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BIO2310 General and Molecular Genetics

5. Structure and Replication of DNA

Key questions:• How was DNA shown to be the genetic material? How about RNA?• How was the structure of DNA determined to be the double helix?• What is the mechanism of DNA replication?

Outlines• DNA as genetic material

o The discovery of transformation – Frederick Griffitho DNA is the transforming principle – Avery, MacLeod, and McCartyo Hershey-Chase experiment

• RNA as genetic material• Building blocks of nucleic acids• Watson and Crick Model – the double helix• DNA replication

o DNA replication in E. colio DNA replication in eukaryotes

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DNA as genetic material

Experiments demonstrated DNA as the genetic material

The discovery of transformation – Frederick Griffith, 1928

The non-virulent bacteria strain was converted to a virulent strain by some chemicals of avirulent strain.

Streptococcus pneumoniae

Bacteria strain cell morphology colonies pathologyIII S with polysaccharide capsule Smooth Cause pneumonia, LethalII R No polysaccharide capsule Rough Not causing any disease

The non-virulent II R strain wastransformed into virulent strain.Griffith suggested that there wassome compound from the III Sstrain could transform the live II Rstrain from non-virulent tovirulent.

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Demonstrating that DNA is the transforming principle – Avery, MacLeod, andMcCarty, 1944

Further demonstrating DNA is the genetic material

Hershey-Chase experiment

Demonstrated that only the DNA of the bacteriophage T2 entered thehost cell and controlled the host cell to produce the T2 phageprogenies.

Bacteriophage (bacterialvirus) T2 consist of aprotein coat and a DNAmolecule. When thephage infects the E. colihost cell, it inject its DNAmolecule into the host celland leave the protein coatoutside. The phage DNAmolecule alone cancontrol the host cell toproduce its phageparticles and eventuallylyses the cell and releasethe phage particles.

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RNA as the genetic material

Some organisms, like viruses, use RNA as their genetic materials.

Demonstrating RNA as genetic material – Heinz Fraenkel-Conrat and B. Singer

Tobacco mosaic virus (TMV) and Holmes ribgrass (HR) viruses are plant viruses. Theyconsist of RNA and protein molecules.In 1956, scientist demonstrated that only the purified RNA molecules from TMV could causeinfection by spreading on the tobacco leaves.Heinz Fraenkel-Conrat and B. Singer were able to separate and purify the RNA and proteinmolecules from these viruses. They could reconstitute the viruses using TMV RNA and HRprotein or HR RNA and TMV protein to form hybrid viruses. These hybrid viruses couldproduce viral progeny according to their RNA origin, that is TMV viral progeny from TMVRNA–HR protein hybrid virus, or vice versa.

Building blocks of nucleic acids

Nucleotides are the building blocks of all nucleic acids.

Each nucleotide consists of three essential components:a nitrogenous basea pentose sugar (deoxyribose in DNA; oxyribose in RNA)a phosphate group

Base + sugar = nucleosideBase + sugar + phosphate = nucleotide RNA

Ribonucleosides RibonucleotidesAdenosine Adenylic acidCytidine Cytidylic acidGuanosine Guanylic acidUridine Uridylic acid

DNADeoxyribonucleosides DeoxyribonucleotidesDeoxyadenosine Deoxyadenylic acidDeoxycytidine Deoxycytidylic acid

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Joining up of the four different deoxyribonucleotidesmakes up a DNA strand.

The Structure of DNA

Watson and Crick Model – the double helix

Leading to the double helix model:

• EM study of the DNA molecule: 20 Å in diameter, unbranched structure

• Chain structure of DNA: nucleotides are linked together by 5’-3’phosphodiester bonds

• The base ratio: A = T, G = C

• X-ray crystallography: reveal the helix structure

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The Waston and Crick model

The DNA molecule is not always a perfect double helix.The structure of the helix can be affect by:

water molecules in solution: B formrelatively unhydrated: A form

base composition poly A/T: mixed A and B forms, usually unable to bind normally with

histone purine-pyrimidine dinucleotide repeats:

in solution, high [Na+] Z formlow [Na+] B form

Main points of the model:

• Two polynucleotide chains coiled around a central axis, forming a right-handeddouble helix.

• Two chains are antiparallel.• The bases of both strand are lying inside of the structure and stacking flat on

one another, 3.4Å (0.34 nm) apart.• The two strands are holding together by hydrogen bonds.• One complete turn of the helix is 34 Å long and contains 10 bases.• There are major grooves and minor grooves along the axis.• The double helix measures 20 Å in diameter.

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methylation methylation favors B to Z transition

DNA replication

Semiconservative replication

Meselson-Stahl experiment

E.coli was cultured in medium containing heavy isotope nitrogen (15N).This isotope was inserted into the nitrogen bases, which then wereincorporated into newly synthesized DNA strands. After many generations,the DNA molecules of the E. coli cells were labeled with the heavyisotope. The E. coli were then cultured in normal nitrogen (14N, light).

Prediction:Mode of replication DNA strands

1st generation 2nd generationConservative 14N/14N, 15N/15N N/N, N/N

1:1 3:1

semiconservative 14N/15N N/N, N/N1 1:1

dispersive all mix all mixlighter even lighter

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Visualization of the semiconservative replication of chromosome during mitosis

The cells were cultured with bromodeoxyuridine (BudR) instead of normal thymidine. Aftertwo rounds of cell division, the chromosomes were stained with fluorescent dye andGiemsa. For semiconservative mode of replication, one sister chromatid was fluorescent.

Mechanism of DNA replication

Riplication in E. coli

• Start at a particular site called origin of replication• The two strands are open up by topoisomerase and helicase.• Short RNA primers (~30 nucleotide long) are synthesized by RNA polymerase or

primase.• RNA primers are primed on the DNA strands.• DNA polymerase III starts the new DNA strand synthesis towards the opening of the

fork. This strand is called leading strand.• DNA plymerase I starts the new strand synthesis in opposite direction of the fork

opening. This strand is called lagging strand.• When the two strands open up more, RNA primers are primed on the single strand

and DNA polymerase I starts to make another lagging strand.• The DNA replication in E. coli is bi-directional and rotating around the axis of the

helix.• Eventually, the lagging strands are joined together by ligase.

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Note the proof reading property of the DNA polymerases (3’ � 5’ exonuclease activity).

DNA replication in eukaryotes

The mechanism of DNA replication in eukaryotes is similar to E. coli.Replication starts at many sites along the chromosome.Replication is bi-directional.

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Since the DNA molecule of the eukaryotes is a linear structure, DNA polymerase cannotreplicate the terminal DNA segment of the lagging strand. The very far end will leave a3’ protruding single strand. This single-stranded end will be removed by some exonuclease.If there is no mechanism to replicate the terminal DNA segment of the lagging strand, thechromosome will become shorted and shorted in each round of replication.To overcome this problem, the telomere (the end) of the chromosome will be extended usingan enzyme called telomease. The telomease contains a RNA molecule, which serve as thetemplate for the extension of the parental strand.