DNA Replication Stages

• DNA replication is a fundamental process in molecular biology, ensuring the accurate duplication of DNA for cell division and inheritance

Stages of DNA Replication

• DNA replication starts with OriC that must be fully methylated
• Topoisomerase enzyme separates parental DNA strands
• Topoisomerase enzyme stabilizes them in a single-stranded state and creates a replication bubble
• DNA replication begins with the recognition of the replication origin by DnaA, a DNA replication initiator protein
• DNA-ATP binds to short, repeated sequences and forms an oligomeric complex that melts the DNA
• DnaC (helicase inhibitor) binds to DnaB helicase in a 1:1 ratio
• The DnaC/DnaB complex loads onto the DNA unwinding element (DUE) on each strand
• Primase (DnaG) displaces DnaC and stimulates the helicase activity of DnaB
• DnaB helicase uses chemical energy from ATP hydrolysis to move in the 5'-3' direction
• Once a single-stranded DNA (ssDNA) region is exposed, DnaG primase synthesizes an RNA primer, facilitating the recruitment of Pol III holoenzyme
• DNA replication can now begin
• Synthesis of daughter strands is initiated at the replication fork

Blocking Multiple Replication Events

• Multiple DNA replication events blocked in E. coli by three systems
• SeqA protein temporarily inactivates oriC immediately after initiation
• SeqA protein preferentially binds to the hemimethylated form of oriC that is produced by replication
• DnaA titration on the datA locus of the chromosome prevents extra initiations, limiting DnaA molecules available to oriC
• DnaA protein is inactivated soon after initiation
• DnaA protein forms complex with ATP or ADP, only ATP-bound form is active in initiation
• Hydrolysis of DnaA-bound ATP yields the ADP-bound inactive form
• The B-subunit of pol III holoenzyme promotes this reaction in vitro
• Hda protein mediates interaction; this inactivation coordinates switching from initiator activity to replication

DnaA Structure and Function

• DnaA has four structural domains and functions as both an initiator of chromosome replication and a transcription factor
• DnaA initiates replication by opening the origin of replication (ori) through binding to specific DNA sequences called DnaA boxes
• As a transcription factor, DnaA autoregulates its own transcription
• DnaA regulates the expression levels of other global regulators involved in cell cycle progression and developmental processes
• Temperature-sensitive dnaA mutants of E. coli show that DnaA has a positive role in chromosome replication and a negative role in regulating its own synthesis
• In vitro, DnaA directly binds to its promoter region
• Increased DnaA levels decrease activity of the dnaA promoter, decreased levels increase activity of the dnaA promoter
• DnaA's ATP-dependent molecular switch modulates transcription factor and replication initiator, is tightly bound to ATP (Kd 30 nM) or ADP (Kd 100 nM)
• Protein Hda and chromosomal loci datA promote hydrolysis of DnaA-ATP to DnaA-ADP in E. coli
• E. coli cell cycle has fluctuations in cellular ratio for DnaA-ATP to DnaA-ADP, can only open ori and initiate replication in E. coli
• Acts as either repressor/activator depending on bound nucleotide, mediating synthesis of DNA substrates through regulation of nrdAB expression
• AGATCT is the ATP specific DNA box sequence, TTATCCACA is the Autoregulation motif

Screening for Protein Function

• Targeted perturbations of cellular networks, such as gene deletions, can improve microbial production strains, but are not feasible if the gene is essential
• Genes which are vital to cell growth/ division should be studied as conditional lethals
• Quick stop mutants stop replication immediately when there is an increase in temperature
• This is typically due to a defect in replication elongation enzymes or related precursors
• Slow stop mutants complete one replication round then stop- defective in replication initiation events

The Replication Process

• DNA double helix acts as a template for its own duplication
• The nucleotide A successfully pairs only with T, G only with C
• Each strand of DNA serve as a template to specify nucleotide sequence - DNA can be copied precisely
• DNA synthesis involves the addition of a deoxyribonucleotide to 3' end of a polynucleotide chain (primer strand), guiding formation of new DNA strand with complementary nucleotide sequence
• DNA polymerase facilitates stepwise addition of a deoxyribonucleotide to 3'-OH end of polynucleotide chain, known as primer strand; pairs with template strand
• Newly synthesized DNA strand polymerizes in the 5'-to-3' direction and occurs via Okazaki fragments
• DNA polymerase is an enzyme which undertakes semiconservative replication
• The fidelity of copying relies on complementary base pairing by proofreading to correct errors

DNA Polymerase and Proofreading

• DNA polymerase fidelity has about 1 mistake in every 109 nucleotides
• Complementary base pairing & proofreading are responsible for high fidelity DNA replication
• Proofreading occurs just before a new nucleotide is added to the chain
• If correct nucleotide in place, molecule can correctly base-pair with the template
• Exonucleolytic proofreading happens immediately after
• DNA Polymerases have 3′-5′ exonuclease actvity to excise incorrectly paired bases, fidelity of replication is improved by proofreading by ~100x

DNA Polymerase Activities

• Has 5′-3′ exonuclease activity to combine with DNA synthesis
• RNA primer is needed at the start of replication on the leading strand, with a base paired chain for new nucleotides
• DNA primase synthesizes short RNA primers on the lagging strand
• DNA ligase joins the new DNA fragments on the lagging strand
• A repair system erases the old RNA primer and replaces it with DNA, creates continuous DNA chain due to the ligase joining
• NOTE: Task for next class is to figure out what enzyme removes the RNA primers during DNA replication & to research erasable primer preference

Opening the DNA Helix

• DNA double helix must be opened up ahead of the replication fork for DNA synthesis
• DNA double helix is stable and for this to happen it must be seperated from complementary strand
• Replication proteins required in opening the double helix (DNA helicases and SSB proteins)
• Process of mechanically unraveling the dsDNA -DNA helicases use ATP to propel themselves rapidly along a DNA single strand -DNA helicases encounter double helix, move along prying apart at rates up to 1000 nucleotide pairs/second
• SSB proteins are unable to open a long DNA helix directly, but they aid helicases by stabilizing the unwound
• SSB cooperative coats and straightens regions of single stranded DNA on lagging-strand template, preventing short hairpin helices

Beta Clamp

• Keeps the DNA polymerase associated with the DNA template when it is moving, allowing them to synthesise nucleotide string
• Otherwise DNA polymerase recycle the Okazaki fragment on the lagging strand for next synthesis
• Clamp keeps polymerase firmly on the DNA moving when it is moving and releases when it is double stranded

Replication Fork

• The moving DNA polymerase on leading-strand template is tightly bound to clamp, and remain associated for a very long time
• Each time the polymerase reaches Okazaki fragment on the lagging-strand template, polymerase is released
• This polymerase molecule then associates with a new clamp on the RNA primer
• Replication proteins ( large unit of total MW > 106 daltons) linked together moves rapidly, enables DNA to be synthesized both sides of the fork efficiently
• As a replication fork moves along dsDNA, it creates “winding problem”
• DNA topoisomerases are swivels that release the winding
• Strand-directed mismatch repair in eukaryotes occur if there is an error

DNA Polymerase Holoenzyme and Eukaryotes

• E. coli DNA polymerase III catalytic core has three subunits ( catalytic and proofreading subunit)
• DNA Pol III holoenzyme (two catalytic cores, processivity clamp, dimerization clamp-loader complex)
• Eukaryotes include bacterial/mammalian machinery
• Bacterial is E. coli DNA polymerase III
• Mammalian DNA primase= subunit of a lagging-strand DNA polymerase + bacterial associated DNA helicase + primosome
• Polymerase (with associated primase) does chains with RNA, hands over to second polymerase (elongates)
• Major mammalian helicase moves along leading strand, not on the lagging-strand template
• Table Summary: There is Helicase, Primase, Ligase etc
• Replication requires: Helicase, Loading Helicase/Primase, Single-strand maintenance, Priming sliding clamp, clamp loading (ATPase), catalysis, holoenzyme dimerization, RNA removal / Ligation
• A replication fork has one complex of DNA polymerase α/primase, one complex of DNA polymerase δ, and one complex of DNA polymerase ɛ
• Strand Synthesis: Initiates the synthesis of both DNA strands and Elongates lagging strands;
• Second DNA polymerase elongates leading strand

High-Fidelity DNA Synthesis

• Replication step has errors for each nucleotide
• 5'→3' polymerization has 1 × 10^5
• 3'→5' exonucleolytic proofreading has 1 × 10^2
• Strand-directed mismatch repair has 1 × 10^2
• Total errors = 1 × 10^9