DNA Structure and Replication

• DNA encodes genetic information for RNA and proteins
• Double helix
• Sugar-phosphate backbone and nucleotide bases
• Complementary strands are anti-parallel
• Runs from 5' to 3'
• Nucleotide bases are connected by hydrogen bonds
• Adenine (A) pairs with Thymine (T)
• Guanine (G) pairs with Cytosine (C)
• Purines (Adenine and Guanine) hydrogen bond with pyrimidines
• Pyrimidines are Thymine and Cytosine
• In RNA, Uracil (U) replaces Thymine (T)
• Guanine and Cytosine forms three hydrogen bonds; Adenine and Thymine forms two
• The bonds between Guanine and Cytosine is harder to break and require more energy

Mnemonic for Nucleotides

• "CUT the Py" to remember that Cytosine, Uracil, and Thymine are pyrimidines
• "Pure As Gold" to remember that Purines are Adenine and Guanine

Phosphodiester Bonds

• Phosphodiester bonds join the 3' -OH group of one nucleoside to the 5' phosphate group of an incoming nucleoside

Replication Theories

• Conservative: Entire DNA molecule is copied into a second, completely new DNA molecule
• Semi-conservative: DNA strands separate, each acting as a template to copy a new strand
• Dispersive: DNA is cleaved into small fragments, fragments are copied and then re-attached

Meselson-Stahl Experiment.

• Designed by Matthew Meselson and Franklin Stahl in Escherichia Coli (E. coli) to determine how DNA replication occurs
• E. coli was grown in "heavy" nitrogen (15N) for all the bacterial DNA to become labelled with 15N
• DNA was extracted and centrifuged which resulted in all DNA becoming heavy
• Cells are transfered to "light" nitrogen (14N) for one replication cycle
• After DNA extraction and centrifuge all DNA= 50% "heavy"/ 50% "light" -This excludes the conservative theory
• DNA replicates in "light" nitrogen (14N) one more time ○ When DNA centrifuged, either 50% "heavy"/ 50% "light" OR all "light"

DNA Replication in Cells

• It is semi-conservative
• The origin of replication in prokaryotes has a special name called Ori

DNA synthesis steps

• Helicase unwinds DNA leaving antiparallel strands and a replication fork
• Single stranded binding proteins (ssb) keep strands apart
• Topoisomerase is responsible for "relaxing" the supercoiling
• DNA Polymerase III grabs new nucleotides and matches them to create a new strand
• It can only bind to the parental DNA and start creating a new strand with an RNA primer, created by primase
• Reads DNA in the 3' to 5' direction and creates a new strand in the 5' to 3' direction
• DNA goes both ways (3' to 5' to 5' to 3')
  • Polymerase moves towards the replication fork (leading strand), and away from the replication fork (lagging strand)
• Sliding clamp tethers DNA polymerase to the strand
• Replication continues until the adjacent replication bubble is met.
• Leading strand: DNA polymerase III adds nucleotides to the newly forming strand as helicase unwinds the DNA
• Lagging strand: parent DNA requires multiple primers and polymerases, creating Okazaki Fragments.
• DNA polymerase I removes the primer and replaces deoxyribonucleotides
• DNA Ligase moves along the lagging strand and ties the Okazaki fragments

Prokaryotes vs Eukaryotes

Telomerase

• RNA primers cannot be added to the 3' end of the DNA (lagging strand)
• Telomerase extends the 3' end of the lagging strand and a DNA polymerase III replicate the 3' end
• Prokaryotic cells don't have telomerase and have circular chromosomes

Mutations

• Occur when there is a change in the normal DNA sequence
• Point mutation means that only one base pair is mutated
• Errors in DNA replication
• Environmental exposure to damaging agents like UV light, radiation, etc.
• Harmful chemicals/ toxins and byproducts of normal cell metabolism

Point mutations

• Nonsense: premature stop codon

• Missense: amino acid change

• Silent: no change in amino acid sequence

• Beneficial: improves the fitness of an organism

• Benign: doesn't change the overall fitness of an organism

• Harmful: negatively impacts the fitness of an organism

• Base substitution is when a nucleotide is replaced by another

• Point mutations are base substitutions

• Transitions are purine for purine, pyrimidine for pyrimidine

• Transversion is purine for pyrimidine and vice-versa

• Frameshift mutations cause changes in the reading frame

• Result from mutations such as insertions/deletions of DNA

DNA Slippage

• DNA polymerase falls off the template strand and when it comes back together
• • The newly synthesized strand "slips" backward resulting in a 1 bp insertion.
• • The template strand "slips" forward resulting in a 1 bp deletion

Tautomeric Shifts

• DNA nucleotides have inherent instability and exists in equilibrium between two forms
• Thymine & Guanine = keto vs. enol
• Adenine & Cytosine = Amino vs. imino
• Equilibrium lies heavy towards usual base-pairing: C-G & A-T
• In alternate form, base pairing changes to T-G & C-A
• 5-bromouracil are in an equal equilibrium between two forms
• Source of single nucleotide polymophisms

Copy Number Variations

• Different number of copies of genes/gene regions
• Unequal cross-over during homologous recombination in prophase I of meiosis
• Non-homologous end joining causes loss of large chromosome regions
• Mobile elements

Transposons

• Regions of the genome are trapped viruses
  • Retain the ability to move within the genome, but cannot leave the cell
  • Do not increase copy number variations if they just "jump"
  • Increase copy number variations if they move through a copy & paste mechanism

Alu Elements

• in the human genome are called Alu elements
• LINES = Long interspersed elements
• SINEs = Short interspersed elements
• Represent about 11% of the human genome
• Move through a retrotransposon copy & paste mechanism
• Paste causes insertional mutagenesis and disrupt genes
• Causes changes in gene expression or regulation, and recombination events to occur where they shouldn't
• Diseases attributed to Alu elements: Hemophilia, leukaemia, muscular dystrophy

Repair of DNA Mutations

• Point mutations (base-pair substitutions)
• Insertion and deletion mutations (addition or removal of base pairs)

DNA Polymerase Exonuclease Activity

• Polymerase III can proofread, prevent mutations, Synthesizes 5' -> 3' and Corrects 3' -> 5'.
• Exonuclease activity breaks phosphodiester bonds to correct mistakes
• Bases are Improper placements by checking geometry to polymerize copy strand

DNA Mismatch Repair

• If DNA Polymerase does not correct mismatched bases during DNA polymerization, other mechanisms are in place
• Specific DNA repair enzymes (endonucleases) recognize incorrect geometry
• and repair the DNA by excising the incorrect nucleotide
  • The gap is filled in later by DNA polymerase
  • Nick is sealed by DNA ligase
• Bacteria's parent strand usually has methylated adenines

Nucleotide Excision Repair

• Mechanism repairs damaged DNA rather than incorrectly paired ones
  • Environmental mutagens damages like ultraviolet (UV) radiation, create covalent bonding of two pyrimidines within DNA
• Enzymes (endonucleases) detect the improper geometry of the DNA
  • Remove the damaged nucleotides from the DNA
  • DNA polymerase adds new nucleotides
  • DNA ligase bonds them

Repair of DNA Strand Breaks

• Double-stranded breaks in DNA
• Incorrect joining of the strands reorganizes of the genes affecting chromosome structure.
• Two main systems for repairing double stranded DNA breaks: non-homologous end joining and homologous recombination
• Non-homologous End Joining (NHEJ) joins two non-homologous ends of DNA together

Non-homologous End Joining Problems

• Base pairs from either ends of the DNA fragments are lost
• Genes can end up joined forming chimeric genes
• this can change cell function or cause cancer

Homologous Recombination

• Damaged DNA sequence is copied from an undamaged or highly similar copy
• Two strands exchange to repair the break through recombination

Polymerase Chain Reaction

• In vitro method of replicating/amplifying DNA that uses the same concepts of DNA replication to amplify a target DNA
• Components in tube: thermostable DNA polymerase (Taq polymerase), deoxyribonucleotides, DNA template, and primers
• A thermocycler is used
• 3 steps involves: Denaturation, Annealing, Elongation

Distinctions from DNA replication in the cell

• Heat is used to unwind DNA strands instead of helicase
• Primers are artificially designed, made of DNA instead of RNA, and dictates the replication
• Only a small region is amplified
• Millions of copies of the target region are produced
• No lagging or leading strands are involved

Primes

• Are in order to only amplify the required region of the genome
• These primers must flank to the DNA that to desired area and be complementary to the strands.
• Have around 18-22 base pairs