Lecture 1 & 2: Structure of DNA and Replication

the branch of biology that deals with the structure and function of the macromolecules (e.g. proteins and nucleic acids) essential of life

34 Å, (3,4 nm)

10,5 base pairs

Central dogma

1. Replication

2. Transcription

3. Translation

DNA -> RNA -> Protein

20 Å

0 Å (2 nm)

Minor groove: 12 Å (1,2 nm)

Major groove: 22 Å (2,2 nm)

0.34 nm (3,4 Å)

By condensation reactions of

• phosporic acid

• 2’-deoxyribose

• base

React to form dAMP (nucleotide)

Adenine & guanine

Base + deoxyribose + phosphate

Cytosine, thymine

Hydrogen bonds + base-stacking interactions

Adenine is a purine Base

2’-deoxyadenosine

2’-deoxyadenosine 5’-phosphate

In DNA, adenine and cytosine prefer the amino form, while guanine and thymine prefer the keto form. Rare imino or enol tautomers cause mispairing and base-substitution mutations.

also: Tautomerie depens on the pH-value (changes charge distribution)

Base flipping is the process in which a single nucleotide base is rotated out of the double helix, allowing enzymes to access it directly.

It is essential for DNA repair (base excision repair), methylation, demethylation, and recognition of damaged or modified bases.

• Definition: Nucleosides can rotate around the glycosidic bond, placing the base either above the sugar (syn) or away from the sugar (anti).

1. syn-conformation:

   • Base sits over the ribose sugar.

   • Much more common in purines (A, G) due to their larger double-ring structure.

   • Can occur in left-handed Z-DNA (purines in syn).

2. anti-conformation:

   • Base extends away from the ribose.

   • Default conformation in right-handed B-DNA (all bases anti).

   • Pyrimidines (C, T) almost exclusively anti because their C2-oxygen causes steric hindrance that prevents rotation into syn.

     
     

     
     

     Biological relevance:

   • syn/anti patterns influence DNA helix geometry, Z-DNA formation, and base recognition.

Right-handed DNA (B-DNA, A-DNA) twists clockwise and is the normal DNA form, with all bases in anti-conformation.

Left-handed DNA (Z-DNA) twists counterclockwise, has a zig-zag backbone, and contains purines in syn- and pyrimidines in anti-conformation.

• DNA is generally in B-form

• RNA-DNA duplexes assume A-form

• Z-form forms alternating purine-pyrimidine sequences and is promotes by high salt, specific base modifications (i.e. methylation) and negative supercoiling

Base pairs in B-DNA are not planar; the two bases are twisted against each other like propeller blades.

This propeller twist enhances helix stability by improving base stacking, reducing water accessibility, and compensating for the weaker A–T pairs.

• pyrimidines have an oxygen at the C2-position

• The C2 oxygen atom produces significant hinderance to the rotation of the glycosidic bond between pyrimidine and ribose

The nucleoid is the compact, organized region of the bacterial cell containing the chromosome. It lacks a membrane but has a highly structured DNA organization.

It is arranged into negatively supercoiled looped domains, stabilized by nucleoid-associated proteins (NAPs) such as H-NS and Fis.

H-NS: histone-like nucleoid-structuring protein

Fis: factor for inversion stimulation

Exponential phase:

many looped, highly supercoiled domains, numerous transcription factories.

Stationary phase:

fewer loops, relaxed DNA, reduced transcriptional activity.

H-NS binds widely across the genome, helps compact the chromosome, and often represses transcription of many genes.

Fis binds to AT-rich consensus sequences, bends DNA, and regulates transcription, replication, and recombination. Levels peak in exponential growth and drop in stationary phase.

Clusters of strongly transcribed genes (especially rRNA operons) with high concentrations of RNA polymerase and nucleoid-associated proteins (NAPs) such as H-NS and Fis, forming superstructures in exponential growth.

Topoisomerases relieve DNA supercoiling by introducing controlled single-strand (Type I) or double-strand (Type II) breaks and resealing them.

This prevents DNA tangling and is essential for replication, transcription, and chromosome segregation.

SMC (Structural Maintenance of Chromosomes) proteins are ATPases with long coiled-coil arms.

They mediate chromosome condensation, DNA organization, and higher-order chromatin structure in bacteria and eukaryotes. (Examples: condensin, cohesin.)

ATP binding triggers SMC dimerization, bringing two coiled-coil arms together.

ATP hydrolysis promotes closing and locking of the SMC ring around DNA, enabling chromosome condensation.

An SMC monomer contains:

• a conserved N-terminal ATP-binding cassette (ABC)

• a C-terminal ATPase domain (Walker motifs)

• two long coiled-coil domains

• a central hinge domain that allows dimerization

An SMC dimer embraces two DNA segments, forming a large protein ring.

This allows tethering of distant DNA regions and creation of looped domains, essential for chromosome compaction.

DNA binding stimulates ATP hydrolysis which triggers condensation.

Also:

• Interaction between two SMC dimers generate ring with even larger diameter

• ATP-induces polymerisation of SMC leads to lare nucleo-rotein complexes

3.000.000 kB, 20.000-25.000 Genes

DNA length: 2 km

1. Double helix (2 nm)

2. Pearlchain chromatin (11 nm)

3. Chromatin fiber (nucleosomes, 30 nm)

4. Part of chromosome (despiraled, 300 nm)

5. Part of chromosome (condensed, 700 nm)

6. Complete chromosome (1400 nm)

“Metachromosomes” (State during metaphase, max. condensation)

• two chromatids, linked by centromere

• Centromere: Region between two chromatides

• Telomere: single stranded ends of chromosome with short sequence repeats

Semiconservative replication

• After replication each newly synthesized double helix is a combination of one old (or original) and one new DNA strand

• 3 Steps:

  • Initiation -> Elongation -> Termination

Telomers are protective caps at the end of chromosomes

• maintain chromosomal stability and prevent chromosomal degradation

• they are regions of repetetive DNA which prevent “copy loss” of the ends of chromosomes

• After many cell cycles (division, replication) telomeres are getting shorter -> cell is aging

Telomerase: Enzyme; Ribonucleoprotein

• reverse transcriptase where RNA is used as matrix, priming the 3’-OH-End of G+T rich telomere strands

• prevents telomer shortening

3000x TTAGGG

• Helicase

dissociates H-H bonds between base pairs

• Topoisomerase

Removes supercoils

Afterwards: singe strand binding proteins (SSB)

prevent re-annealing of the DNA strands and stabilize the single-stranded DNA that is exposed after helicase unwinds the double helix.

oriC is the chromosomal origin of replication in E. coli. It contains conserved sequence motifs essential for initiation:

• Three 13-bp AT-rich repeats (unwinding region)

• Four 9-bp DnaA-boxes (binding of DnaA) At least nine proteins cooperate to initiate replication at oriC.

The 9-bp repeats are DnaA-binding sites (consensus TTATCCACA).

Cooperative DnaA binding bends the DNA and initiates opening of the adjacent 13-bp AT-rich region.

The three AT-rich 13-bp sequences form the DNA-unwinding element (DUE). (Consensus: GATCTNTTNTTTT)

Because AT pairs have only two H-bonds, DnaA can easily melt this region to open the helix and begin replication

9 proteins

Elongation (Initiation -> Elongation -> Termination)

1. Leading strand synthesis

   DNA-Polymerase III synthesizes DNA continously in 5’3’, starting from the primer

2. Lagging strand synthesis

   discontinously as short fragments (Okazaki, 100-1000 bp)

3. Primer replacement

   DNA polymerase I removes RNA primers and replaces them with DNA using its 5′→3′ exonuclease activity

4. Sealing nicks with DNA Ligase

The first step ist the Initiation.

Steps:

1. Origin recognition

   • Replication starts at a specific Sequence (e.g. OriC in E coli) where initiator proteins (DnaA) binds to unwind.

   • Complex of 20 DnaA molecules binds to the 4-9 bp repeats

   • recognition of A/T rich sequences in the 13 bp repeats requires ATP and the HU protein

2. Loading of helicase

   • DnaB Helicase (aided by DnaC) is loaded onto the ss by the primosome.

   • Hexamers of the helicase DnaB binds with the help of DnaC to the separated DNA strands and separates more DNA

3. Primase recruitment

   • Primase (DnaG) synthesizes short RNA primers complementary to the ss to provide 3’-OH group for DNA polymerases to begin synthesis

4. ss-stabilization

   • ss-binding proteins (SSB) bind to ss-DNA to prevent re-annealing and protect from nucleases

The primosome is the DnaB helicase–primase (DnaG) complex at the replication fork.

DnaB unwinds the DNA; primase synthesizes RNA primers for Okazaki fragments.The primosome drives lagging-strand initiation and coordinates discontinuous DNA synthesis.

• Type I Topoisomerase

  makes a nick in one strand of a DNA molecule, passes the intact strand through the nick and reseals the gap using energy from ADP

  -> reversible & energy efficient

  • tyrosine at the active site, covalently attaches to a DNA phosphate, thereby breaking a phosphodiester linkage in one strand

  • the two ends of the DNA double helix can then rotate relative to each other, relieving the accumulated strain

  • the original phosphodiester bond energy is stored in the phosphotyrosine linkage, making the reaction reversible

• Type II Topoisomerases

  makes a double-stranded break in the double helix, creating a gate through which a second segment of the helix is passed.

Bacterial monomer helicase PcrA

Consists of 4 domains:

• A1: binds ATP (P-loop)

• A2

• B1 binds ssDNA (together with A1)

• B2

• A single matrix strand is required as a template

• Primer with a 3’OH group is required

  
  

DNA elongation occurs when the 3'-OH group of the growing DNA strand performs a nucleophilic attack on the α-phosphate of an incoming dNTP.

This forms a new phosphodiester bond and releases pyrophosphate (PPi).

PPi = pyrophosphate, which consists of two linked phosphate groups. PPi is immediately hydrolyzed into 2 Pi (inorganic phosphates), making the reaction irreversible and driving DNA synthesis forward.

Its template runs 5′→3′ toward the fork, so Pol III must synthesize away from the fork.

As the fork opens, primase must repeatedly add RNA primers, producing Okazaki fragments.

The leading strand can be synthesized continously,because its template runs 3′→5′ toward the replication fork, allowing Pol III to extend the new strand 5′→3′ in the same direction as fork movement without interruption.

Pol III functions as a dimer, synthesizing both leading and lagging strands simultaneously. The τ-subunits link the two Pol III cores and connect them to:

• DnaB helicase

• the β-clamp

• This coordination enables continuous leading-strand synthesis and repeated cycling on the lagging strand for Okazaki fragments.

  4 β-subunits per enzyme form two ring clamps

Pol III is a holoenzyme with 18 subunits, forming an asymetric dimer.

These 3 form the core polymerase:

• α-subunit: polymerase activity (5′→3′ synthesis)

• epsilon ε-subunit: 3′→5′ exonuclease → proofreading

• theta θ-subunit: stabilizes the ε-subunit

Additionally:

• 4 beta-subunits per enzyme form two ring clamps

• 2 beta-subunits form 12 symetric helices (-> ring)

DNA polymerase III proofreads using the 3′→5′ exonuclease activity of its ε-subunit.

A mispaired nucleotide destabilizes the interaction with the template strand, causing the newly synthesized DNA to shift from the polymerase active site to the exonuclease site.

The ε-subunit cleaves the incorrect nucleotide, and the α-subunit then resumes correct 5′→3′ DNA synthesis.

50.000 nucleotides / min polymerized (high)

Beta clam form a ring around the DNA, increasing processitivity by preventing the enzyme from dissociating during enlongation

• Binding of the correct complementary dNTP induces a conformational change in the “finger” domain of DNA polymerase III, forming a tight binding pocket in which only A–T or G–C pairs fit.

  • This increases selectivity and reduces misincorporation.

    
    

• Combined with proofreading, Pol III inserts only 1 wrong nucleotide in 10⁹–10¹⁰ bp.

  • Since the E. coli genome is 4.6 × 10⁶ bp, this corresponds to one error every 1,000–10,000 replication cycles.

General:

• Purine+Purine -> too wide

• Pyrimide + Pyrimidine -> too tight

DNA polymerase I removes RNA/DNA primers using its 5′→3′ exonuclease activity (“nick translation”).

It cleaves nucleotides ahead of the nick while simultaneously synthesizing DNA with its 5′→3′ polymerase activity.

As Pol I degrades the RNA primer, it fills the gap with DNA.

When Pol I leaves a final nick, DNA ligase seals it.

by ligase activity

• A phosphodiester bond is formed between 3`-OH of one DNA fragment and the 5´phosphate of the next.

• This reaction requires energy: ATP in eukaryotes and archaea, NAD+ in bacteria.

1. Adenylation

   Ligase enzyme reacts with ATP/NAD+, becoming covalently bound to AMP

2. Activitation

   AMP is transferred to 5’-phosphate of DNA at the nick

3. Nick sealing (by AMP displacement)

   3’-OH attacks activated 5’-phosphate, releasing AMP and sealing backbone

DNA-Ligase can not join two ssDNA molecules. It repairs single strand breaks

in dsDNA.

Replication ends at the termination region, which contains multiple Ter sites (each ~20 bp).

Ter sites are binding sites for the Tus protein, that blocks DnaB helicase upon binding.

This creates two “fork traps” that stop clockwise and counterclockwise replication forks exactly within the termination zone.

Tus binds to Ter sequences and acts as a one-way barrier for helicase movement.

From one direction, DnaB can displace Tus; from the opposite direction, Tus locks onto the DNA and halts the fork.

This ensures that the two replication forks meet in a controlled region rather than overrunning each other:

it creates a Clockwise and a counter-clockwise fork trap.

The duplicated circular chromosomes become catenanes (interlinked rings).

These are resolved by Topoisomerase IV, a type II topoisomerase that cuts both strands, passes one duplex through, and reseals it.

This decatenation allows the two daughter chromosomes to segregate.