the genetic code and DNA replication

the transforming principle

• The heritable material that transfers genetic traits between cells

• Transformation still occurred after destroying proteins and RNA

• Transformation stopped only when DNA was destroyed

discovery of DNA

• X ray crystallography unveiled the double helix structure 

Stable, double stranded, not very reactive 

Heritable, passed down from cell to cell 

Information transfer from DNA to protein 

the central dogma

• Gene -> protein, not protein to gene 

• DNA -> RNA (transcription) 

• RNA -> protein (translation) 

  • DNA (information repository of the cell, double stranded deoxyribonucleotide polymer, stable) 

  • RNA (information carrier, intermediate between DNA and protein, less stable (single stranded) 

  • Protein (amino acid polymers, enzymes, structural, encoded by DNA/RNA base pair sequence, codon = 3 base pairs = 1 amino acid) 

the genetic code

• 20 amino acids (64 combinations of codons - degenerate) 

• Mutations (alterations to the genome) 

  • Substitution 

  • Deletion 

  • Addition 

• Results of mutation in protein-coding DNA sequence 

  • No change in amino acid sequence of protein 

  • Change in amino acid sequence of protein that does not affect its function 

  • Change in amino acid sequence that affects its function 

DNA structure

• ATCG 

• Phosphate sugar backbone 

• 10 base pairs per turn 

• Right handed helix 

• Antiparallel strands 

• Bond between c and g is stronger (as more hydrogen bonds – 3 vs 2 for a and t) 

difference between DNA and RNA

• Ribose = OH group, deoxyribose = H group 

• Thus ribose is more reactive 

 

fundamental units of DNA and RNA

• Base 

• Adenine 

• Cytosine 

• Guanine 

• Thymine 

• Uracil 

• Base + sugar = nucleoside 

• Base + sugar + Pi = nucleotide 

• Base + ribose sugar = ribonucleic acid 

• Base + deoxyribose sugar = deoxyribonucleic acid 

nucleotides from bases (purine, pyrimidine)

• Adenine + sugar + Pi = adenosine 

• Cytosine + sugar + Pi = cytidine 

• Guanine + sugar + Pi = guanosine 

• Thymine + sugar + Pi = Thymidine 

• Uracil + sugar + Pi = uridine 

nucleotide triphosphate

• NTP = nucleotide triphosphate 

• DNTP = deoxynucleotide triphosphate 

nucleotide polymerisation

• Incoming dNTP has PPPi on the 5' carbon atom in the ribose group 

• Phosphodiester bond forms with 3' OH group on the end of the DNA chain 

• As a nucleotide is formed (phosphodiester bond made), a diphosphate is ejected 

• Catalytic energy comes from hydrolysis of PPi 

DNA replication

• Semi-conservative 

• Happens in 5' -> 3' 

• Leading strand 

  • Synthesised continuously from a replication fork 

  • Starts with a RNA primer 

• Lagging strand 

  • Synthesised discontinuously 

  • Okazaki fragments form (100-200 base pairs long) 

  • Each fragment starts with a RNA primer 

eukaryote DNA replication

• Have many origins as DNA is very long (unzipped in multiple places) 

stages of DNA replication

• Initiation 

• Elongation 

• Termination 

initiation

• Proteins bind to DNA and open the double helix 

• Prepare DNA for complementary base pairing 

• RNA primers bind first to start the process 

• DNA polymerase can only add a nucleotide to a pre-existing strand (thus RNA primer is made first) 

elongation

• Proteins join incoming nucleotides together into continuous new strands 

• Always polymerise 5' -> 3' so one strand is replicated discontinuously 

• Proof-reading takes place to ensure fidelity of sequence 

• RNA primer is replaced with DNA 

termination

• Proteins release the replication complex 

enzymes involved in DNA replication

• DNA helicase – breaks hydrogen bonds between base pairs at origins of replication 

• DNA primase – assembles and catalyses synthesis of short RNA primers on the DNA template 

• DNA polymerase – forms phosphodiester bonds between DNA and RNA, forming the sugar-phosphate backbone 

• DNA ligase – joins discontinuous strands (e.g. Okazaki fragments, RNA primers replaced by DNA) 

• DNA topoisomerase – break and reform phosphodiester bonds in the sugar phosphate backbone to relieve physical tension (supercoiling) that could damage the DNA 

sliding clamp and single strand binding proteins

• Single strand binding proteins (SSB) 

  • Prevent ssDNA from base-pairing with the other template strand 

• Sliding clamp 

  • Keeps DNA polymerase from falling off of the strand 

  • Assembled at the replication fork by a clamp-loader complex 

  • Ensures efficient replication 

DNA supercoiling during replication

• DNA topoisomerases make cuts in the DNA and put it back together to reduce tension 

 

proof reading in DNA replication

• When the wrong base pair is added, it causes a conformational change in shape, causing the daughter stand to flip to the endonuclease domain 

  • This is due to incorrect hydrogen binding between base pairs slowing the enzymatic process, allowing the nucleotide to diffuse away 

  • Mis-matched bases impair polymerase reaction 

• The exonuclease removes the mis-paired base, allowing the strand to flip back up to be paired