Section 1.2 DNA Replication

Theories surrounding mechanism of DNA replication

Conservative replication: Yields an original intact fully parental DNA and one fully daughter DNA

Semi-conservative replication: Yields two DNAs, each with one parent and daughter strands

Dispersive replication: Yields two DNA molecules that are hybrids/mixtures of parental and daughter DNA

Meselson and Stahl Experiment

Grew E. coli in ¹⁵N (tracer isotope) medium, then grew them in consecutive ¹⁴N mediums

Isolate and centrifuge DNA in CsCl gradient; Heavier ¹⁵N¹⁵N DNA would be at the bottom, then ¹⁵N¹⁴N, then ¹⁴N¹⁴N.

If ______ was correct, what should have happened?

Conservative: After first and consecutive replications, only ¹⁵N¹⁵N and ¹⁴N¹⁴N would be shown

Semi-conservative: First replication yields only ¹⁵N¹⁴N, consecutive replication shows ¹⁵N¹⁴N and ¹⁴N¹⁴N

Dispersive: After first and consecutive replication, only ¹⁵N¹⁴N would be shown

DNA polymerase

Uses dNTPs; Catalyzes extension of DNA strand one dNMP at a time; synthesizes 5’ → 3’; requires a template and DNA/RNA primers

DNA synthesis reaction

Phosphoryl group transfer; dephosphorylates dNTP to dNMP; requires two Mg²⁺ ions at the active site, held by D to stabilize intermediate form

3’-OH of 3’ nucleotide attacks α phosphate of dNTP, releasing PP_i

Based on “Watson-Crick-Franklin” base pairing rules

DNA pol nomenclature in bacteria vs in eukaryotes

Bacteria: Roman numerals; DNA pol I, Pol IV

Eukaryotes: Greek letters; DNA pol α, DNA pol ε

Accuracy of replication/DNA pol

Error in replication can introduce mutation into genome permanently, passing it onto subsequent daughter cells

Average E. coli (4.6 × 10⁶ bp) has mutation rate of 1bp in 10⁹-10¹⁰ bp (1 error per 1,000-10,000 replications)

DNA pol active site restricts base pairing to “Watson-Crick-Franklin” bp; referred to as Presynthetic error control (Does this nt fit into this active site?)

DNA pol adds wrong base every 10^-4/-5

What enzyme removes nucleotides? What are the types of this enzyme?

Nucleases is the general term; DNase is specific for DNA; RNase is for RNA

Two types:

• Exonucleases only breaks phosphodiester bonds at one end of a polynucleotide chain; Can work both 5’ → 3’ or 3’ → 5'

• Endonucleases only breaks phosphodiester bonds within a polynucleotide chain

  • May be sequence-independent or -specific

  • Can either induce single-strand (nick) or double-strand breaks

How do DNA pols proofread during replication

High-fidelity DNA polymerases can proofread; All DNA pol for genome replication for example, are high-fidelity

They contain two active sites:

1. Typical catalytic site for DNA synthesis

2. 3’ → 5’ exonuclease site for removing mis-incorporated nucleotides

DNA synthesis is 5’ → 3’ while proofreading occurs 3’ → 5’

DNA synthesis begins where?

DNA synthesis begins at an origin of replication sequence; Parent strands separate and bidirectional synthesis is initiated; DNA is synthesized by DNA pol at replication forks

Replication fork; Leading vs lagging strand

Where parent DNA is being used as a template for replication by DNA polymerase

Helicase unwinds strands via ATP hydrolysis

Leading Strand: DNA synthesis occurs continuously 5’ → 3’

Lagging Strand: DNA synthesis occurs discontinuously in Okazaki fragments as a series of 5’ → 3’ reactions

Supercoils

Caused by torsional stress due to under/over-winding of DNA

Overwound B-DNA has <10.5 bp/turn

Underwound B-DNA has >10.5 bp/turn

What enzyme add/remove supercoils?

Topoisomerases adds/removes supercoils by cutting phosphodiester bonds in one or both strand, unwrapping the helix, and resealing the strands

Topoisomerase II (DNA gyrase)

In bacteria; Introduces negative supercoils to compact the genome and also removes positive supercoils in front of replication forks

Clinical application of topoisomerases; Selective toxicity

DNA gyrase is an essential supercoiling enzyme that only exists in bacteria

Fluoroquinolones (Ex, ciprofloxacin) are a classification of drugs that target DNA gyrase by blocking its ability to reseal DNA

This type of targeting is called selective toxicity; we specifically target bacterial enzymes to treat conditions such as UTIs, respiratory infections, and gastrointestinal infections

The more selective a drug is, the fewer side effects it has for humans

Initiation of replication in bacteria

Replication occurs once per cycle of division, with initiation being the most regulated step (bc DNA replication is energy-intensive)

Replication begins at the origin of replication “oriC”, a unique 245bp sequence

DNA unwinding element (DUE) is an AT-rich segment where separation occurs

Note:

R1-5 and I1-3 are binding sites for the DnaA protein

IHF and FIS are binding sites for replication initiation factors

Steps of E. coli DNA replication initiation (Pathway)

1. Many DnaA proteins bind ATP and become active, they bind to oriC and create a helical shape, causing a positive supercoil that results in DNA denaturation at DUE

2. DnaC binds ATP and loads a DnaB helicase at both ends of the replication bubble; Helicase leads replication fork 5’ → 3’ via ATP; DnaC dissociates

3. DNA polymerase and additional proteins are added to DnaB helicase

4. ATP on DnaA is hydrolyzed and it dissociates; DnaA is very slow to release ADP (regulation of initiation)

Methylation as a regulator of replication initiation in bacteria

oriC is methylated by Dam methylase (DNA adenine methylation), which methylates the N⁶ position of A within (5’) GATC sequence

After DNA replication, DNA is hemimethylated (Half-methylated); hemimethylated oriC associates with plasma membrane and is sequestered; replication begins only after it is fully methylated (Which takes a while for Dam methylase)

Important Prokaryotic DNA Polymerases

3’ → 5’ exonuclease serves as a proofreader in bacteria

Polymerization rate is how fast it catalyzes nt addition

Processivity is how many nt it adds before dissociating

DNA polymerase I: Has both 3’ → 5‘ and 5’ → 3’ exonuclease, a polymerization rate of 10-20 nt/s and a processivity of 3-200

DNA polymerase III: Only has 3’ → 5’ exonuclease, a polymerization rate of 250-1,000 nt/s and a processivity of >500,000

They both play a role in genomic replication (High-fidelity) and DNA repair. DNA pol III is the main replicator

DNA pol I is the only enzyme with 5’ → 3’ exonuclease

Holoenzyme

Main/core enzyme plus its accessory proteins

DNA polymerase III Holoenzyme

Core polymerase (Pol III) catalyzes DNA synthesis

Clamp loader: Acts as a scaffold for DNA polymerase III complex; assembles β clamp onto DNA via ATP; coordinates the replication fork by interacting with DnaB helicase through τ subunit

β clamp (sliding clamp) tethers the core pol to DNA; Decreases dissociation and increases processivity

Primase

DNA pol III requires a primed DNA template (3’-OH)

Primase is an RNA polymerase; DNA template-dependent, Primer-independent; synthesizes <9nt RNA primer at the beginning of leading strand and each Okazaki fragment

Lagging Strand Synthesis Pathway in bacteria

1. DnaB helicase travels along the lagging template strand in the 5’ → 3’ direction and unwinds DNA

   • Single-strand DNA-binding protein (SSB) binds single-stranded DNA (Stabilization)

   • DnaG primase occasionally associates with DnaB and synthesizes a short RNA primer; this occurs at the replication fork right as the core polymerase is almost done with an Okazaki fragment

2. A new β clamp is loaded onto the lagging strand at each new RNA primer by the clamp loader

   • Clamp loader binds ATP, then the sliding clamp, and opens the clamp at one subunit interface

   • ATP hydrolysis closes the clamp and allows the loader to dissociate

3. DNA pol III synthesis of Okazaki fragment is complete when it reaches the previous primer

   • Lagging strand core pol stops, releases its β clamp, and is then transferred to the new β clamp, leaving the old one behind for lagging strand processing and in DNA repair pathways

4. Lagging strand core polymerase initiates synthesis of the next Okazaki fragment

5. Clamp loader acquires a new β clamp and opens it to prep loading for the next primer

Lagging strand processing in bacteria

DNA pol I uses its 5’ → 3’ exonuclease to remove primer and synthesizes DNA to fill the gap

DNA ligase repairs the nick between fragments, linking them into a single DNA strand (Bacteria use NAD⁺ for DNA ligase)

DNA ligase

Links two existing DNA chains together by forming a phosphodiester linkage; resulting in a continuous 5’ → 3’ strand

In vivo, its primary role is to seal single-strand breaks (nicks)

Completion of circular DNA molecule in bacteria

Bidirectional, semiconservative replication yields two identical DNA molecules

After replication, circular chromosomes are linked like links in a chain (catenated state); separation of catenated circles in E. coli requires topoisomerase IV

Quick comparison of prokaryotic vs eukaryotic DNA replication

Similarities:

• Eukaryotes also have many DNA polymerases (~15) with specialized functions like DNA repair;

• DNA synthesis is still semiconservative, bidirectional, template- and primer-dependent, in the 5’ → 3’ direction, continuous in the leading strand, and discontinuous in the lagging strand;

• Some polymerases are high fidelity with proofreading abilities

Differences:

• Eukaryotic chromosomes are linear and can be very long; DNA polymerase complex “replicase” architecture studies are not yet complete

• Replicase has both DNA pol ε and DNA pol δ

• Primase is in a complex with DNA pol ⍺

• Replication initiation is very different; Eukaryotes have multiple origins of replication; coordination of regulation requires “licensing”

• Replication elongation is slower with smaller Okazaki fragments, and the removal/replacement of RNA primers is different

• Telomeres are unique to eukaryotes

Eukaryotic origin of replication

Human chromosomes have ~30,000 - 50,000 origins of replication

On average, ~25,000 bp apart, though the locations used may vary depending on cell type

Usually an AT-rich element and is associated with actively transcribed genes

Our DNA is wrapped around proteins to help condense the genome (They unbind during replication)

Licensing coordination in eukaryotic replication initiation; How does this prevent reactivation of replication?

Origin of replication complexes (ORC) binds tightly to DNA in G1 phase and recruits proteins

• Cell division cycle (CDC6) and chromatin licensing and DNA replication factor (CDT1) join to load the helicase Mini Chromosome Maintenance (MCM) (x2), which translocates 3’ → 5’ along the leading strand template using ATP

In S phase, replication is initiated by phosphorylation of ORC proteins by a cyclin-dependent kinase (CDKs); the replisome is assembled, and bidirectional DNA synthesis is initiated

How does this prevent reactivation of replication?

Cdc and Cdt are phosphorylated to begin replication, meaning they can’t reattach to the complex post-dissociation

Licensing can also only occur during G1 phase

Meier-Gorlin Syndrome

DNA replication disorder: Mutation in the MCM5 gene, which encodes a component of the helicase complex. This makes helicase MCM inefficient, slowing replication/cellular division/development

Parts of The Eukaryotic Replisome

• MCM in helicase

• DNA pol ε synthesizes leading strand; High processivity and proofreads

• DNA pol δ synthesizes lagging strand; Proofreads

• DNA pol ⍺-primase complex contains primase for RNA primer synthesis and a separate DNA synthesis activity

  • ⍺-primase makes the primer, then its DNA pol portion comes in to extend the primer with a bit of DNA

• Replication factor C (RFC) is the clamp loader

• Proliferating cell nuclear antigen (PCNA) is the sliding clamp; Increases processivity

• Replication protein A (RPA) is the single-stranded DNA-binding protein (SSB)

Lagging Strand Processing in Eukaryotes

Replication elongation is very similar to prokaryotes

Differences: Replisome uses two different core enzymes; completion of lagging strand is also different

DNA pol δ makes the lagging strand; Also called “Strand-displacing” DNA polymerase; The enzyme pushes the previous template off the template as it synthesizes DNA; This process leaves behind an overhang of the RNA primer

Flap endonuclease-1 (FEN1) clips off the overhang and DNA ligase repairs the nick to join the Okazaki fragments

Synthesis of linear chromosomes in humans

Humans have 46 linear chromosomes

Ends of DNA molecules are substrates for DNA repair enzymes and nucleases (Basically they can cause unnecessary repair and breakage)

These ends also shorten after every round of replication because primers are needed for replication but templates are not available at the ends of the chromosomes

Telomeres

Telomeres are DNA structures at the end of eukaryotic chromosomes that protects the chromosome

Consists of a repetitive short sequence; In human it’s TTAGGG (TG strand); this strand is always longer than the complementary strand (CA strand)

No genes or important sequences exist within the telomere, so if they shorten as a result of replication, no genes are lost

T-Loops in Telomeres

T-loops are specialized structures that sequester the single-stranded end of the telomere by base pairing, which protects the 3’ ends from nucleases and repairing enzymes

Proteins are bound to the telomere to form the T-loop; Shelterin proteins protect the single-stranded 3’ end in the DNA duplex; TTAGGG repeat factor, TRF1 and TRF2 bind the looped DNA

Telomere length shortens with age, why?

Shortening of telomeres in somatic cells helps to keep track of the cell’s “age”

• When telomeres reach a critical length, the cell no longer divides, enters G0 permanently (senescence), and goes through apoptosis (programmed cell death

• Prevents the loss of important DNA and cancer development

Rapid aging phenotypes of diseases like Progeria are associated with shorter telomeres

Germ and stem cells do not have shortening of telomeres

Telomere synthesis (Only RT covered)

Only occurs in stem and germ cells

Telomerase is a specialized reverse transcriptase; also classified as a ribonucleoprotein (RNA and protein); contains an internal RNA with CA-rich repeat that serves as a template

Template RNA anneals to existing TG sequence at a telomere; Telomerase catalyzes 5’ → 3’ DNA synthesis of the TG strand; then shifts so template RNA can anneal to the newly synthesized DNA; process repeats

Complementary CA strand is synthesized by DNA pol ⍺-primase

After the RNA primer is removed, the overhang 3’ end base pairs to the CA strand and forms a T-loop