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DNA

The Search to Identify the Genetic Material

Discovery of Nucleic Acids - Friedrich Miescher, 1869

Miescher isolated the nuclei of white blood cells obtained from pus cells. His experiments revealed that nuclei contained a chemical that contained nitrogen and phosphorus but no sulfur. He called the chemical nuclein because it came from nuclei. It later became known as nucleic acid.

Proteins Produce Genetic Traits - Archibald Garrod, 1909

Garrod noticed that people with certain genetic abnormalities (inborn errors of metabolism) lacked certain enzymes. This observation linked proteins (enzymes) to genetic traits.

Genetic Material can Transform Bacteria - Frederick Griffith, 1931

When Streptococcus pneumoniae (pneumococcus) bacteria are grown on a culture plate, some produce smooth shiny colonies (S) while others produce rough colonies (R). This is because the S strain bacteria have a mucous (polysaccharide) coat, while R strain does not.

Mice infected with the S strain die from pneumonia infection but mice infected with the R strain do not develop pneumonia.

Griffith was able to kill bacteria by heating them. He observed that heat-killed S strain bacteria injected into mice did not kill them. When he injected a mixture of heat-killed S and live R bacteria, the mice died. Moreover, he recovered living S bacteria from the carcasses.

He concluded that some substance needed to produce the mucous coat was passed from the dead bacteria (S strain) to the live ones (R strain); they became transformed.

This must be due to a change in the genotype associated with the transfer of the genetic material.

The transforming material is DNA - Oswald Avery, Colin MacLeod, and Maclyn McCarty, 1944

Prior to the work of Avery, MacLeod, and McCarty, the genetic material was thought to be protein. Avery, MacLeod, and McCarty worked to determine what the transforming substance was in Griffith's experiment (above).

They purified chemicals from the heat-killed S cells to see which ones could transform live R cells into S cells. They discovered that DNA alone from S bacteria caused R bacteria to become transformed.

They also discovered that protein-digesting enzymes (proteases) and RNA-digesting enzymes (RNAse) did not affect transformation, so the transforming substance was not a protein or RNA. Digestion with DNase did inhibit transformation, so DNA caused transformation.

They concluded that DNA is the hereditary material, but not all biologists were convinced.

Discovery of the Structure of DNA

Erwin Chargaff, 1940’s and early 50's

DNA was thought to contain equal amounts of A, T, T, and C. Chargaff found that the base composition of DNA differs among species.

His data showed that in each species, the percent of A equals the percent of T, and the percent of G equals the percent of C. so that 50% of the bases were purines (A + G) and 50% were pyrimidines (T + C)

Chargafff’s rule: The amount of A = T and the amount of G = C.

M.H.F. Wilkins and Rosalind Franklin, early 50’s

Wilkins and Franklin studied the structure of DNA crystals using X-rays.

They found that the crystals contain regularly repeating subunits.

Structures that are close together cause the x-ray to bend more than structures that are further apart. The X pattern produced by DNA suggested that DNA contains structures with dimensions of 2 nm, 0.34 nm, and 3.4 n. The dark structures at the top and bottom indicate that some structure was repeated, suggesting a helix.

James Watson and Francis H.C. Crick, 1953

Watson and Crick used Chargaff and Franklin’s X-ray diffraction data to construct a model of DNA.

The model showed that DNA is a double helix with sugar-phosphate backbones on the outside and the paired nucleotide bases on the inside, in a structure that fit the spacing estimates from the X-ray diffraction data.

Chargaff's rules showed that A = T and G = C, so there was complementary base pairing of a purine with a pyrimidine, giving the correct width for the helix.

The paired bases can occur in any order, giving an overwhelming diversity of sequences.

Properties of Genetic Material

DNA is an ideal genetic material because it can store information, is able to replicate, and is able to undergo changes (mutate).

Structure of DNA

DNA is composed of units called nucleotides. Each nucleotide contains a phosphate group, a deoxyribose sugar, and a nitrogenous base.

The nucleotides joined together to form a chain. The phosphate end of the chain is referred to as the 5' end. The opposite end is the 3' end.

DNA is composed of two chains of nucleotides linked together in a ladder-like arrangement with the sides composed of alternating deoxyribose sugar and phosphate groups and the rungs being the nitrogenous bases as indicated by the diagram below.

The "A" of one strand is always paired with a "T" on the other. Similarly, the "G" of one strand is paired with a "C" on the other.

The two strands are held together by hydrogen bonds (electrostatic attraction). Two hydrogen bonds hold adenine to thymine. Three bonds attach cytosine to guanine as indicated in the diagram above.

During the process of cell division, the DNA becomes tightly coiled, forming structures called chromosomes. The diagram below is a portion of a double-stranded chromosome showing the centromere and a portion of the base sequence. The diagram does not show the extensive looping and coiling and the proteins associated with coiling. Notice that the base sequence in the two chromatids is identical.

How is Information Stored?

The diagram below shows that one strand of the DNA double-helix serves as a template for the construction of mRNA. The sequence of nucleotides in this DNA strand is complimentary (opposite) the sequence in mRNA. The diagram also shows that the sequence of nucleotides in mRNA determines the amino acids in the protein. For example GUG in mRNA (or CAC in DNA) codes for valine (see below).

The strand of DNA that contains the genetic code is called the anti-sense strand. It is often referred to as the template strand. The other strand (the sense strand) is not used. Notice that the sense strand has the same base sequence as mRNA except that mRNA has U instead of T.

The codes in DNA are copied to produce mRNA. Each three-letter code in mRNA (called a codon) codes for one amino acid. The sequence of amino acids in proteins is therefore most directly determined by the sequence of codons in mRNA, which in turn, are determined by the sequence of bases in DNA.

There are four letters in the genetic alphabet (A, T, G, and C) and each codon contains three letters. It is therefore possible to have 64 different codons. Because there are only 20 different amino acids and 64 possible codons, some amino acids have several different codons.

Terminators are codes that indicate the end of a genetic message (gene).

An initiator codon (usually AUG) indicates where the genetic information begins.

DNA replication

DNA helicase unwinds the DNA molecule by breaking hydrogen bonds. 

The area in a DNA molecule where unwinding is occurring is called a replication fork. In the diagram, it looks like an upside-down Y.

RNA primase adds an RNA primer.

The direction of synthesis is from 5 to 3.

The red color represents RNA.

The enzyme that will synthesize new DNA is called DNA polymerase. It cannot initiate a new strand, it can only elongate a strand that is already present. Synthesis of new DNA therefore cannot begin until a short strand of nucleotides is added. This short strand is called a primer. Primase creates an RNA primer and then DNA polymerase lengthens this strand by adding DNA nucleotides. The RNA primer will later be removed and replaced by DNA.

DNA polymerase III lengthens the strand that is being synthesized by adding nucleotides that are complimentary to those on the template strand (A paired with T and G paired with C).

The arrows indicate the direction of synthesis: 5' to 3'.

It proofreads the new strand as it synthesizes it. Incorrectly paired bases are removed and the correct one is inserted (discussed below).

The DNA polymerase molecule on the right cannot proceed further.

This diagram shows that helicase continues to unwind the DNA.

DNA polymerase on the right side is able to continue lengthening the strand continuously.

DNA polymerase cannot synthesize DNA on the right side of the diagram because it needs an RNA primer. Recall that DNA polymerase can lengthen a strand but it cannot initiate synthesis. The RNA primer is synthesized by primase, indicated by the green structure in the diagram above.

DNA polymerase can now attach and add nucleotides.

 

After a primer is synthesized (red), DNA polymerase can lengthen the strand.

The next two diagrams show that another molecule of DNA polymerase, called DNA polymerase I, removes the RNA primer and replaces it with DNA.

Notice that there is a gap between the two DNA strands on the right.

Because the direction of synthesis is from 5' to 3', the strand on the left in the diagram is synthesized continuously but the strand on the right is synthesized in fragments. The strand that is synthesized continuously is called the leading strand. The strand that is synthesized in fragments is called the lagging strand.

The fragments are called Okazaki fragments.

DNA ligase catalyzes the formation of covalent bonds between the Okazaki fragments.

 

Link

The link below may be a helpful summary.

http://www.johnkyrk.com/DNAreplication.html

Replication Forks

DNA synthesis occurs at numerous different locations on the same DNA molecule (hundreds in a human chromosome).

These form bubbles of replication with a replication fork at the growing edge.

The replication rate of eucaryotic DNA is 500 to 5000 base pairs per minute.

A human cell typically requires a few hours to duplicate the 6 billion base pairs.

Repair Enzymes

Changes in the DNA code are called mutationsRepair enzymes repair most of the errors that occur in DNA. 

Errors During DNA Replication

One in 100,000 bases are mismatched. 

Several enzymes including DNA polymerase proofread and remove mismatched bases. Mismatching causes replication to pause while the mismatch is removed and replaced with the correct nucleotide. 

After proofreading, the error rate is 1 in 1 billion base pairs.

Other Mutations

A number of environmental agents such as radiation (UV, X-rays, radioactive elements) and chemicals (pesticides, cigarette smoke) can cause mutations (changes) in DNA. 

A number of enzymes monitor the DNA and repair these changes. For example, excision repair occurs when a mutated segment of DNA is removed and replaced with a new segment.

A common type of mutation caused by ultraviolet radiation occurs when two thymines become bonded to each other, forming a kink in the DNA molecule. It is important that this be fixed because during replication, incorrect nucleotides could be added to the newly synthesized strand. To repair this mutation, the segment is excised and new DNA is synthesized.

Xeroderma pigmentosum is a genetic disease in which some repair enzymes do not function.

Organization of DNA

Chromosome Structure

Chromosomes are structures composed of condensed DNA and associated proteins. When DNA condenses, the molecule becomes wrapped around proteins called histones. The histones are then arranged in a coiled pattern to produce a larger fiber. This larger fiber is further compacted by looping to produce looped domains. The looped domains are coiled and compacted to produce chromosomes.

Heterochromatin and Euchromatin

Chromatin is DNA and its associated protein. Heterochromatin is DNA that is coiled and condensed. In this state, it is not transcribed. Euchromatin is less condensed and is actively transcribed.

During interphase, looped domains may be attached to protein supporting structures on the inside of the nuclear membrane. Some of the DNA is coiled and compacted but other parts are not.

Unknown Function

Approximately 1.5% of human DNA codes for protein. The function of the remaining DNA is not known but perhaps much of it has no function.