Genetic Information Transfer

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DNA RNA protein

transcription translation replication

reverse transcription

Central dogma

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• 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

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DNA Replication

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Section 1

General Concepts of DNA Replication

DNA replication

• A reaction in which daughter DNAs are synthesized using the parental DNAs as the template.

• Transferring the genetic information to the descendant generation with a high fidelity

replication

parental DNAdaughter DNA

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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.

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The DNA backbone

• Putting the DNA backbone together– refer to the 3 and 5 ends

of the DNA

OH

O

PO4

base

CH2

O

base

OPO

C

O–O

CH2

1

2

4

5

1

2

3

3

4

5

Phosphodiester bond formation

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Template: double stranded DNA

Substrate: dNTP

Primer: short RNA fragment with a free 3´-OH end

Enzyme: DNA-dependent DNA polymerase (DDDP),

other enzymes,

protein factor

DNA replication system

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Characteristics of replication

Semi-conservative replication

Bidirectional replication

Semi-continuous replication

High fidelity

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§1.1 Semi-Conservative Replication

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Semiconservative replication

Half of the parental DNA molecule is conserved in each new double helix, paired with a newly synthesized complementary strand. This is called semiconservative replication

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Semiconservative replication

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Experiment of DNA semiconservative replication

"Heavy" DNA(15N)

grow in 14N medium

The first generation

grow in 14N medium

The second generation

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Significance

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

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§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.

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3'

5'

5'

3'

5'

3'

5'3'

direction of replication

Replication fork

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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 syntheses continue.

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Bidirectional replication

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Replication of prokaryotes

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

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Replication of eukaryotes

• Chromosomes of eukaryotes have multiple origins.

• The space between two adjacent origins is called the replicon, a functional unit of replication.

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origins of DNA replication (every ~150 kb)

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§1.3 Semi-continuous Replication

The daughter strands on two template strands are synthesized differently since the replication process obeys the principle that DNA is synthesized from the 5´ end to the 3´end.

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5'

3'

3'

5'

5'

direction of unwinding3'

On the template having the 3´- end, the daughter strand is synthesized continuously in the 5’-3’ direction. This strand is referred to as the leading strand.

Leading strand

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Semi-continuous replication

3'

5'

5'3'

replication direction

Okazaki fragment

3'

5'

leading strand

3'

5'

3'

5'replication fork

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• Many DNA fragments are synthesized sequentially on the DNA template strand having the 5´- end. These DNA fragments are called Okazaki fragments. They are 1000 – 2000 nt long for prokaryotes and 100-150 nt long for eukaryotes.

• The daughter strand consisting of Okazaki fragments is called the lagging strand.

Okazaki fragments

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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

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Section 2

Enzymology

of DNA Replication

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

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• 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

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

• All of them possess the following biological activity.

1. 53 polymerizing

2. exonuclease

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DNA-pol of E. coli

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DNA-pol I

• Mainly responsible for proofreading and filling the gaps, repairing DNA damage

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Klenow fragment

• small fragment (323 AA): having 5´→3´ exonuclease activity

• large fragment (604 AA): called Klenow fragment, having DNA polymerization and 3´→5´exonuclease activity

N end C end

caroid

DNA-pol Ⅰ

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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

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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

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Structure of DNA-pol III

α ： has 5´→ 3´ polymerizing activity

ε ： has 3´→ 5´ exonuclease activity and plays a key role to ensure the replication fidelity.

θ: maintain heterodimer structure

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39

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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

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§2.2 Primase

• Also called DnaG

• Primase is able to synthesize primers using free NTPs as the substrate and the ssDNA as the template.

• Primers are short RNA fragments of a several decades of nucleotides long.

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• Primers provide free 3´-OH groups to react with the -P atom of dNTP to form phosphoester bonds.

• Primase, DnaB, DnaC and an origin form a primosome complex at the initiation phase.

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§2.3 Helicase

• Also referred to as DnaB.

• It opens the double strand DNA with consuming ATP.

• The opening process with the assistance of DnaA and DnaC

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§2.4 SSB protein

• Stand for single strand DNA binding protein

• SSB protein maintains the DNA template in the single strand form in order to

• prevent the dsDNA formation;

• protect the vulnerable ssDNA from nucleases.

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§2.5 Topoisomerase

• Opening the dsDNA will create supercoil ahead of replication forks.

• The supercoil constraint needs to be released by topoisomerases.

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• The interconversion of topoisomers of dsDNA is catalyzed by a topoisomerase in a three-step process:

• Cleavage of one or both strands of DNA

• Passage of a segment of DNA through this break

• Resealing of the DNA break49

• Also called -protein in prokaryotes.

• It cuts a phosphoester bond on one DNA strand, rotates the broken DNA freely around the other strand to relax the constraint, and reseals the cut.

Topoisomerase I (topo I)

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• It is named gyrase in prokaryotes.

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

• It can change dsDNA into the negative supercoil state with consumption of ATP.

Topoisomerase II (topo II)

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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

§2.6 DNA Ligase

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• 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.

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§2.7 Replication Fidelity

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

• Enzymes use two mechanisms to ensure the replication fidelity.

– Proofreading and real-time correction

– Base selection

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• 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

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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

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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

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• The replication starts at a particular point called origin.

• The origin of E. coli, ori C, is at the location of 82.

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

§3.1 Replication of prokaryotes

a. Initiation

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Genome of E. coli

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• Three 13 bp consensus sequences• Two pairs of anti-consensus repeats

Structure of ori C

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Formation of preprimosome

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• DnaA recognizes ori C.

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

• DnaA is replaced gradually.

• SSB protein binds the complex to stabilize ssDNA.

Formation of replication fork

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• Primase joins and forms a complex called primosome.

• Primase starts the synthesis of primers on the ssDNA template using NTP as the substrates in the 5´- 3´ direction at the expense of ATP.

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

Primer synthesis

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• The supercoil constraints are generated ahead of the replication forks.

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

• The negatively supercoiled DNA serves as a better template than the positively supercoiled DNA.

Releasing supercoil constraint

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Dna ADna B Dna C

DNA topomerase

5'3'

3'

5'

primase

Primosome complex

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

• The core enzymes ( 、、 and ) catalyze the synthesis of leading and lagging strands, respectively.

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

b. Elongation

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• 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.

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• 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 ligase.

Lagging strand synthesis

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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

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• 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

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§3.2 Replication of Eukaryotes

• DNA replication is closely related with cell cycle.

• Multiple origins on one chromosome, and replications are activated in a sequential order rather than simultaneously.

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Cell cycle

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• 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

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• DNA replication and nucleosome assembling occur simultaneously.

• Overall replication speed is compatible with that of prokaryotes.

b. Elongation

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3'

5'

5'

3'

3'

5'

5'

3'

connection of discontinuous

3'

5'

5'

3'

3'

5'

5'

3'

segment

c. Termination

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• The terminal structure of eukaryotic DNA of chromosomes is called telomere.

• Telomere is composed of terminal DNA sequence and protein.

• The sequence of typical telomeres is rich in T and G.

• The telomere structure is crucial to keep the termini of chromosomes in the cell from becoming entangled and sticking to each other.

Telomere

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• The eukaryotic cells use telomerase to 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

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

Significance of Telomerase

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Section 4

Other Replication Modes

§4.1 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.

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Viral infection of RNA virus

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Reverse transcription

Reverse transcription is a process in which ssRNA is used as the template to synthesize dsDNA.

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Process of Reverse transcription

• Synthesis of ssDNA complementary to ssRNA, forming a RNA-DNA hybrid.

• Hydrolysis of ssRNA in the RNA-DNA hybrid by RNase activity of reverse transcriptase, leaving ssDNA.

• Synthesis of the second ssDNA using the left ssDNA as the template, forming a DNA-DNA duplex. 88

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Reverse transcriptase

Reverse transcriptase is the enzyme for the reverse transcription. It has activity of three kinds of enzymes:

• RNA-dependent DNA polymerase

• RNase

• DNA-dependent DNA polymerase

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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. 91

Significance of RT

• This discovery enriches the understanding about the cancer-causing theory of viruses. (cancer genes in RT viruses, and HIV having RT function)

• Reverse transcriptase has become a extremely important tool in molecular biology to select the target genes.

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§4.2 Rolling Circle Replication

5'3'

5'

3'

5'

3'

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§4.3 D-loop Replication

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