Part 3 Genetic Information Transfer

The biochemistry and molecular biology department of CMU

Central dogma
replication transcription translation

DNA

RNA

protein

reverse transcription

 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

Chapter 10
DNA Replication

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 DNA daughter DNA

Daughter strand synthesis

Chemical formulation:

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

Phosphodiester bond formation

DNA replication system

Template: Substrate: Primer: Enzyme: double stranded DNA dNTP short RNA fragment with a free 3-OH end DNA-dependent DNA polymerase (DDDP), other enzymes, protein factor

Characteristics of replication

Semi-conservative replication Bidirectional replication Semi-continuous replication High fidelity

1.1 Semi-Conservative Replication

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

Semiconservative replication

Experiment of DNA semiconservative replication

"Heavy" DNA(15N) grow in 14N medium The first generation grow in 14N medium The second generation

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

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.

Replication fork
5' 3' 3' 5' 5' 3' 5'

direction of replication

3'

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.

Bidirectional replication

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

origins of DNA replication (every ~150 kb)

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 3end.

Leading strand
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.
3' 5' 3'
direction of unwinding

3'

5' 5'

Semi-continuous replication
3' 5' replication fork

3' 3' replication direction 5' 5' 3' 5' Okazaki fragment
3' 5' leading strand

Okazaki fragments
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.

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.

Section 2
Enzymology of DNA Replication

Enzymes and protein factors

protein Dna A protein Dna B protein Dna C protein DNA pol Dna G protein SSB DNA topoisomerase 60,000 75,600 400,000 1 4 4 Mr 50,000 300,000 29,000 # 1 6 1 function recognize origin open dsDNA assist Dna B binding Elongate the DNA strands synthesize RNA primer single-strand binding release supercoil constraint

2.1 DNA Polymerase

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

 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. 5 3 polymerizing 2. exonuclease

DNA-pol of E. coli

DNA-pol I
Mainly responsible for proofreading and filling the gaps, repairing DNA damage

Klenow fragment
N end DNA-pol caroid C end

small fragment (323 AA): having 53 exonuclease activity large fragment (604 AA): called Klenow fragment, having DNA polymerization and 35exonuclease activity

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

Structure of DNA-pol III

has 5 3 polymerizing activity has 3 5 exonuclease activity and plays a key role to ensure the replication fidelity. : maintain heterodimer structure

DNA-pol of eukaryotes
DNA-pol : initiate replication and synthesize primers DNA-pol : replication with low fidelity DNA-pol : polymerization in mitochondria DNA-pol : elongation DNA-pol : proofreading and filling gap DNA-pol III DNA-pol I DnaG, primase repairing

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.

 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.

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

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.

2.5 Topoisomerase
Opening the dsDNA will create supercoil ahead of replication forks. The supercoil constraint needs to be released by topoisomerases.

 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 break

Topoisomerase I (topo I)
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 II (topo II)

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.

2.6 DNA Ligase

3' 5' 5' 3'

RNAase 3' 5'

dNTP 3' 5' P

OH

5' 3'
5' 3'

DNA polymerase

ATP 3' 5'

DNA ligase 5' 3'

 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.

2.7 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

Proofreading and correction

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.

Exonuclease functions
53 exonuclease activity 35 exonuclease activity

cut primer or excise mismatched excise mutated nuleotides segment 3' 5' C T T C A G G A

3'

G A A G T C C G G C G

5'

Section 3
DNA Replication Process

Sequential actions
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

3.1 Replication of prokaryotes

a. Initiation
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.

Genome of E. coli

Structure of ori C
Three 13 bp consensus sequences Two pairs of anti-consensus repeats

Formation of preprimosome

Formation of replication fork

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.

Primer synthesis
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.

Releasing supercoil constraint

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.

Primosome complex

Dna B Dna A 5' 3' DNA topomerase

Dna C
primase

3'

5'

b. Elongation
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.

 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.

Lagging strand synthesis

Primers on Okazaki fragments are digested by RNase. The gaps are filled by DNA-pol I in the 53direction. The nick between the 5end of one fragment and the 3end of the next fragment is sealed by ligase.

3' 5'

5' 3'

RNAase 3' 5'

dNTP 3' 5' P

OH

5' 3'

DNA polymerase 5' 3'

ATP 3' 5'

DNA ligase 5' 3'

c. Termination
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.

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.

Cell cycle

Initiation
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.

b. Elongation
DNA replication and nucleosome assembling occur simultaneously. Overall replication speed is compatible with that of prokaryotes.

c. Termination
3' 5' 3' 5' 5' 3' 5' 3'
connection of discontinuous segment 5' 3' 5' 3'

3' 5' 3' 5'

Telomere
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.

Telomerase
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.

Inchworm model

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

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.

Viral infection of RNA virus

Reverse transcription

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

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.

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

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

Significance of RT
This discovery enriches the understanding about the cancercausing 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.

4.2 Rolling Circle Replication

3' 5' 3'

5'

3'

5'

4.3 D-loop Replication

Section 5
DNA Damage and Repair

5.1 Mutation

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 Changes of genotype, but no effect on phenotype

5.2 Causes of Mutation

UV radiation

Chemical modification

Physical factors

carcinogens DNA damage

infection spontaneous mutation

T G

viruses

evolution

Physical damage
O R P R N O N CH3 CH3 O N O R O N N O CH3 CH3 R N O N O

UV
N

(TT)

Mutation caused by chemicals

Carcinogens can cause mutation. Carcinogens include:
Food additives and food preservatives; spoiled food Pollutants: automobile emission; chemical wastes Chemicals: pesticides; alkyl derivatives; -NH2OH containing materials

5.3 Types of Mutation

a. Point mutation (mismatch)
Point mutation is referred to as the single nucleotide alternation. Transition: the base alternation from purine to purine, or from pyrimidine to pyrimidine. Transversion: the base alternation between purine and pyrimidine, and vise versa.

Transition mutation

Hb mutation causing anemia

Single base mutation leads to one AA change, causing disease.
HbS chains mRNA AA residue 6 in chain CAC GUG Val HbA CTC GAG Glu

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

c. Rearrangement
It is an exchange of large DNA fragments. It can be either reverse the direction or recombination between chromosomes. 1. Site-specific recombination 2. Homologous genetic recombination 3. DNA transposition

5.4 DNA Repairing

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.

Light repairing
O R P R N O N CH3 CH3 O N O R O N N O CH3 CH3 R N O N O

UV
N

(TT)

Excision repairing
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 : synthesize the DNA segment to fill the gap. DNA ligase: seal the nick.

Xeroderma pigmentosis (XP)

XP is an autosomal recessive genetic disease. Patients will be suffered with hyper-sensitivity to UV which results in multiple skin cancers. The cause is due to the low enzymatic activity for the nucleotide excisionrepairing process, particular thymine dimer.

Excision repairing

Recombination repairing
It is used for repairing when a large segment of DNA is damaged. Recombination protein RecA, RecB and RecC participate in this 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.