DNA and the Molecular Basis of Heredity

The Search for the Basis of Heredity

• his group showed that genes are located on chromosomes
• two molecules became the top candidates for the molecule of heredity
  • protein, the most likely candidate, since there were 20 amino acids
  • DNA, the less likely candidate, since there are only 4 nucleotides
  • a third theory, DNA + protein, was also considered

• British Medical Officer, served in WWI
• worked on Streptococcus pneumoniae
• Two strains of bacteria - Rough (R) and Smooth (S)
  • Rough - bacteria had a rough appearance in culture, non-virulent (doesn't kill)
  • Smooth - bacteria had a  smooth appearance in culture, virulent (kills)
• Performed several experiments whose results can be summarized as follows:
  • Mouse + Live S >> Died. Autopsy revealed infestation of S strain bacteria
  • Mouse + Live R >> Lived a happy life
    • These two experiments illustrate the effects of normal R and S strains
  • Mouse + Heat-killed S >> Lived a happy life
  • Mouse + Heat-killed R >> Lived a happy life
    • These two experiments illustrate that dead bacteria by themselves are harmless
  • Mouse + Heat-killed S + Live R >> Died. Autopsy revealed infestation of S strain
  • Apparently something from the heat-killed S changed the live R to make them virulent - this was called transformation
    • Griffith didn't know what it was that transformed the R strain into the S strain, but he demonstrated that it could be done

• Performed the same experiment, only they used tissue cultures growing in petri dishes
  • Heat-killed S + Live R + protease >> Live R and S colonies
  • Heat-killed S + Live R + DNAse >> No growth
  • Technically, this does not prove DNA is the carrier of genetic information, but it provided strong circumstantial evidence
• Other experiments - purified each chemical class associated with transformation and subjected bacteria to each one individually
  • Only DNA produced transformation
• Their results were met with much resistance - even if DNA was accepted as the molecule of heredity, very little was know about it and no one could possibly imagine how such a uniform molecule could possibly contain all of the information necessary to make you and me!

• Worked with radiolabled T2 bacteriophages
  • Bacteriophage - composed only of protein coat and DNA - injected something inside host bacterial cell which contained its genetic information
  • Was the injected material DNA or protein?

• In the first experiment, illustrated on the left, the "hot" substance was the protein coat.
• In the second experiment, illustrated on the right, the "hot" substance was the DNA.
• After the bacteriophage had injected the genetic material into the cell, the solutions were centrifuged and each reaction flask was measured for radioactivity.
• In the first experiment, all of the radioactivity was in the fluid that the cells were originally suspended.
• In the second experiment, all of the radioactivity was in the bacteria.
• It was concluded that since the radioactivity in the second experiment was in the bacteria, the genetic information was contained in the DNA and not the protein.

• Prior to mitosis, eukaryotic cells exactly double their DNA
  • This DNA is distributed exactly between the two daughter cells
• Diploid cells have exactly twice as much DNA as the haploid gametes found in eukaryotes
• Chargaff's Rule
  • The DNA composition differs between organism to organism and species to species
  • In ALL organisms, there are equal numbers of A and T.  Likewise, there are equal numbers of C and G.
  • The A/G, A/C, T/G, T/C ratios, however, differ from person to person, species to species

There was a race to elucidate the 3-D structure of DNA

• Linus Pauling in California proposed a triple helix
• Maurice Wilkins and Rosalind Franklin in London were also hot on the trail
• However, the first scientists to properly describe the structure of DNA were two relatively unknown Americans, James Watson and Francis Crick
  • Read the actual paper here - note, there ain't much there, is there?
  • Read a Time Magazine blurb on the dynamic duo here
  • More info on the people involved in this study here

• DNA is composed of Four Nucleotides, Adenine (A), Guanosine (aka Guanine, G), Thymine (T) and Cytosine (C)

• Adenine and Guanine are  Purines - they are larger and have a double ring
• Thymine and Cytosine are Pyrmidines - they are smaller and  have a single ring
• The nucleotides have three components, a pentose sugar, an amino base (the A, T, C, G), and a phosphate group
  • The deoxyribose sugar of the DNA backbone has 5 carbons and 3 oxygens.
  • The carbon atoms are numbered 1', 2', 3', 4', and 5' to distinguish from the numbering of the atoms of the purine and pyrmidine rings.
  • The hydroxyl groups on the 5'- and 3'- carbons link to the phosphate groups to form the DNA backbone.
  • Deoxyribose lacks an hydroxyl group at the 2'-position when compared to ribose, the sugar component of RNA.

Structure of Deoxyribose

• The Nucleotides are arranged in an anti-parallel double helix with Adenine pairing with Thymine and Guanine pairing with Cytosine via hydrogen bonds
  • A purine always pairs with a pyrmidine (A-T has two H-bonds, G-C has three H-bonds)
  • One strand runs 5' to 3' while the other runs 3' to 5'

DNA Replication - is DNA replication conservative, semiconservative, or dispersive?

Conservative - old strand acts as a template One daughter strand is the original template while the other strand is composed entirely out of new nucleotides  Dispersive Model Each strand of both daughter molecules contains a mixture of old and newly synthesized DNA parts Semiconservative - old strand splits apart and acts as a template  Both daughter strands are composed of one of the old strands and one comprised out of new nucleotides Isotopes were used to prove that DNA replication was semiconservative  How was this done?

The Meselson - Stahl Experiment

• E. coli bacteria were cultured for several generations in heavy nitrogen (¹⁵N - normal nitrogen is ¹⁴N)
  • The bacteria incorporated ¹⁵N into their nucleotides and thus, their DNA
• Meselson and Stahl then transferred the bacteria to a medium containing ¹⁴N,
  • Thus, any DNA that the bacteria synthesized would be lighter than the "old" DNA made with the ¹⁵N medium
• The DNA was extracted from the cells and centrifuged in a cesium chloride density gradient for 20 hours at 40,000rpm.
  • The DNA migrated to a point that was equivalent to their density.
  • Results from the parental generation contained only a single high density band - all DNA molecules contained the "heavy" nitrogen.
  • DNA taken from the two generations after the switch contained an intermediate-density band - DNA contained a "heavy" DNA strand from the parent and a complementary "light" DNA strand.
  • Density results from generation 3, displayed two bands. They included an intermediate density band, composed of one parental "heavy" strand and a new light band, composed of only DNA strands with "light" nitrogen.
  • Mixing the first and third generations showed all three types of DNA molecules - heavy, light and intermediate.
  • For a diagram of the experimental procedure, click here
  • Self test: what were the predicted results of the experiment for conservative, semiconservative, and dispersive
    • Answer?  Click here

DNA Replication

The Enzymes of DNA Replication

• Topoisomerase is responsible for eliminating supercoiling in DNA.  The tension holding the helix in its coiled and supercoiled structure can be broken by nicking a single strand of DNA. Try this with string. Twist two strings together, holding both the top and the bottom. If you cut only one of the two strings, the tension of the twisting is released and the strings untwist.
• Helicase accomplishes unwinding of the original double strand, once supercoiling has been eliminated by the topoisomerase. The two strands very much want to bind together because of their hydrogen bonding affinity for each other, so the helicase activity requires energy (in the form of ATP ) to break the strands apart.
• DNA polymerase proceeds along a single-stranded molecule of DNA, recruiting free dNTP's (deoxy-nucleotide-triphosphates) to hydrogen bond with their appropriate complementary dNTP on the single strand (A with T and G with C), and to form a covalent phosphodiester bond with the previous nucleotide of the same strand. The energy stored in the triphosphate is used to covalently bind each new nucleotide to the growing second strand. There are different forms of DNA polymerase , but it is DNA polymerase III that is responsible for the processive synthesis of new DNA strands. DNA polymerase cannot start synthesizing de novo on a bare single strand. It needs a primer with a 3'OH group onto which it can attach a dNTP. DNA polymerase is actually an aggregate of several different protein subunits, so it is often called a holoenzyme. The holoenzyme also has proofreading activities, so that it can make sure that it inserted the right base, and nuclease (excision of nucleotides) activities so that it can cut away any mistakes it might have made.
• Primase is actually part of an aggregate of proteins called the primeosome. This enzyme attaches a small RNA primer to the single-stranded DNA to act as a substitute 3'OH for DNA polymerase to begin synthesizing from. This RNA primer is eventually removed by RNase H and the gap is filled in by DNA polymerase I.
• Ligase can catalyze the formation of a phosphodiester bond given an unattached but adjacent 3'OH and 5'phosphate. This can fill in the unattached gap left when the RNA primer is removed and filled in. The DNA polymerase can organize the bond on the 5' end of the primer, but ligase is needed to make the bond on the 3' end.
• Single-stranded binding proteins are important to maintain the stability of the replication fork. Single-stranded DNA is very labile, or unstable, so these proteins bind to it while it remains single stranded and keep it from being degraded.

A portion of the double helix is unwound by a helicase.  A molecule of DNA polymerase binds to one strand of the DNA and begins moving along it in the 3' to 5' direction, using it as a template for assembling a leading strand of nucleotides and reforming a double helix.  Because DNA synthesis can only occur 5' to 3', a second DNA polymerase molecule is used to bind to the other template strand as the double helix opens. This molecule must synthesize discontinuous segments of polynucleotides (called Okazaki fragments). Another enzyme, DNA ligase then stitches these together into the lagging strand

Speed of Replication
 

• Prokaryotes - The single molecule of DNA that is the E. coli genome contains 4.7 x 106 nucleotide pairs. DNA replication begins at a single, fixed location in this molecule, the replication origin, proceeds at about 1000 nucleotides per second, and thus is done in no more than 40 minutes. And thanks to the precision of the process (which includes a "proof-reading" function), the job is done with only about one incorrect nucleotide for every 109 nucleotides inserted. In other words, more often than not, the E. coli genome (4.7 x 106) is copied without error!

• Eukaryotes - The average human chromosome contains 150 x 106 nucleotide pairs which are copied at about 50 base pairs per second. The process would take a month (rather than the hour it actually does) but for the fact that there are many replication origins on the eukaryotic chromosome. Replication begins at some origins earlier in S phase than at others, but the process is completed for all by the end of S phase. As replication nears completion, "bubbles" of newly replicated DNA meet and fuse, finally forming two new molecules

Click here to see a shockwave graphic on DNA replication

Enzymes Proofread DNA During Its Replication and Repair Damage in Existing DNA

We cannot attribute the accurate of DNA replication solely to the specificity of base pairing

• Base pairing specificity has an error rate of one in 10,000 base pairs
• However, errors only occur in one in 1,000,000,000 base pairs (one in a billion)
• Mismatch repair - during DNA replication, DNA polymerase proofreads the DNA as soon as it is added to the strand searching for mismatches
  • When found, the polymerase removes the mismatched nucleotide, adds a correct nucleotide, and continues synthesizing DNA
• DNA is also constantly exposed to environmental hazards (UV, X-rays, carcinogens, mutagens, etc.)
  • Enzyme systems constantly moniter DNA looking for altered DNA
  • Example - UV radiation causes two adjacent Thymines to form a Thymine dimer
    • When found, nuclase enzymes remove the TT dimer and a few surrounding nucleotides
    • DNA polymerase fills in the gap
    • ligase seales the remaining nick
  • Over 100 DNA repair enzyme are known in E. coli alone!

For linear DNA (i.e. eukaryotic chromosomes) the fact that DNA polymerase can only add nucleotides to the 3' end of a preexisting polynucleotide presents a serious problem

• Prokaryotes - they have circular DNA so this is not a problem
• Eukaryotes have a special DNA sequences called a telomeres on the terminal regions of their chromosomes
  • Telomeres do not contain genes; instead, they contain repeating units of one short nucleotide sequence, usually TTAGGG
  • Telomeric DNA protects the organism's genes from being eroded through successive rounds of DNA replication
  • Telomeric DNA aids in preventing separate chromosomes from fusing
  • Telomeric DNA and associated proteins prevent the ends from activating the cell's systems for monitoring DNA damage
    • A DNA molecule detected as broken may trigger signal-transduction pathways leading to cell-cycle arrest or death
• Telomerase - a special enzyme that catalyzes the lengthening of telomere
  • Telomerase is special in having a piece of RNA associated with its protein
  • This RNA serves as a template so DNA Polymerase can extend the end of the telomere
  • Since the telomeric units are repeating, this can occur many times