fidelity

• Replication based on the principle of base pairing is crucial to the high accuracy of the genetic information transfer.

• Enzymes use three mechanisms to ensure the replication fidelity.

1×101×10-5-5

　　1×101×10-2-2

1×101×10-2-2

1×101×10-9-9

§3.2 Replication of Eukaryotes

• DNA replication is closely related with cell cycle: S- phase.

• Multiple origins on one chromosome.

• Cell cycle

DNA-pol of eukaryotes

DNA-pol : elongation DNA-pol III

DNA-pol : initiate replication and synthesize primers

DnaG, primase

DNA-pol : replication with low fidelity

DNA-pol : polymerization in mitochondria

DNA-pol : proofreading and filling gap

DNA-pol I

repairing

• The eukaryotic origins are shorter than that of E. coli.

• Requires DNA-pol (primase activity) and DNA-pol (polymerase activity and helicase activity).

• Needs topoisomerase and replication factors (RF) to assist.

Initiation

• DNA replication and nucleosome assembling occur simultaneously.

• Overall replication speed is compatible with that of prokaryotes.

b. Elongation

3

5

53

Leading strand

3

5

3

5

Lagging strand

primernucleosome

3'

5'

5'

3'

3'

5'

5'

3'

connection of discontinuous

3'

5'

5'

3'

3'

5'

5'

3'

segment

c. Termination

The End Replication Problem:Telomeres shorten with each S phase

Ori

3'5'

3'5'

5'

5' 3'3' 5'

• Telomere: the terminal structure of eukaryotic DNA of chromosomes.

• composed of terminal DNA sequence and protein.

• Function: keep the termini of chromosomes in the cell from becoming entangled and sticking to each other.

Telomere

shoelace

Repetitive DNA sequence(TTAGGG in vertebrates)

Form a 'capped' end structure

The Nobel Prize in Physiology or Medicine 2009

Elizabeth Blackburn

Carol Greider Jack Szostak

"for the discovery of how chromosomes are protected by telomeres and the

enzyme telomerase"

• Telomerase: the enzyme that essentially builds new telomeres, maintain the integrity of DNA telomere.

• The telomerase is composed of telomerase RNA telomerase association protein telomerase reverse transcriptase • It is able to synthesize DNA using RNA

as the template.

Telomerase

Inchworm modelinchworm

• Telomerase is highly active in the embryo, and after birth it is active in the reproductive and stem cells.

• Telomerase may play important roles in cell aging and cancer cell biology.

Significance of Telomerase

In most somatic tissues, telomerase is expressed at verylow levels or not at all -- as cells divide, telomeres shorten

Telomerase and Senescence

Short telomeres signal cells to senesce (stop dividing)

cellular clock

Telomerase and Cancer•Strong evidence to suggest that the absence of senesence in cancer cells is linked to the activation of the telomerase.•Telomerase is an attractive target for cancer chemotherapy.

SUMMARY

Telomeres are essential for chromosome stability

Telomere shortening occurs owing to the biochemistry ofDNA replication

Short telomeres cause replicative senescence

Telomerase prevents telomere shortening andreplicative senescence

Section 4

Reverse Transcription

Reverse Transcription

• The genetic information carrier of some biological systems is ssRNA instead of dsDNA (such as ssRNA viruses).

• The information flow is from RNA to DNA, opposite to the normal process.

• This special replication mode is called reverse transcription.

Viral infection of RNA virus

Reverse transcription

•Reverse transcription is a process in which ssRNA is used as the template to synthesize dsDNA. •Synthesis of ssDNA complementary to ssRNA, cDNA, forming a RNA-DNA hybrid. •Hydrolysis of ssRNA: RNase activity of reverse transcriptase, leaving ssDNA.•Synthesis of the second ssDNA, forming a DNA-DNA duplex.

David Baltimore

• In 1970• Discover RNA-dependant DNA polymerase

which later known as reverse transcriptase.

• 1975 Nobel Prize in Physiology or Medicine

Howard M. Temin

Significance of RT

• An important discovery in life science and molecular biology

•RNA plays a key role just like DNA in the genetic information transfer and gene expression process.

•RNA could be the molecule developed earlier than DNA in evolution.

•RT is the supplementary to the central dogma.

Section 5

DNA Damage and Repair

•Definition: mutation is a change of nucleic acids in genomic DNA of an organism. •The mutation could occur in the replication process as well as in other steps of life process. • Consequences of mutation

•To create a diversity of the biological world; a natural evolution of biological systems •To lead to the functional alternation of biomolecules, death of cells or tissues, and some diseases as well

§5.1 Mutation

§5.2 Causes of Mutation

DNA damage

UV radiation

viruses

carcinogensPhysical factors

evolution

infection

T

G

spontaneous mutation

Chemical modification

N

N O

O

CH3

R

PN

N O

O

CH3

R

N

N O

O

CH3

R

P

N

N OR

UV

O

CH3

( T T )

)

Physical damage

Mutation caused by chemicals

• Carcinogens can cause mutation.

• Carcinogens include: • Food additives and food preser

vatives; spoiled food • Pollutants: automobile emissio

n; chemical wastes • Chemicals: pesticides; alkyl deri

vatives; nitrous acid(HNO2)

• Transition: the base alternation from purine to purine, or from pyrimidine to pyrimidine.

• Transversion: the base alternation between purine and pyrimidine, and vise versa.

Point mutation is referred to as the single nucleotide alternation.

a. Point mutation (mismatch)

§5.3 Types of Mutation

• Nitrous acid (HNO2): react with base that contain amino groups, deaminates C to produce U, resulting in G•C A•U

• Nitrous acid formed by digestion of nitrites (preservatives) in foods.

Consequences of point mutations

• Silent mutation: The code containing the changed base may code for the same amino acid. UCA, UCU, all code for serine.

• Missense mutation: the changed base may code for a different amino acid. UCA for serine, ACA for threonine.

• Nonsense mutation: the codon with the altered base may become a termination codon. UCA for serine, UAA for stop codon.

HbS HbA

chains CAC CTC

mRNA GUG GAG

AA residue 6 in chain Val Glu

Hb mutation causing anemia

•Single base mutation leads to one AA change, causing disease.

b. Deletion and insertion

• Deletion: one or more nucleotides are deleted from the DNA sequence.

• Insertion: one or more nucleotides are inserted into the DNA sequence. Deletion and insertion can cause the reading frame shifted.

Frame-shift mutation

Normal

5´… …GCA GUA CAU GUC … …

Ala Val His Val

Deletion C

5´… …GAG UAC AUG UC … …

Glu Tyr Met Ser

• DNA repairing is a kind response made by cells after DNA damage occurs, which may resume their natural structures and normal biological functions.

• DNA repairing is a supplementary to the proofreading-correction mechanism in DNA replication.

§5.4 DNA Repairing

N

N O

O

CH3

R

PN

N O

O

CH3

R

N

N O

O

CH3

R

P

N

N OR

UV

O

CH3

( T T )

)

Photoreactivation repair (or lignt repair)

300~600nm

• One of the most important and effective repairing approach.

• UvrA and UvrB: recognize and bind the damaged region of DNA.

• UvrC: excise the damaged segment.

• DNA-pol Ⅰ: synthesize the DNA segment to fill the gap.

• DNA ligase: seal the nick.

Excision repairing

UvrA UvrBUvrC

OH P

DNA-pol ⅠOHP

DNA ligase NAD+

• XP is an genetic disease.• Patients will be suffered w

ith hyper-sensitivity to UV which results in multiple skin cancers.

• The cause is due to the low enzymatic activity for the nucleotide excision-repairing process, particular thymine dimer.

Xeroderma pigmentosum (XP)

• The most obvious, and often important part of treatment is avoiding exposure to sunlight.

• It is used for repairing when a large segment of DNA is damaged.

• Recombination protein RecA, RecB and RecC participate in this repairing.

Recombination repairing

SOS repairing

• It is responsible for the situation that DNA is severely damaged and the replication is hard to continue.

• If workable, the cell could be survived, but may leave many errors.

• In E. coli, uvr gene and rec gene as well as Lex A protein constitute a regulatory network.

PointsI. General characteristics• Semi-conservative; Specific origins; Bidirectional; Semidiscontinuous repli

cation

II. Bacterial ReplicationA. Polymerization

1. template, primer, dNTP, proceed in 5` to 3` direction2. Pol I, Pol II, Pol III

3. other replication proteins at the replication fork – SSB, helicase, topoisomerase

B. Semidiscontinuous replication: leading strand and lagging strand synthesis 1. RNA primer synthesized by the primases

2. polymerization by Pol III3. completion by Pol I and ligase

4. Okazaki fragment

Points (continue)Ⅲ. Eukaryotic Replication• S phase; Telomere and Telomerase

Ⅳ. Reverse transcription• Definition; Significance

Ⅴ. Mutation, DNA damage and repair• Point mutation; insertion and deletion, Frameshift mutati

ons• Physical and chemical damage; • photoreactivation repair; excision repair• Xeroderma pigmentosum (XP)