DNA Replication & Genetic Information Flow

• Focus is on of the replication of genetic material and the flow of genetic information in organisms
• Key point is important proteins required for DNA replication and DNA replication pathway,
• Focus on the role of DNA polymerases, proofreading and repair pathways, and DNA recombination

The Central Dogma

• "Central truth" is the flow of genetic information in the cell
• DNA leads to RNA which leads to Protein
• Replication is the process of duplication of DNA, creating RNA
• Transcription uses a DNA template to form RNA and uses RNA to make proteins
• Translation uses mRNA to create proteins, reflecting the mRNA sequence

DNA Replication Overview

• DNA is the hereditary genetic material that must be maintained and replicated
• Replication is performed by various enzymes
• Replication differs between prokaryotes and eukaryotes because of DNA structure
• Focus is on prokaryotic DNA replication, but the fundamentals remain the same

Semi-Conservative Replication

• During semi-conservative replication, the DNA double-helix separates, and a new strand is built using the original strand as a template
• Each daughter cell receives a DNA strand with one parental strand and one daughter strand, conserving half of the original DNA

Conservative Replication

• A new double-helix of DNA is built using a parental strand as a template
• One cell contains the original strands, and one contains the newly formed strands

Dispersive Replication

• The original double helix strands break apart into fragments
• These fragments then act as templates to create a new strand

Replication Fork

• DNA replication begins at a specific point called the origin of replication
• DNA strands are usually synthesized in both directions, known as bidirectional replication
• At any origin of replication, there will be two replication forks
• Replication fork is where new polynucleotide (DNA) chains are formed

Initiating DNA Replication

• DNA helicase is a helix-destabilizing protein that promotes unwinding at the replication fork by breaking hydrogen bonds between DNA base-pairs
• Single-stranded binding proteins (SSB) stabilize the single-stranded DNA regions by binding tightly, preventing the bases from re-biding
• DNA gyrase prevents overwinding of the DNA ahead of the replication fork and inserts negative supercoils to "under wind” the DNA
• Primase is an enzyme that makes a short piece of RNA, complementary to the DNA sequence acting as a "primer" for DNA synthesis and providing a 3' OH for DNA polymerase to build on
• Primosome is a complex at the replication fork consisting of the RNA primer, primase, and helicase
• DNA polymerase builds the new DNA strand, which is complementary to the template strand

DNA Polymerase Mechanism

• DNA polymerase creates DNA by adding dNTPs (deoxynucleotide triphosphates) to the growing DNA chain
• The 3' OH group of the DNA strand acts as a nucleophile, resulting in the formation of a covalent, phosphodiester bond between the oxygen and phosphate groups
• Because this has built-in energy, it does not need extra ATP
• Addition of the base is located at the 3' end of DNA meaning DNA is synthesized in a 5'-3' direction in viruses, bacteria, and eukaryotes
• The diphosphate is removed, in an energetically favorable event

DNA Polymerase Types

• In E. coli, there are three main types of DNA polymerase that have been studied
• DNA polymerase I is a single polypeptide chain that fills in gaps left by RNA primers
• DNA polymerase III is a multi-subunit polypeptide that is far more processive than the other polymerases and it stays attached the DNA for a longer period, enhancing the speed of replication and is considered the main DNA polymerase in replication

DNA Proofreading

• Both DNA polymerase I and III contain 3' → 5' exonuclease activity
• This activity enables DNA proofreading to remove incorrect nucleotides during DNA replication
• When an incorrect base is added it does not hydrogen bond correctly
• The distortion causes the DNA polymerase to notice the incorrect base and exonucleases cleave incorrect peptide bonds from the end of a polypeptide chain while adding new dNTPs until the correct once is bound

Errors and Mutation

• Complete DNA replication happens once each generation in each cell
• Errors in replication can result in mutations, which may be fatal to the cell
• Mutation is a change in the DNA sequence that can result in mutated RNA, which in turn makes mutated proteins
• These mutated proteins may not function correctly, leading to cellular death
• DNA proofreading results in an error rate of ~10-8 to 10-11 per base pair added
• Errors can still happen after proofreading

Semi-Discontinuous Synthesis

• DNA runs anti-parallel with one strand in the 5' – 3' direction and the other in the 3' – 5' direction
• DNA is opened in the 5' – 3' direction by DNA helicase
• One strand can be synthesized continuously from 5' – 3' and is the leading strand
• The other strand is synthesized discontinuously in Okazaki fragments and is the lagging strand
• New primers are added by primase as more of the 5' is exposed on the lagging strand, and DNA polymerase fills in the gaps

Primer Removal

• The RNA primers used in DNA synthesis must be removed and replaced with DNA
• DNA polymerase I removes the primer via 5' → 3' exonuclease activity, which is unique to it
• DNA polymerase I fills in the gap left by the primer, resulting in dsDNA, but the phosphate backbone is not continuous
• DNA ligase connects the gaps in the phosphate backbone after primers are removed forming a phosphodiester bond between adjacent phosphates

Mutagens and DNA Repair

• Mutagens cause a mutation in the DNA
• Mutagens include ultraviolet light, which causes neighboring bases to become covalently bound
• Ionizing radiation causes double-stranded breaks in the DNA
• Chemical agents cause bases to change or alter DNA sequence
• Free radicals bind bases, causing incorrect base pairing

Base Excision Repair

• DNA mismatch occurs when base pairs are incorrectly paired together
• In base excision repair, only the damaged base is initially removed, leaving an abasic site (missing a base) by an endonuclease
• Next the abasic site and surrounding region is removed by an excision exonuclease
• Finally, DNA polymerase I and DNA ligase fill the gap, removing small chunks

Nucleotide Excision Repair

• Commonly used for DNA lesions caused by chemicals or UV
• The section containing the lesion is removed by an ABC exonuclease
• DNA polymerase I and DNA ligase seal the gap, removing big chunks

Double-Stranded Breaks (DSB)

• Double-stranded breaks occur when both strands of the DNA molecule are broken, posing a stability threat to the genome
• The DNA cannot be replicated or expressed, therefore, it must be fixed or it will die
• Two repair mechanisms include non-homologous end-joining and recombination

DNA Recombination

• Genetic recombination is the natural process in which genetic information is rearranged
• Homologous recombination involves a reaction between homologous sequences and requires two copies of a region of DNA where one strand is used as a template to repair the other
• Nonhomologous recombination is a combination of different nucleotide sequences (better to be wrong than dead)