DNA Replication: Accuracy and Timing

• DNA replication is essential for cell division across all organisms, ensuring that genetic information is accurately transferred.

DNA Replication Necessity

• For unicellular organisms, it enables the formation of new organisms.
• Multicellular organisms rely on DNA replication for the creation of new cells as part of tissue growth and repair.

Genetic Information

• Genetic information is stored and transferred with 100% accuracy.

Timing of Replication Start

• Eukaryotes initiate DNA replication during the S phase of the cell cycle, when DNA synthesis occurs.
• Prokaryotes start replication based on protein amount and cell size.
• Replication begins when DnaA protein reaches a certain level in prokaryotes.

Genetic Information Storage

• Genes, the units of genetic information, are contained within chromosomes.
• DNA or RNA carries genetic information.

Nucleic Acid Building Blocks

• Nucleotides are the monomers of nucleic acids.
• Nucleotides consist of a pentose sugar, a nitrogenous base, and a phosphate molecule.
• The pentose sugar can be either ribose (in RNA) or deoxyribose (in DNA).
• The nitrogenous base can be a purine (Adenine and Guanine) or a pyrimidine (Cytosine, Thymine or Uracil).

Central Dogma and Gene Expression

• Genetic information stored in DNA is transferred to RNA.
• Messenger RNA (mRNA) carries genetic information from DNA to ribosomes.
• Transfer RNA (tRNA) translates mRNA sequences into amino acids.
• Ribosomal RNA (rRNA) has catalytic and structural roles in ribosomes.
• Replication involves copying DNA, transcription involves mRNA synthesis from DNA, and translation involves protein synthesis from mRNA, which occurs in ribosomes.

Bacterial Replication

• In bacteria, one mRNA molecule can encode multiple proteins.
• Bacteria lack a nucleus.
• Replication, transcription, and translation are closely linked and occur in the nucleoid region.
• Translation starts before the completion of mRNA synthesis in bacteria through the process of coupled transcription and translation.

DNA Structure

• DNA consists of complementary strands that are double-stranded and anti-parallel.
• Weak hydrogen bonds hold the double-stranded structure together.
• Specific base pairing occurs: Adenine pairs with Thymine (A-T) via two hydrogen bonds, while Guanine pairs with Cytosine (G-C) via three hydrogen bonds.
• The size number of nucleotide bases or base pairs.
• 1,000 bases of DNA equal 1 kilobase (kb) of DNA.
• Double-stranded DNA is measured in kilobase pairs (kbp).
• 5,000 base pairs is equal to 5 kbp.
• 1 million base pairs make up 1 Megabase pair (Mbp).

DNA size in E. Coli

• The E. coli chromosome is several hundred times longer than the cell itself.
• DNA is supercoiled by topoisomerases.

DNA Replication

• DNA replication is necessary for cell division.
• It allows unicellular organisms to create new organisms and multicellular organisms to produce new cells.
• Genetic information from the parent cells gets transferred to daughter cells with 100% accuracy.
• DNA replication is a precise process.
• Chromosome replication in E. coli takes 40 minutes.
• A replicon is the DNA where replication occurs, with the entire E. coli genome forming one replicon.

DNA Features

• DNA is a double helix composed of two complementary strands.
• Replication is semi-conservative, producing two new double-stranded DNA molecules, each with one new and one old strand.
• The template strand is the parent DNA strand, and the complementary strand is the newly synthesized daughter strand.
• Before each mitotic division, DNA molecules are duplicated to form two exact copies.
• Replicated DNA molecules are passed to the daughter cells and replication occurs during the synthesis (S) phase of the cell cycle.
• Semi-conservative replication means each new DNA molecule contains one parental strand and one newly synthesized strand.

DNA Replication Process

• During replication, each parental DNA strand acts as a template for synthesizing a new, complementary daughter strand.
• The process is considered semi-conservative because each new double helix contains one original and one newly synthesized strand.

Semi-Conservative Nature of DNA

• The semi-conservative nature of DNA replication was discovered by Watson and Crick and general model demonstrated this by Watson-Crick.
• This model leads to theoretical predictions about how the original and newly synthesized DNA strands are distributed after one round of replication.

DNA Synthesis

• The precursor for new nucleotides in a DNA strand is deoxyribonucleoside 5'-phosphate
• Two terminal phosphates are removed during the replication process.
• The remaining phosphate links to the 3' end of the growing DNA chain.
• DNA replication always proceeds in the 5' to 3' direction.
• The 5'-phosphate of the incoming nucleotide binds to the 3'-OH of the previously added nucleotide.

Key Enzymes and Molecules

• DNA polymerases are nucleotide polymerizing enzymes.
• E. coli has five DNA polymerases (DNA Pol I-V).
• DNA Pol III is the main enzyme for DNA polymerase, synthesizing DNA in the 5'-3' direction, adding nucleotides to the 3'-OH group, and unable to initiate synthesis de novo.
• Primase synthesizes an RNA primer of 11-12 nucleotides to initiate replication, later removed and replaced with DNA.
• DNA polimeraz I, discovered by Arthur Kornberg et, was the first enzyme isolated in E. coli and is involved in DNA synthesis.

Bacterial DNA Polymerases

• Mutant E. coli experiments showed that replication can still occur without DNA polymerase I, but DNA repair is impaired.
• DNA polymerase I has 5'-3' exonuclease activity, can remove nucleotides from the sequence, is found in greater amounts in the cell, is more stable than DNA polymerase III, and removes the RNA primer and fills teh gap with DNA.
• DNA pol catalyzes the joining of nucleotides to form the growing DNA chain.
• DNA polymerization requires a single-stranded DNA template, using free nucleotides (deoxyribonucleoside triphosphates; dNTPs) as substrates.

DNA Polymerase III

• DNA polymerase III, the holoenzyme, has subunits, and the core enzyme contains α, ε, and θ subunits for catalysis.
• The α subunit is responsible for DNA synthesis, the ε subunit has 3'-5' exonuclease activity for proofreading, and the theta subunit aids core assembly.

DNA Replication Process

• It requires DNA polymerase, a single-stranded DNA template, and dNTPs (dATP, dTTP, dGTP, dCTP).
• Replication begins at origins of replication, specific nucleotide sequences, also requires the following;
• Origin recognition.
• Unwinding of dsDNA using helicase.
• Formation of the replication fork.

Prokaryotes

• They and eukaryotes initiate replication from specific nucleotide sequences, origins of replication.
• The replication apparatus recognizes these origins through sequence-specific dsDNA binding proteins, the initiator proteins.
• In Saccoromyces cerevisia, replication origins have eleven nucleotide sequences.

Replication Origins

• Prokaryotes have a single replication origin.
• Higher eukaryotes have many replication origins (30,000 ORC in the human genome).
• ORIc, contains 245 base pair DNA with 9 base pair and 13 base pair repeat sequences, is rich in AT content, which helps open the helix.
• The unwinding of the double helix is greatly aided via the DnaA and DnaA binds open the DNA strands in this region.
• DNA helicase can then binds and unwinds the double helix.

Replication Fork

• Helicase unwinds and relaxes the double helix, using energy from ATP; this is Dna B.

The Replisome

• Consists of Helikase, Primase and single strand binding protein.
• Helikase (2 copies) unwinds the helix.
• Primase (2 copies) is a type of RNA polymerase that synthesizes RNA primer.
• SSBP stablizes the single stranded DNA.
• In ORIc DnaA opens the DNA at specific sites and binds, opening double helix then helicase binds (DnaB), this is maintained by DnaC.
• Then the DNA pol III does nueclotide polymerixation wich leads to chain elongation.
• Finally, DNA gyrase unwinds the supercoiled double-stranded DNS by making nicks.

The DnaA Protein

• Is the initiator protein for replication and opens up the DNA by binding to dnaA boxes at the oriC region.
• This region contains 245 base pair DNA with nine bp repeat pattern.

Leading and Lagging Strands

• On the Leading Strand DNA synthesis is continuous and always runs in 5'-3' direction, there is always a free 3'-OH end and DNA strand replication is set in place.
• But on the Lagging Strand Replication is discontinuous and there is no free 3'-OH end, and synthesis as "okazaki fragments", a type DNA fragments 1000-2000 nucleotide long.

DNA Polymerases

• DNA Pol III synthesizes most of DNA.
• DNA Pol I synthesizes, with exonuclease activity, removes RNA primer and replaces with DNA..
• DNA Ligase, seals the gaps because phosphodiester bond is closed by ligase
• For DNA repair use DNA Pol I, DNA ligase, DNA pol II

Bidirectional Replication

• Replication proceeds bidirectionally from the origin.
• Each chromosome has two replication forks moving in opposite directions.
• Bidirectional synthesis forms a theta structure.
• Replication initiation begins at the oriC site for DNA polymerase A.
• Terminating the replication by using Terminus of replication (Ter) containing Tus protein, recognizes this region and halts replication.

Replisome Complex Composition

• DNA Pol III
• DNA gyrase
• DNA helicase
• DNA primase
• Single strand binding proteins

Accuracy

• During proofreading, DNA Pol I/II accurately adds bases according to base pairing rules.
• Low rate of mutation due to proofreading.
• DNA Pol I ve DNA Pol III have mismatch detecting 3′-5′ exonuclease activity which removes mismatches and ithen adds the correct nucleotides in.