5. Structure and Replication of DNA

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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?

1 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 Griffith o DNA is the transforming principle Avery, MacLeod, and McCarty o 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. coli o DNA replication in eukaryotes 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 a virulent strain. Streptococcus pneumoniae Bacteria strain cell morphology colonies pathology III S with polysaccharide capsule Smooth Cause pneumonia, Lethal II R No polysaccharide capsule Rough Not causing any disease The non-virulent II R strain was transformed into virulent strain. Griffith suggested that there was some compound from the III S strain could transform the live II R strain from non-virulent to virulent. 1

Demonstrating that DNA is the transforming principle Avery, MacLeod, and McCarty, 1944 Further demonstrating

entered the host cell and controlled the host cell to produce the T2 phage progenies.

coli host cell, it inject its DNA molecule into the host cell and leave the protein coat outside.

2 Demonstrating that DNA is the transforming principle Avery, MacLeod, and McCarty, 1944 Further demonstrating DNA is the genetic material Hershey-Chase experiment Demonstrated that only the DNA of the bacteriophage T2 entered the host cell and controlled the host cell to produce the T2 phage progenies. Bacteriophage (bacterial virus) T2 consist of a protein coat and a DNA molecule. When the phage infects the E. coli host cell, it inject its DNA molecule into the host cell and leave the protein coat outside. The phage DNA molecule alone can control the host cell to produce its phage particles and eventually lyses the cell and release the phage particles. 2

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.

3 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. They consist of RNA and protein molecules. In 1956, scientist demonstrated that only the purified RNA molecules from TMV could cause infection by spreading on the tobacco leaves. Heinz Fraenkel-Conrat and B. Singer were able to separate and purify the RNA and protein molecules from these viruses. They could reconstitute the viruses using TMV RNA and HR protein or HR RNA and TMV protein to form hybrid viruses. These hybrid viruses could produce viral progeny according to their RNA origin, that is TMV viral progeny from TMV RNA 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 base a pentose sugar (deoxyribose in DNA; oxyribose in RNA) a phosphate group Base + sugar = nucleoside Base + sugar + phosphate = nucleotide DNA Deoxyribonucleosides Deoxyadenosine Deoxycytidine Deoxyribonucleotides Deoxyadenylic acid Deoxycytidylic acid RNA Ribonucleosides Adenosine Cytidine Guanosine Uridine Ribonucleotides Adenylic acid Cytidylic acid Guanylic acid 3 Uridylic acid

Joining up of the four different deoxyribonucleotides makes up a

Å in diameter, unbranched structure Chain structure of DNA:

4 Joining up of the four different deoxyribonucleotides makes 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 4

The Waston and Crick model Main points of the model: Two polynucleotide chains coiled around a central axis, forming a right-handed double helix. Two chains are antiparallel.

5 The Waston and Crick model Main points of the model: Two polynucleotide chains coiled around a central axis, forming a right-handed double 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. The DNA molecule is not always a perfect double helix. The structure of the helix can be affect by: water molecules base composition in solution: B form relatively unhydrated: A form poly A/T: mixed A and B forms, usually unable to bind normally with histone purine-pyrimidine dinucleotide repeats: in solution, high [Na+] Z form low [Na+] B form 5

6 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 were incorporated into newly synthesized DNA strands. After many generations, the DNA molecules of the E. coli cells were labeled with the heavy isotope. The E. coli were then cultured in normal nitrogen (14N, light). Prediction: Mode of replication DNA strands 1 st generation 2 nd generation Conservative 14N/14N, 15N/15N N/N, N/N 1:1 3:1 semiconservative 14N/15N N/N, N/N 1 1:1 dispersive all mix all mix lighter even lighter 6

7 Visualization of the semiconservative replication of chromosome during mitosis The cells were cultured with bromodeoxyuridine (BudR) instead of normal thymidine. After two rounds of cell division, the chromosomes were stained with fluorescent dye and Giemsa. 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. 7

8 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. 8

If there is no mechanism to replicate the terminal DNA segment of the lagging strand, the chromosome will become shorted and shorted in each round of replication.

9 Since the DNA molecule of the eukaryotes is a linear structure, DNA polymerase cannot replicate the terminal DNA segment of the lagging strand. The very far end will leave a 3 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, the chromosome will become shorted and shorted in each round of replication. To overcome this problem, the telomere (the end) of the chromosome will be extended using an enzyme called telomease. The telomease contains a RNA molecule, which serve as the template for the extension of the parental strand. 9

Genetic material must be able to:

Genetic material must be able to: Contain the information necessary to construct an entire organism Pass from parent to offspring and from cell to cell during cell division Be accurately copied Account