Biology Chapter 14, DNA: Genetic Material

Mc G hill

Genes

Scientists knew chromosomes were primarily made of protein and DNA but did not know which actually made up genes

DNA composed of 4 nucleotides, proteins contained 20 distinct amino acids suggesting proteins had greater capacity for storing information

Genes

A series of experiments in

1920-1950s determines DNA is the genetic material

Fredrick Griffith- 1928

Studied

Streptococcus Pneumoniae, a pathogenic bacterium causing pneumonia

Two strains: “S” strain is Virulent, “R” strain is nonvirulent

Griffith infected mice with both strains hoping to understand the difference between the strains

Griffith’s Results

Injection of, live virulents (S)  and live nonvirulent (R)

S-Strain cells killed the mice 

R- Strain cells did not kill the mice 

Griffith’s Result

injections 

Heat killed virulent (S) strain cells did not kill the mice 

Heat killed virulent (S) strain + lives nonvirulent (R) strain cells killed the mice 

Transformation

Griffith called the transfer of virulence form the dead S strain cells into the live R strain cells transformation

Did not know the mechanism for movement of genetic information

Transformation

Modern interpretation is that genetic material was 

Physically transferred between the cells 

Avery MacLeod & MacCarty 1944

Repeted Griffith experiments experiment using purified cell extracts

Removel of all protein form the transforming material did not destroy its ability to transform R strain cells

DNA digesting enzymes destory all transformign ability


Supported DNA as the genetic material at least in bacteria

Hershey & Chase 1952

Investigated genetic material using bacteriophages ( also called phages) Virus that infect bacteria


Wanted to determine which of molecules is the genetic material is the genetic material that is injected into the bacteria

Bacteriophages are composed of 

Only DNA and protein 

Hershey and chase experiment

Bacteriophage DNA was labeled with

Radioactive phosphorus

Hershey & Chase Experiment

Bacteriophage proteins was labeled with

Radioactive Sulfur

Hershey & Chase Experiment

Radioactive molecules

Were tracked

Hershey & Chase Experiment

Only the bacteriophage DNA entered the

Bacteria and was used to prodice more bacteriophage

Hershey & Chase Experiment

Conclusion

DNA is the genetic material

DNA Structure 

DNA is a 

Nucleic acid composed of Nucleotides:

Deoxyribose (5 carbon sugar) with a Phosephate group attached 

Nitrogenous base: Adnine, Thymine, Cytosine, Guanine 

Free Hydroxyl Group ( attached to 3’ carbon of sugar)

Nucleotide subunits of DNA and RNA, Structure

Phosphodiester Bond

Bound between adjacent nucleotides

Formed between the phosphate group of one nucleotide and the 3’ -OH of the next neucleotide

The Chain of nucleotides has 5’-to-3’ orientation

Phosphodiester Bond

Chargaff’s Rule

Erwin Chargadd determined that

Always an equal proportion of two ringed purines (A & G ) and a single ringed pyrimidines (C & T)

Chargaff’s Rule

Amount of Adenine =

Amount of Thymine

Chargaff’s Rule

Amount of Cytosine =

Amount of Guanine

Chargaff’s Rule

Ratio of A-T and G-C

Varies by species

Rosalind Franklin

Preformed X- Ray diffraction studies to identify the 3-D structure

Discovered that DNA is helical

Using Maurice Walkins DNA fibers, discovered that the molecule has a diameter of 2nm and makes a complete turn of the helix every 3.4 nm

James Watson and Francis Crick 1953

Deduced the structure of DNA using evidence form Charguff, Franklin and others 

They did not preform a single experiment themselves related to DNA

Key insight of their model was each DNA molecule was made of two intertwined chains of nucleotides that is a double helix structure

Strucutre of a single DNA strand

Phosphodiester back bone repeating

Sugar and phosphate units joined by phosphodiester bonds

A single strand extends in a 5’ to 3’ direction

The double helix

Two strands arrange as a double helix

Forms two groves the larger major groove and the smaller minor groove

The double helix 

Strands connect via hydrogen bonds between bases on opposite strands 

Result is specific base pairs : A-T and G-C 

Helix has a consistent diameter is stable because of addictive property of thousands of low energy hydrogen bonds

Base pairing

Pattern of base pairing is complementary

A forms two H bonds with T

G forms 3 H bonds with C

Base pairing

Two strands of single DNA molecule are not identical

Each strand specifies the other rby base pair complementarity

Antiparallel configuration 

each phosphodiesterase strand has inherent polarity base on orientation of sugar phosphate backbone 

One end terminates in 3’ OH 

One end terminates in 5’ PO 

Antiparallel Configuration

Strands are referred as having

5’-to-3’ or 3’-to-5’ polarity

Antiparallel Configuration

The two strands of a single DNA molecule have

Opposite polarity to one another

Three possible models of DNA Replication

1. Conservative

2. Semiconservative

3. Dispersive

DNA replication models

Conservative Model

Both strands of parental DNA remain intact, new DNA copies consist of all new molecules 

DNA replication model

SemiConvservative model

Daughter stands each consist of one parental strand and one new strand

DNA replication model

Dispersive Model

New DNA is dispersed throughout each strand both daughter molecules after replication

DNA replication models

Conservative Model, Image

DNA replication models

SemiConservative Model, Image

DNA replication model

Dispersive Model, Image

Meselson and Stahl 1958

Bacterial cells were grown in a

Heavy isotope of Nitrogen, 15^N

Meselson and Stahl 1958

After Several generation the DNA of

These Bacteria was denser than normal DNA

Meselson and Stahl 1958 

Cells were switched to

Media Containing lighter 14N

Meselson and Stahl 1958

DNA was Extracted form the cells at

Various time intervals and centrifuged to separate out by weight

Meselson and Stahl’s Results

Conservative Model

Is rejected

Two density bands were not observed after round 1

Meselson and Stahl Results 

Semiconservative model

is Supported 

Consistent with all observations 

One band after round 1 

Two bands after round 2 

Meselson and Stahl Results

Dispersive Model

Is rejected

1st Round Results consistent

2nd Round, did not observed one band

The Meselson Stahl experiment,

Diagram

DNA Replication

Requirements 

1. Something to copy, parental DNA molecule 

2. Something to do the copying, Enzymes 

3. Building Blocks to make copy, Nucleotide Triphosphates 

DNA Replication

Something to copy

Parental DNA Molecule

DNA Replication

Something to do the copying

Enzymes 

DNA Replication

Building Blocks to make copy

Nucleotide Triphosphates

Stages of DNA replication

Initiation

Replication Begins

Stages of DNA Replication 

Elongation

New strand of DNA are synthesized by DNA Polymerase

Stages of DNA replication

Termination

Replication is Terminated

Action of DNA polymerase

Diagram

DNA Polymerase

Match existing DNA bases with

Complementary nucleotides and links them, That is build new DNA strands

DNA Polymerase

All have several common features

Add new base to 3’ end of existing strands

Synthesize in 5’- to-3’ direction

Require a primer of RNA

RNA Polyermerase makes primer to DNA polymerase extends primer, image

Prokaryotic Replication 

E. coli used as model systems for Understanding universal attributes of replication

Single circular molecule of DNA

Replication in both directions around the chromosome

Replicon

Replicon 

DNA controlled by an origin 

replication is bidirectional form a unique origin , Diagram

E. coli has three DNA polymerase

DNA polymerase |

Acts on lagging strand to remove primers and replace them with DNA

E. coli has three DNA polymerase

DNA polymerase ||

Involved in DNA repair processes 

E. coli has three DNA polymerase

DNA polymerase |||

Main replication enzyme

E. coli has three DNA polymerase

All 3 have 3’-to- 5’ exonuclease activity: proofreading

DNA Pol | has 5’-to- 30 exonuclease activity remivug RNA primers: removing RNA primers

DNA Polyemerase activity

In addition to adding nucleotides to a growing DNA strand

Some polymerase molecules can remove nucleotides acting as nucleases 

DNA Polyemerase activity

Can be

Endonucleases : Cut DNA internally

Exonuclease: Remove neucleotides form end of DNA

DNA Polymerase activity

All three E. Coli DNA polymerase have

3’-to-5’ Exonuclease activity- Proofreading

DNA polymerase activity

DNA Pol | has

5’-to-3’ Exonuclease activity : removing RNA primers 

Enzymes unwind DNA

Helicases

Use energy form ATP to unwind DNA

Enzymes unwind DNA

single strand binding proteins (SSBs)

Coats strands to keep them apart

Enzymes unwind DNA

Unwinding of DNA introduces Torsional strand in the molecules that can lead to 

Additional twisting of the helix called supercoiling

Enzymes unwind DNA

Topoisomerases

Are enzymes that prevent supercoiling

DNA gyrase is the Topoisomerase involved in DNA replication that relives the torsional strand

Unwinding the helix causes torsional strand, Supercoiling diagram

Unwinding the helix causes torsional strand,  No Supercoiling diagram

Replication is semi discontinuous

DNA polymerase can only synthesize in the

5’-to- 3’ direction

Replucation is semi discontinuous

Antiparallel nature of DNA means

New DNA strand must be synthesized in oppsite directions

Replication is semi discontinuous 

Leading  strand synthesized  

Continuously form an initial primer

Replication is semi discontinuous

Lagging strand synthesized

Discontinuously form an initial primer

DNA fragments on the lagging strand are called OKazaki fragments must be connected together 

Lagging is synthesized in pieces, Diagram

Synthesis occurs at the replication fork

Replication Fork

Is partial opening of helix formed where doubles stranded DNA is being unwound

Synthesis occurs at the replication fork 

DNA Primase

RNA Polymerase that makes RNA primer 

RNA will be removed and replaced with DNA later 

Leading strand synthesis

Single priming event

Leading strand synthesis

strand extended by DNA Pol |||

Processivity the ability of polymerase to stay attached

β subunit forms “sliding clamp” to keep DNA Pol ||| attached to DNA ( high processivity)

Lagging strand synthesis requires additional enzymes 

Discontinuous synthesis, requiers multiple Enzymes 

DNA Pol ||| - like leading strand 

Primase - Makes RNA primer for each Okazaki fragment 

DNA Pol  |- Removes all RNA primer and replaced with DNA 

DNA ligase- joins Okazaki fragments to form complete strands 

Lagging strand synthesis requires additional enzymes

Termination occurs at specific site 

DNA Gyrase unlinks two copies 

Lagging strand synthesis, Diagram latter

Replisome

Is a macromolecular assembly of enzymes involved in DNA replication

Replisome

Two main components  

Primosome - Primase, helicase, accessory proteins 

Complex of two DNA Pol ||| - one for each strand

Model of the structure of the replication fork diagram

Eukaryotic Replication

More complex than in prokaryotes due primarily to

Large amount of DNA in multiple chromosomes 

Linear structure ( versus circular chromosomes) 

Eukaryotic Replication uses multiple origins

Basic Enzymology is similar

Requiers new enzymatic activity for dealing with ends only

Eukaryotic Replication uses multiple origins

Multiple replicons, multiple origins of replications for each chromsome

Not sequence specific can be adjusted

Example: early in development when cells divide rapidly more origins can be used

Eukaryotic replication fork is more complex

Before S phase

Helicase are loaded onto possible replication origins but not activated

Eukaryotic replication fork is more complex

During S phase

Subset of these are activated and the rest of the replisome assembled

priming uses a complex of both DNA polymerase α and Primase

DNA polymerase epsilon (Pol ε) synthesizes leading strand 

DNA polymerase Delta (Pol  δ) synthessizes lagging strand 

Archaeal and eukaryotic replication proteines are evolutionarily related 

Enzymes that are similar between Eukaryotes and Archaea but different form those in Prokaryotes

DNA polymerases

Replicative helicase 

Primases

Linear chromosomes have specialized ends

Telomeres

Specialized structures found on the ends of eukaryotic chromosomes

Composed of specific repeat sequences

Linear chromosomes have specialized ends

Protect ends chromosomes form nucleases

Maintain the integrity of linear chromosomes 

Not made by replication complex 

Replication of the end of linear DNA presents a problem, diagram