Medic_Summaries (IMS)
Nucleic Acids
Evidence for DNA’s biological role: seminal experiments in 1920s-1950s
- Griffith: 1928
- Streptococcus pneumoniae is a cause of fatal pneumonia in humans
- There are two major strains of Streptococcus pneumoniae:
- Rough (harmless)
- Smooth (deadly)
- Inject with rough strain - SURVIVES (non-virulent)
- Inject with smooth strain - DEATH (virulent)
- Inject with heat-killed strain - SURVIVES
- Inject with rough and heat-killed smooth strain - DEATH
- Conclusion:
- Live smooth and rough strains were isolated from the dead mouse
- SEMI CONSERVATIVE REPLICATION
- Smooth strain ‘came back to life’
- Avery: 1944
- Recognised something in the heat-killed deadly strain could transform the rough
strain into the deadly strain
- DNA was that transforming agent
- Purified DNA, RNA, protein, lipid, and carbohydrates were present
- Only DNA from heat-killed virulent strain could induce virulence from non-virulent
- Hershey and Chase: 1952
- Bacteriophage are made up of protein and DNA
- Protein and DNA of bacteriophage were labelled with radioactive molecules
- Bacteriophage attach to bacteria and inject their DNA, which then replicates
- Sulphur is not found in DNA; phosphorus is not found in protein
- Sulphur labelled protein capsule (no sulphur in cells, found in supernatant)
- Phosphorus labelled DNA core (phosphorus in cells)
- Therefore DNA, not protein, must contain genetic information
Chargaff: structure of DNA (1952)
- Base composition of DNA studied in different species (variation between species)
- Concluded that the ratio of G:C and A:T was always ca. 1:1
- This discovery aided Watson and Crick with their DNA model (BASE PAIRS)
Wilkins and Franklin: X-ray crystallography data
- Sugar-phosphate backbone (adjacent deoxyribose linked by phosphodiester bonds)
- Base pairs are complementary
- AT = two H-bonds; GC = three H-bonds
- DNA single unit is called a NUCLEOTIDE (base + sugar (deoxyribose) + phosphate)
- DNA is directional (5’-3’)
- DNA is tightly coiled and packaged into chromosomes (along with histone = CHROMATIN)
- Active genes are loosely coiled (takes less energy to uncoil, so easy access)
- Interphase (ca. 90%): chromatin appears disordered (non-condensed)
- Chromatin condenses into ‘classical’ chromosome during cell cycle
- Helped Watson and Crick with their antiparallel strands of DNA in the double-helix
Meselson and Stahl: semiconservative replication
- E coli was cultured in an N15 medium (DNA synthesised therefore contained N15)
- E coli was then shifted to an N14 environment
- DNA was isolated at various time intervals, corresponding to replication cycles
- After 1 cycle: 100% intermediate density DNA (ruled out conservative replication model)
- After 2 cycles: two bands were observed, one of intermediate and one of light density
- After 3+ cycles: light density band gradually increases as N14 only E coli replicates
1 Adapted from Lectures at the University of Leeds Medical School
, Medic_Summaries (IMS)
DNA replication:
- Each double-strand contains an original strand and a newly synthesised strand
- DNA synthesis is initiated at the origin of replication (utilising the replication fork)
- At the replication fork:
- HELICASES unwinds the double strand
- Single-stranded binding protein stabilise denatured DNA (stop it from reforming
the original DNA double helix)
- DNA primase synthesises a short RNA primer (allows replication to begin)
- DNA polymerase carries out elongation of a new strand of DNA by pairing bases
to the template strand
- dNTPs (deoxynucleoside triphosphates) needed as building blocks for new DNA strands
- The addition of a new nucleotide requires a free 3’-OH (RNA PRIMER)
- DNA polymerase’s active site is the site for addition of new nucleotides
- DNA is synthesised in one direction (5’-3’) from short fragments (OKAZAKI FRAGMENTS)
- DNA ligase seals the gaps between fragments
- DNA polymerase catalyses the formation of new phosphodiester bonds
- DNA polymerase I replaces RNA primers with DNA (DNA ligase seals the gaps)
- DNA polymerase III is involved in DNA replication in prokaryotes
Summary: DNA synthesis is initiated at the origin of replication and utilises the replication fork. At
the replication fork, helicases unwind double-stranded DNA to allow replication to occur. Single-
stranded binding proteins stabilise denatured DNA. DNA primase synthesises short RNA primers
to allow replication to commence and DNA polymerase carries out elongation of new strands of
DNA, which form by complementary base pairing to a template strand.
2 Adapted from Lectures at the University of Leeds Medical School
Nucleic Acids
Evidence for DNA’s biological role: seminal experiments in 1920s-1950s
- Griffith: 1928
- Streptococcus pneumoniae is a cause of fatal pneumonia in humans
- There are two major strains of Streptococcus pneumoniae:
- Rough (harmless)
- Smooth (deadly)
- Inject with rough strain - SURVIVES (non-virulent)
- Inject with smooth strain - DEATH (virulent)
- Inject with heat-killed strain - SURVIVES
- Inject with rough and heat-killed smooth strain - DEATH
- Conclusion:
- Live smooth and rough strains were isolated from the dead mouse
- SEMI CONSERVATIVE REPLICATION
- Smooth strain ‘came back to life’
- Avery: 1944
- Recognised something in the heat-killed deadly strain could transform the rough
strain into the deadly strain
- DNA was that transforming agent
- Purified DNA, RNA, protein, lipid, and carbohydrates were present
- Only DNA from heat-killed virulent strain could induce virulence from non-virulent
- Hershey and Chase: 1952
- Bacteriophage are made up of protein and DNA
- Protein and DNA of bacteriophage were labelled with radioactive molecules
- Bacteriophage attach to bacteria and inject their DNA, which then replicates
- Sulphur is not found in DNA; phosphorus is not found in protein
- Sulphur labelled protein capsule (no sulphur in cells, found in supernatant)
- Phosphorus labelled DNA core (phosphorus in cells)
- Therefore DNA, not protein, must contain genetic information
Chargaff: structure of DNA (1952)
- Base composition of DNA studied in different species (variation between species)
- Concluded that the ratio of G:C and A:T was always ca. 1:1
- This discovery aided Watson and Crick with their DNA model (BASE PAIRS)
Wilkins and Franklin: X-ray crystallography data
- Sugar-phosphate backbone (adjacent deoxyribose linked by phosphodiester bonds)
- Base pairs are complementary
- AT = two H-bonds; GC = three H-bonds
- DNA single unit is called a NUCLEOTIDE (base + sugar (deoxyribose) + phosphate)
- DNA is directional (5’-3’)
- DNA is tightly coiled and packaged into chromosomes (along with histone = CHROMATIN)
- Active genes are loosely coiled (takes less energy to uncoil, so easy access)
- Interphase (ca. 90%): chromatin appears disordered (non-condensed)
- Chromatin condenses into ‘classical’ chromosome during cell cycle
- Helped Watson and Crick with their antiparallel strands of DNA in the double-helix
Meselson and Stahl: semiconservative replication
- E coli was cultured in an N15 medium (DNA synthesised therefore contained N15)
- E coli was then shifted to an N14 environment
- DNA was isolated at various time intervals, corresponding to replication cycles
- After 1 cycle: 100% intermediate density DNA (ruled out conservative replication model)
- After 2 cycles: two bands were observed, one of intermediate and one of light density
- After 3+ cycles: light density band gradually increases as N14 only E coli replicates
1 Adapted from Lectures at the University of Leeds Medical School
, Medic_Summaries (IMS)
DNA replication:
- Each double-strand contains an original strand and a newly synthesised strand
- DNA synthesis is initiated at the origin of replication (utilising the replication fork)
- At the replication fork:
- HELICASES unwinds the double strand
- Single-stranded binding protein stabilise denatured DNA (stop it from reforming
the original DNA double helix)
- DNA primase synthesises a short RNA primer (allows replication to begin)
- DNA polymerase carries out elongation of a new strand of DNA by pairing bases
to the template strand
- dNTPs (deoxynucleoside triphosphates) needed as building blocks for new DNA strands
- The addition of a new nucleotide requires a free 3’-OH (RNA PRIMER)
- DNA polymerase’s active site is the site for addition of new nucleotides
- DNA is synthesised in one direction (5’-3’) from short fragments (OKAZAKI FRAGMENTS)
- DNA ligase seals the gaps between fragments
- DNA polymerase catalyses the formation of new phosphodiester bonds
- DNA polymerase I replaces RNA primers with DNA (DNA ligase seals the gaps)
- DNA polymerase III is involved in DNA replication in prokaryotes
Summary: DNA synthesis is initiated at the origin of replication and utilises the replication fork. At
the replication fork, helicases unwind double-stranded DNA to allow replication to occur. Single-
stranded binding proteins stabilise denatured DNA. DNA primase synthesises short RNA primers
to allow replication to commence and DNA polymerase carries out elongation of new strands of
DNA, which form by complementary base pairing to a template strand.
2 Adapted from Lectures at the University of Leeds Medical School
