Genetics 3 - DNA metabolism

• Semi conservative replication is when two strands of DNA unwind from each other, and each strand acts as a template for the synthesis of a new, complementary strand. This results in two DNA molecules with one original strand and one new strand.
• Advantages of semi conservative replication:

  • proof reading
  • repair
• In Eukaryotes when new strand is synthesised it has 'nicks' which are single strand breaks
• In prokaryotes after replication the old strand is methylated at GATC sites
• Eukaryotic initiation:

  Contains linear chromosomes and multiple origins of replication

  Steps of replication:

  During G1 phase:

  1. Origin recognition complex (ORC) binds to the replication origin
  2. Helicase loading proteins, Cdc6 and Cdt1 , attach to ORC
  3. Mcm hexameric helicase is loaded to form prereplicative complex with proteins and ORC

  During S phase:

  4. Cyclin-dependant kinases (Cdks) phosphorylate replication proteins within the prereplicative complex causing it to disassembles

  5. Helicases unwind DNA and DNA polymerase is recruited for DNA replication
• Prokaryotic intiation

  • single origin of replication
  • hemimethylated origins are resistant to initiation
• Prokaryotic intiation steps:

  1. Intiator proteins bind to DnaA
  2. Helicase binds
  3. loading of Helicase onto DNA
  4. DNA unwinds and melts and DNA primase is recruited
  5. RNA primer is synthesised
  6. DNA polymerase is recruited for replication
  7. Two replication forks move in opposite directions
• DNA primase

  • Found on both leading and lagging strands
  • Adds RNA nucleotides
  • Allows DNA synthesis via DNA polymerases
• Prokaryotic DNA polymerases

  There are 5 types of polymerases:

  • I - DNA replication - initiation, proofreading, repair, RNA primer removal
  • II - DNA Repair
  • III - Main DNA polymerase
  • IV & V - Repair under specific conditions
• Eukaryotic DNA polymerases

  There are 5 types of polymerases:

  • α - DNA replication - Initiation
  • β - DNA Repair
  • γ - Mitochondrial DNA replication and repair
  • δ - Main DNA polymerase - lagging strand
  • ε - Main DNA replication - leading strand and repair
• DNA extension:

  • primed with RNA
  • added to 3' -OH
  • 5' to 3' direction of chain growth
• DNA polymerase -reads template bases -> changes shape -> detects correct nucleotide.
• Replication fork

  Okazaki fragment elongation

  • lagging strand
  • from RNA primer to previous primer
  • RNAse H removes previous RNA Primer
  • DNA Ligase attaches fragments together
  • In prokaryotes fragment length is 1,000 - 2,000 bp
  • In eukaryotes fragment length is 100 - 200 bp
  • Eukaryotic replication forks x10 slower
• DNA helicase - separates DNA strands
• Single Stranded DNA binding proteins (SSB) - prevent DNA hairpins
• Sliding clamp- holds DNA polymerase on the DNA

  • a new sliding clamp will bind at the RNA primer and recruit the polymerase
• Enzymes required for DNA replication

  A) Topoisomerase II

  B) DNA ligase

  C) DNA poll I

  D) helicase

  E) DnaA

  F) SSB

  G) ORC

  H) primase

  I) Rnase H

  
• Proof reading:

  • Correction of errors
  • Only 1 mistake every 10^9 nucleotides copied

  Checks:

  1. Original DNA polymerase - requires base pair check, conformational change leads to catalysis
  2. 3′-5′ Exonucleolytic proofreading - removes last wrong base preformed originally by DNA polymerase
  3. Strand-directed Mismatch repair

  • error is recognised
  • ATP hydrolysis is used to change shape of mobile clamp
  • MutLa is recruited
  • PCNA-activated MutL nicking of daughter strand
  • error removed and strand is resynthesised
  • Ligation occurs
• Eukaryotic telomeres

  • tandem repeats
  • Human repeat : GGGTTA
  • repeated 1,000 times

  Primed by telomerase on lagging strand

  • RNA/Protein hybrid
  • Binds parental lagging strand telomere

  extended end filled by DNA polymerase
• Telomere length is regulated by telomerase activity:

  Function of regulation:

  • tissue development
  • ageing
  • reduce mutations
• DNA mismatch repair (MMR)

  Damage: base pair mismatches

  Proteins:

  • Recognition - MSH2/MSH6
  • Strand breaks - PMS2/MLH1
  • Removal - EXO1, DNA polymerase δ or ε

  Repair : Large section replaced by DNA polymerase δ
• Base excision repair (BER)

  Damage: Base modification (deamination, oxidation , alkylation), bulky helix-distorting

  Proteins

  • Recognition - DNA glycosylases
  • Base removal - DNA glycosylases
  • Backbone removal - AP endonucleases

  Repair

  • Short patch BER (Single Nucleotide) Polymerase β and ligase
  • Long-patch BER (2-10 nucleotides) Polymerase β, flap endonucleases and ligase
• Nucleotide excision repair (NER)

  • Global genomic (GG-NER)
  • or transcription coupled (TC-NER) - stalled RNA Polymerase

  Damage: Damage induced by ultraviolet light (UV)e.g. Thymine dimers

  Proteins:

  • Recognition:
  • TC-NER – XPG/CSB
  • GG-NER – XPC/Rad23B, XPE/DDB2
  • Strand breaks - DNA Endonuclease (XGP & XPF/ERCC1)
  • Base removal - DNA Helicase

  Repair - DNA Polymerase β and ligase

  • Other strand as template
• Non-Homologous end joining:

  • double strand break recognised
  • Exonucleases cause break of strand and loss of ends
  • Ligase joins ends together
• Homologous recombination in newly replicated DNA:

  • Double strand break in one out of two sister chromatid
  • exonucleases cleave off ends
  • Ends are processed and cleaved chromatid forms base pairs with same strand on other sister chromatid and DNA polymerase catalyses synthesis of new strand filling in the gaps
  • resulting in accurate repair
• Werner syndrome

  • recessive mutations in WRN
  • Werner protein - maintenance and repair of DNA
  • shorter protein -> fails to enter nucleus
  • life expectancy : late 40s, cancer, atherosclerosis
• Bloom syndrome:

  • recessive mutation in BLM
  • RecQ Helicase- Unwinds DNA in repair & Prevent excess sister chromatid exchange
  • Loss of function→ Excess sister chromatid exchanges during chromosomal recombination →More chromosomal breakage
• Reverse Transcriptase

  • From retro viruses (e.g. HIV)
  • Uses RNA as template
  • Generates DNA strand copy

  Reverse transcriptase used to make cDNA

  • DNA copies of mRNA molecules
  • Quantify mRNA in tissues
  • Clone mRNA from a gene
  • Use Poly T Prime
• Reverse transcriptase action:

  1. Lyse cells and purify mRNA from tissue
  2. mRNA sequence is hybridised by Poly T primer
  3. Reverse transcriptase uses mRNA as template to generate cDNA
  4. Rnase H digests strand
  5. DNA polymerase joins strands together to form double stranded cDNA copy or original mRNA
• Polymerase chain reaction (PCR)

  • Region of interest in template DNA molecule is heated to 98 degrees to separate nucleotides
  • Temperature is reduced to between 48-72 degrees to allow annealing of primers to template strands
  • Temperature is then increased again to between 68 to 72 degrees to allow DNA polymerase to catalyse addition of nucleotides
  • primers face opposite direction but nucleotides are added from 3' end of primer
• Using specific primers during PCR allows for amplification specificity. PCR is repeated multiple times to produce many copies of the region of interest.
• Identifying - Nucleic acid amplification tests (NAATs)

  SARS-CoV-2 Detection

  • Sampling: Nasopharyngeal or oropharyngeal swabs
  • Test :Real-Time Polymerase chain reaction (RT-PCR)
  • RNA virus- uses reverse transcriptase (RNA → DNA compliment)

  SYBR dyes bind to double-stranded DNA

  • Fluorescence increases 20-100 fold
• Agarose gel electrophoresis

  • Separate particles of different sizes/charge
  • e.g. DNA fragments, RNAs, Proteins
• DNA Sequencing - Dideoxy method

  Sanger sequencing - by Frederick Sanger

  • Normal nucleotides (T G C A) and.....
  • Chain-terminating dideoxynucleotides
  • Only one primer
  • Fluorescent dyes (T C A G)
  • primer binds to single stranded DNA with unknown sequence
  • dideoxynucleotides bind to primer on sequence and form a new strand
  • Primer, sequence, DNA polymerase and dideoxynucleotides enter gel electrophoresis
  • DNA is separated, sequence of new strand is read and converted to sequence of template strand
• Primer design:

  • 20 to 40 nucleotides long (Often 21 for sequencing)
  • GC content of 40–60%
  • 3′ ends in two of G or C
  • No more than 1,000 bp for Sanger sequencing- First 40 bp sequenced will be junk
  • For PCR (Non Sequencing reactions)- Primer pairs should not have complementary 3 ́ end