Genetik

• Antiparallel strands with opposite 5’-to-3’-direction that form a double helix

• Sugar-Phosphate backbone connected with phospodiester bonds

• Complementary bases always opposite to each other

• 3.4A base separation (1A=10^-10m)

• Full turn every 3.4A(~10.4 base pairs)

• Forms two grooves (sites where backbone of helix is seperated differently)

  • major groove: wide & deep (mostly proteins interactions)

  • minor groove: narrow and deep

• Nucleic acids: contain genetif info via nucleotides (=monomers of nucleic acids (building blocks))

• DNA (genetic blueprint) and RNA (transcription product): polynucleotides (short segments of nucleic acids (=oligonucleotide (short, single-stranded DNA/RNA))

• Gene: Functional unit of genetic info -> are part of genetic elements like large molecules and/or chromosomes

• Size: expressed in base pairs

  • 1000 base pairs = 1kbp /1 mio. base pairs = 1Mbp

    (e.g. E coli genome = 4.64 Mbp (human genome ~6 Gigabase pairs & about 2m long)

  • Supercoiling compacts DNA

• Pyrimidines to Purines

  • Thymine - Adenine

  • Cytosine - Guanine

• Nucleoside composed of:

  -> nitrogenous base (adenine, guanine, cytosine, thymine)

  -> five carbon sugar

• Nucleotide composed of:

  -> Nucleoside

  -> one or more phosphate groups

1. Overwinding of DNA

2. Both strands have to be copied at the same time but antiparallel

• Helical tension: unwinding of parental strands causes DNA ahead of replication fork to become overwound (positive supercoiling)

• Topological constraint: Cricular DNA (in bacteria/archaea) or anchored loops (in eukaryotes) cannot freely rotate to relieve this stress

Mathematical Description:

• Lk = Tw + Wr (White’s equation)

  • Lk: Total times one strand crosses the other in a closed loop (imaginatory planar projection)

    -> cannot change unless the DNA backbone is broken

  • Tw: Number of helical turns (Watson-Crick Twists not lie in plane)

  • Wr: Number of supercoils (DNA crossing over itsself)

  => As long as molecule is kept flat Lk=Tw

During Replication:

• Unwinding the helix reduces Tw (fewer helical turns)

• To conserve Lk, Wr increases -> DNA forms supercoils to compensate (e.g. if two twists get unwound, as a compensation (for the higher energy state) two superhelical twists are formed)

  -> Problem: supercoils ahead of replication fork creates torsional stress making it harder to separate DNA strands

Solution:

• Toposiomerases (enzymes that regulate supercoiling by temporarly cutting DNA strands):

  • Type I Topoisomerases

    • cut one strand, allowing rotation to relax supercoils, then reseal

    • reduces Lk by 1 per action

    • no need of energy

  • Type II Topoisomerases

    • Cut both strands, pass another DNA segment through the gap, then reseal

    • Can make negative supercoils (to counteract overwinding) or relaxe positive ones

    • reduce Lk by 2 per action

    • Nee energy (ATP, NADH)

• Enzymes that catalyze the polymerisation of desoxynucleotides

• always! synthesizes in 5’-3’ direction

• can only add nucleotides to preexisting 3’-OH group -> need a primer to start a new DNA chain

• Types of Polymerases (I-V) E. coli:

  • Polymerase I: Replicating DNA, removing primer (& DNA repair)

  • Polymerase II/IV/V: DNA repair

  • Polymerase III: Primary enzyme for replication chromosomal DNA (& repair)

site where replication bubble forms

• Initial segment of nucleic acid polymer (11-12 nucleotides, often RNA) that is made by enzyme primase and extended on 3’-OH Group

• gets removed and replaced with DNA at the end -> Important because first nucleotide is very error-prone

• A sequence of DNA or RNA that directs synthesis of complementary sequence

Synthesizes an RNA primer to start DNA replication

• Leading strand: Synthesized continuously in the 5’-3’ direction by DNA Polymerase III

• Lagging strand: synthesized discountiously in short segments (about 1000 nucleotides) => OKAZAKI FRAGMENTS

short DNA segments synthesized by DNA Polymerase III

Enzyme that unwinds DNA, creating a replication bubble with two replication forks

protect single-stranded DNA from damage and prevent double-stranded breaks (important for Mismatch repair)

Enzyme that synthesizes a short RNA primer complementary to the DNA segment -> provides starting point for DNA synthesis

(supercomplex) binds to the replication fork, enabling simultaneous synthesis of both DNA strands

• Enzyme that degrade nucleotides (proofreading)

• removes mismatched nucleotides from the 3’ to end

-> Incorrect base pairing causes slight distortion in topolgy of double helix (less stable)

-> detects the incorecct nucleotide (e.g. A-T)

-> Exonuclease revers direction, removing the mismatched nucleotide from the 3’end

-> DNA Polymerase then resumes adding the correct nucleotide, ensuring the sequence matches the template

• Template Strand: Read 3’-5’

• New Strand synthesized 5’-3’

1. Initiation:

   • begins at origin of replication

   • Helicase unwinds DNA, creating a replication bubble with two replication forks

   • DNA strands are immediatly stabilzed(& covered) by single stranded binding proteins

2. Primer Synthesis

   • Primase synthesizes a short RNA primer complementary to the DNA segment -> starting point for DNA syntehsis

3. Leading Strand Synthesis (5’-3’)

   • DNA Polymerase III extends RNA primer, synthesizing the new DNA strand continuously in the 5’-3’ direction

4. Lagging Strand Synthesis (3’-5’)

   • Primase adds mutiple RNA primers at various points along the lagging strand

   • Okazaki Fragments: DNA Polymerase III synthesizes short DNA segments between the primers

   • DNA Polymerase I (has 2 very specific active sites for both reactions) binds to 3’ end of Okazaki Fragments, replacing RNA primers with DNA and extending the strand

   • DNA Ligase: joins Okazaki Fragments on the lagging strand, sealing the gaps and creating a contiuous strand

5. Topological PROBLEMS: resolution (verwickelung der strands)

   • Topoisomerase I make single-strand cuts to relieve supercoiling

   • Topoisomerase II makes double-strand cuts and uses ATP to introduce negeative supercoild aiding in DNA packaging and stability

6. Proofreading:

   • Proofreading: DNA Polymerase has a proofreading function to correct mismatches

   • Repair Mechanisms: additional enzames recognize and repair DNA damages and errors

https://www.youtube.com/watch?v=IjVLhoyfGAM

Solution: Lagging strand is synthesized in ~ 1000 nuclceotide segments (Okazaki fragments)

• 2 replication forks & bidirectionally

• Origin of replication always at the same spot -> A&T rich recions because they form less hydrogen bonds and are easier to open up

• Polymerases colide and break off at the terminus of replication

• Replication proteins all aggragate to form a large replication complex

• lagging strand loops around so whole replisome moves in same direction along chromosome

Replication machinery

consist of:

• 2 copies of DNA Polymerase III

• Helicase

• Primase

  • Subunit called primosome

• additional elements for stability etc.

Right hand analogy

• Fingers: make up one side of the cleft that binds the DNA

• Thumb: form the other side of the cleft, holding hte DNA in place

• Palm: conatins the active site where the actual chemical reaction accurs

all Polymerase have similar architecture like right hand

• Initial error rate: 1 in 10’000

• Exonuclease reduces error rate to 1 in a million (still 1000 mistakes per copied human cell)

• Spontaneous mutation rate: 10’000 per day per cell

  -> Missmatch excision repair to fix polymerase mistakes

1. DNA polymerase grabs a nucleotide triphospate

2. Nucleotide fits into the cleft formed by the Finger and Thumb

3. Catalysis:

   • 3’-hydroxyl grroup (OH) of the last nucleotide on the growing DNA strand attacks the phophate bond of the new nucleotide - breaks bond between the alpha and beta phosphate of the incoming nucleotide

   • Pyrophosphate (two linked phosphates) is released

   • Ester Bond forms between the 3. OH group of the last nucleotide and the phosphate of the new nucleotide

• base-excision repair

• nucleotide-excision repair

• fixes bulky (=large damages) DNA lesions (e.g. UV-induced damage)

• repairs small, non-bulky DNA lesions (e.g. oxidative damage (reactions with O), deamination (amino group removed))

Process:

1. Damaged base is removed by gylocylase -> abasic site

2. Endonucelase cleave DNA backbone at the abasic site -> single stranded break

3. DNA Polymerase I fills the gap with the correct base and DNA Ligase seals it

• correctis mismatched bases, insertion/deletation loops and other replication errors

Example: Cytosine Deamination (breaks down amino acid)

Cytosine can spontaneoulsy deaminate to uracil => U pairs with A => MUTATION

Repair:

1. recognized by uracil-DNA Glycosylase

2. cleaves bond creating an abasic site (site without a base)

3. endonuclease cleaves DNA at the abasic site -> single-stranded break

4. DNA Polymerase I fills gap with correct base

5. DNA Ligase seals the DNA

=> MMR ensure accurate DNA replication and repair BUT due to addaptive Mutagens (interactive with MMR) it increases mutations (->diseases, cancer)

• Agents that cause DNA mutations, potentially leading to diseases like cancer

Bsp:

• Chemical Mutagens: substances like certain chemical that interact with DNA

• Physical Mutagens: UV-Light, X-rays and other forms of radiation that can damage DNA

• Biological Mutagens: Certain viruses and bacteria that can insert their genetic material into the host DNA, causing mutations

1. Point mutations:

• Transversion: purine base (A,G) is replaced by pyrimidine base (C,T) or vice versa

  • less common

  • can result from chemical or environmental factors

• Transition: purine base is replaced by another purine base (A ->G) or vice vera / pyridine base is replaced by another pyridine base (C->T) or vice versa

  • more common (due similar active sites)

  • often due to sponatneous chemical reactions

1. Insertions/Deletions

   • insertions: adding extra nucleotides to the DNA sequence

   • deletations: removing nucleotides from the DNA seuquence

Problem: Propagation

=> Mutations will be passed on during DNA replication if not corrected