History – pages 172-173 in textbook
• DNA
– Comprised of genes
– In a prokaryote, the DNA consisted of a naked circular loop of DNA (no proteins integrated into
“chromosome”)
– In a eukaryote, in a non-dividing cell nucleus contents exists as chromatin
• Protein/DNA complex – proteins function in organizing, packaging, and scaffolding of DNA.
• Each unduplicated (each containing unreplicated DNA) chromosome contains one DNA molecule. In humans, there are 23 pairs of chromosomes (=46 DNA molecules). After replication of DNA in the nucleus, that leads to the formation of duplicated chromosomes, the number of DNA molecules in the nucleus doubles.
• Among eucaryotic species, the numbers, size, and morphology of chromosomes will vary.
– Chromosomes form during cell division
• Duplicate to yield a full set in daughter cell
DNA is Genetic Material
From Chapter 2 - Panel 2-6
• Nucleic acids are polymers
– Monomers are called nucleotides
– Nucleotides = base + sugar + phosphate
• Base = purine or pyrimidine
– Purines = adenine, guanine
– Pyrimidines = thymine, cytosine, uracil
• Sugar = deoxyribose or ribose
• Phosphate, a single phosphate in DNA
– Sugar of nt 1 is linked to the phosphate of nt 2 by a phosphodiester bond
DNA is a Double Helix – Figures 5-2. 5-6, 5-7 and 5-8 in the textbook.
• Nucleotides
– A, G, T, C
• Sugar and phosphate form the backbone
• Bases lie between the backbone
• Held together by H-bonds between the bases
– A-T – 2 H bonds
– G-C – 3 H bonds
H - Bonds – Figure 5-6
• Base-pairing rules
– A  T only (A  U if DNA-RNA hybrid)
– G  C only
• DNA strand has directionality – one end is different from the other end
• 2 strands are anti-parallel, run in opposite directions
– Complementarity results
– Important to replication

Nucleotides as Language
We must start to think of the nucleotides – A, G, C and T as part of a special language – the language of genes that we will see translated to the language of amino acids in proteins
Genes as Information Transfer – Figure 5-9 and 5-10
• A gene is the sequence of nucleotides within a portion of DNA that codes for a peptide or a functional
RNA
• Sum of all genes = genome
DNA Replication – Figure 6-5
• Semiconservative
• Daughter DNA is a double helix with 1 parent strand and 1 new strand
• Found that 1 strand serves as the template for new strand
DNA Template – Figures 6-3 and 6-10
• Each strand of the parent DNA is used as a template to make the new daughter strand
• DNA replication makes 2 new complete double helices each with 1 old and 1 new strand
Replication Origin – Figure 6-5
• Site where replication begins
– 1 in E. coli
– 1,000s in human
• Strands are separated to allow replication machinery contact with the DNA
– Many A-T base pairs because easier to break 2 H-bonds that 3 H-bonds
• Note anti-parallel chains
Replication Fork – Figure 6-9 and 6-12
• Bidirectional movement of the DNA replication machinery
DNA Polymerase – Figure 6-13 and 6-14
• An enzyme that catalyzes the addition of a nucleotide to the growing DNA chain
• Nucleotide enters as a nucleotide tri-PO
4
• 3’–OH of sugar attacks first phosphate of tri-PO
4
bond on the 5’ C of the new nucleotide
– releasing pyrophosphate (PP i
) + energy
• Bidirectional synthesis of the DNA double helix
• Corrects mistaken base pairings
• Requires an established polymer (small RNA primer) before addition of more nucleotides
• Other proteins and enzymes necessary
How is DNA Synthesized?
• Original theory
– Begin adding nucleotides at origin
– Add subsequent bases following pairing rules
• Expect both strands to be synthesized simultaneously
• This is NOT how it is accomplished
Why DNA Isn’t Synthesized 3’  5’ – Figure 6-15

How is DNA Synthesized? – Figure 6-16 and 6-17
• Actually how DNA is synthesized
– Simple addition of nucleotides along one strand, as expected
• Called the leading strand
• DNA polymerase reads 3’  5’ along the leading strand from the RNA primer
• Synthesis proceeds 5’  3’ with respect to the new daughter strand
• Remember how the nucleotides are added!!!!! 5’  3’
• Actually how DNA is synthesized
– Other daughter strand is also synthesized 5’  3’ because that is only way that DNA can be assembled
– However the template is also being read 5’  3’
• Compensate for this by feeding the DNA strand through the polymerase, and primers and make many short segments that are later joined (ligated) together
– Called the lagging strand
Mistakes during Replication
• Base pairing rules must be maintained
– Mistake = genome mutation, may have consequence on daughter cells
• Only correct pairings fit in the polymerase active site
• If wrong nucleotide is included
– Polymerase uses its proofreading ability to cleave the phosphodiester bond of improper nucleotide
• Activity 3’  5’
– And then adds correct nucleotide and proceeds down the chain again in the 5’  3’ direction
Proofreading – Figure 6-13
Starting Synthesis – Figure 6-16 and 6-17
• DNA polymerase can only ADD nucleotides to a growing polymer
• Another enzyme, primase, synthesizes a short RNA chain called a primer
– DNA/RNA hybrid for this short stretch
– Base pairing rules followed (BUT A-U)
– Later removed, replaced by DNA and the backbone is sealed (ligated)
• Simple addition of primer along leading strand
– RNA primer synthesized 5’  3’, then polymerization with DNA
• Many primers are needed along the lagging strand
– 1 primer per small fragment of new DNA made along the lagging strand
– Called Okazaki fragments
Removal of Primers
• Other enzymes needed to excise (remove) the primers
– Nuclease – removes the RNA primer nucleotide by nucleotide
– Repair polymerase – replaces RNA with DNA
– DNA ligase – seals the sugar-phosphate backbone by creating phosphodiester bond
• Requires Mg 2+ and ATP

Other Necessary Proteins
• Helicase opens double helix and helps it uncoil
• Single-strand binding proteins (SSBP) keep strands separated – large amount of this protein required
• Sliding clamp
– Subunit of polymerase
– Helps polymerase slide along strand
• All are coordinated with one another to produce the growing DNA strand (protein machine)
Polymerase & Proteins Coordinated – Figure 6-17
• One polymerase complex apparently synthesizes leading/lagging strands simultaneously
• Even more complicated in eukaryotes
Completion of DNA sythesis at the ends of a eukaryotic chromosome – Fig. 6-18 (Telomeres and telomerase)
DNA Repair
• For the rare mutations occurring during replication that isn’t caught by DNA polymerase proofreading
• For mutations occurring with daily assault
• If no repair
– In germ (sex) cells  inherited diseases
– In somatic (regular) cells  cancer
Error rates – Table 6-1
Effect of Mutation – Figure 6-21
Uncorrected Replication Errors – Figures 6-21 and 6-22
• Mismatch repair
– Enzyme complex recognizes mistake and excises newly-synthesized strand and fills in the correct pairing
• Eukaryotes “label” the daughter strand with nicks to recognize the new strand
– Separates new from old
Depurination or Deamination – Figures 6-23 and 6-24
• Depurination – removal of a purine base from the DNA strand
• Deamination is the removal of an amine group on Cytosine to yield Uracil
– Could lead to the insertion of Adenine rather than Guanosine on next round
Thymine Dimers – Figure 6-24
• Caused by exposure to UV light
• 2 adjacent thymine residues become covalently linked
Repair Mechanisms – Figure 6-26
• Different enzymes recognize, excise different mistakes
• DNA polymerase synthesizes proper strand
• DNA ligase joins new fragment with the polymer