Prokaryotes vs Eukaryotes and Microscopy

Prokaryotes vs Eukaryotes

• Prokaryotes lack a nucleus, cell wall, and organelles
• Eukaryotes contain a nucleus, no cell wall, and organelles such as mitochondria, chloroplasts, and endoplasmic reticulum

Microscopy and the Gram Stain

• Microscopy is used to examine bacterial cell shape, color (stain), size, and shape
• The Gram Stain is the most important staining method in microbiology
• The Gram Stain differentiates between Gram-positive and Gram-negative bacteria based on differential lipid content of their cell envelopes
• Gram-positive bacteria retain the crystal violet-iodine complex and appear blue
• Gram-negative bacteria lose the crystal violet-iodine complex and appear red after the counterstain with safranin

Bacterial Cell Structure

• Genome: Contains genetic information including chromosome and plasmids
• Cytoplasmic Membrane: Surrounds cytoplasm and acts as an osmotic barrier
• Cell Wall: Rigid layer surrounding the cytoplasmic membrane, provides structure and support
• Outer Membrane (Gram-negative bacteria): Covers the cell wall and acts as a molecular sieve

Bacterial Growth

• Bacteria typically divide by binary fission, a process where the chromosome replicates and divides into two identical copies
• Bacterial growth requires energy sources like carbohydrates, lipids, and proteins, as well as nutrients (water, carbon, nitrogen, inorganic salts, iron), and appropriate environmental conditions (temperature, pH, and oxygen)
• Understanding bacterial genetics is essential for clinical microbiology because it explains the emergence of antibiotic resistance and virulence factors

Bacterial DNA

• Bacterial DNA is a circular molecule of double-stranded DNA
• The genome contains approximately 4,000 genes and 5 million DNA base pairs
• DNA replication involves several enzymes: helicase, polymerase, ligase, and gyrase
• The process of DNA replication involves initiation, elongation, proofreading, and termination

Bacterial Gene Expression

• Gene expression is the process by which bacteria decode genetic information in DNA to produce proteins
• Genes can occur individually or in groups (operons)
• Transcription is the process of converting DNA into RNA, using RNA polymerase
• Translation is the process of decoding mRNA to produce proteins, using ribosomes and tRNA molecules

Plasmids

• Small circular extrachromosomal DNA molecules that replicate independently of the bacterial chromosome
• Can confer phenotypic advantages to the host cell, including antibiotic resistance, virulence factors, and metabolic genes
• Plasmids can be transferred between bacterial cells through conjugation, increasing the spread of antibiotic resistance

DNA Mutations

• Mutations are the most common source of genetic variation in bacteria
• Mutations can occur spontaneously or be induced by mutagens such as radiation or chemicals
• There are three main types of mutations: substitutions, deletions, and insertions

Substitution Mutations

• Single-base changes that usually only affect the amino acid coded by that codon
• May have significant effects on protein function or be silent with no effect

Deletion Mutations

• Removal of one or more bases, which affects the amino acid sequence beyond the deletion point
• Often result in a frameshift and prematurely truncated proteins

Insertion Mutations

• Addition of one or more bases, which also result in a frameshift and often lead to truncated proteins
• Both deletions and insertions can result in non-functional proteins

Eukaryotic DNA Replication

• DNA replication is a key process in the life cycle of a cell.
• It is a complicated process with many proteins involved.
• It is essential for growth, development, and repair.
• The process has significant medical interest, as proteins involved are potential targets for drug design.
• Examples include antibiotics, cancer therapies, and anti-cancer drugs.

Eukaryotic DNA Replication

• Large amounts of DNA to be replicated.
• Chromosomes disassemble and reassemble nucleosomes.
• Old histones are randomly distributed, and new histones are delivered.
• Replication requires numerous proteins and enzymes.
• Replication takes longer in eukaryotic cells compared to yeast cells, ranging from hours to days.
• Some human cells are permanently in a quiescent stage (G0) and don't replicate DNA.

DNA Replication

• Semiconservative replication:
  • The parent DNA unwinds.
  • Both strands act as templates.
  • Watson-Crick base pairing is vital for replication.
  • Each daughter DNA strand contains one parental and one newly synthesized strand.
• For DNA replication to occur, it requires:
  • A single-stranded template
  • Deoxyribonucleotide triphosphates (dNTPs)
    • These are composed of A, G, C, and T, and require Mg2+
  • Replisome - A nucleoprotein complex coordinating replication activities.
    • Contains numerous enzymes and proteins
  • A primer with a free 3’ hydroxyl group.

Initiation

• Replication begins at specific points called 'Origins of Replication'.
• These regions are short AT-rich sequences.
• Eukaryotes have multiple origins of replication.
• Two 'replication forks' move outwards from the origin in opposite directions.
• The replisome (a complex of proteins and enzymes) forms at the origin to initiate replication.
• Each chromosome has multiple origins of replication ( 1 every 3-300Kb).
• Humans have thousands of origins across their 23 chromosomes.
• These multiple origins ensure rapid replication of the genome.
• Replication is bidirectional from each origin.

What are the roles of the Proteins in the replisome?

• Step 1: Unwinding proteins
  • DNA Helicase: Separates DNA strands in an ATP-dependent process.
  • Single-Strand Binding (SSB) proteins: Bind to prevent re-association of the separated strands.
  • Topoisomerase: Regulates DNA twisting (supercoiling).
    • Possesses nuclease and ligase activities.
• Step 2: Enzymes that replicate
  • Primase: Synthesizes RNA primers needed to initiate DNA synthesis.

DNA Polymerase

• DNA polymerase can only replicate DNA in the 5' to 3' direction.
• It uses single-stranded DNA as a template.
• It reads the template strand 3' to 5'.
• It aligns and adds nucleotides to the new strand based on the template.
• It catalyzes the formation of phosphodiester bonds between nucleotides.
• The reaction is driven by hydrolysis of pyrophosphate (PPi).
• (DNA)n + dNTP (DNA)n+1 + Pyrophosphate (PPi)
  • Each dNTP is composed of a deoxyribose sugar, a phosphate group, and one of the four nitrogenous bases (A, G, C, T).

DNA Polymerase

• (1) Highly processive: Can synthesize up to 1,000 bases per second.
• PCNA (Proliferating Cell Nuclear Antigen):
  • A sliding clamp that encircles DNA.
  • Keeps DNA polymerase closely associated with the template as it moves rapidly along the strand.
  • Involved in organizing and orchestrating the replication process on both leading and lagging strands.
  • PCNA also participates in DNA repair, sister chromatid cohesion, chromatin assembly and remodeling, and interacts with proteins involved in cell cycle progression.
• (2) Proofreading activity:
  • Reduces errors by 100-fold.
  • Helps to maintain the high fidelity of DNA replication.
  • 1. Substrate specificity:
    • The active site of DNA polymerase preferentially binds the correct nucleotide.
  • 2. ‘Proof-reading’ : error correction activities:
    • 3’ to 5’ exonuclease activity (in the reverse direction)
    • Removes incorrectly incorporated nucleotides at the 3’ end of the newly synthesized strand.

Semi-discontinuous Replication

• DNA replication is semi-discontinuous, meaning it occurs in both continuous and discontinuous segments.
• The two strands of DNA run in opposite directions (antiparallel).
• One strand (the leading strand) is synthesized continuously.
• The other strand (the lagging strand) is synthesized discontinuously in short fragments called Okazaki fragments.
• This is because DNA polymerase can only build new DNA strands from 5' to 3', but the lagging strand is oriented in the 3' to 5' direction.

Lagging Strand Synthesis

• The lagging strand is synthesized in Okazaki fragments.
• Primase synthesizes short RNA primers at regular intervals on the lagging strand.
• DNA polymerase extends these primers, producing Okazaki fragments.
• Once the DNA polymerase reaches the previous primer, it is blocked.
• The RNA primers are then removed by enzymes (RNase H and Flap endonuclease 1).
• The gaps are filled by DNA polymerase.
• The fragments are joined by DNA ligase.
• Okazaki Fragments are roughly 1000bp long.

Eukaryotic DNA Polymerases

• Humans have multiple DNA polymerases, but three are primarily involved in replication:
  • Pol alpha: Involved in initiating replication.
    • Forms a complex with primase.
    • Synthesizes a short RNA primer (7-10 nucleotides) followed by 15 deoxyribonucleotides
    • Extends the primer in the 5' to 3' direction.
    • Lacks exonuclease activity, so it does not have proofreading ability.
    • Moderately processive.
  • Pol epsilon: Responsible for replicating the leading strand.
  • Pol delta: Responsible for replicating the lagging strand.
  • Pol beta: Involved in DNA repair processes.
  • Pol gamma: Replicates mitochondrial DNA.

Primer removal

• Two enzymes are required for primer removal:
  • RNase H1: Removes most of the RNA primer, leaving a single 5' ribonucleotide at the junction of the primer and the newly synthesized DNA.
  • Flap endonuclease 1 (FEN1): Removes the remaining 5' ribonucleotide at the junction.
    • It is an endonuclease that can remove up to 15 base pairs from the 5' end of annealed DNA.

Replication Termination

• Eukaryotic DNA replication does not terminate at specific sequences, unlike prokaryotes.
• Replication forks meet and merge.
• Replication ends when the synthesis of both strands is complete.

DNA Repair mechanisms

• 1. Non-homologous end joining (NHEJ):
  • Repairs double-stranded breaks in DNA.
  • DNA ends are brought near each other by Ku protein, which holds both strands and leaves the ends accessible to enzymes (nucleases, polymerases, and ligases).
  • The ends are then aligned, trimmed, or filled.
• NHEJ is often error-prone and mutagenic.
  • It can introduce deletions or insertions at the repair site.
  • This can lead to cancer and immunodeficiency syndromes.
• 2. Recombination or Homologous Repair:
  • Uses homologous recombination to repair double-strand breaks.
  • The damaged region of DNA is repaired using the information from a homologous chromosome.
• During the S phase, the sister chromatid is nearby and provides the sequence information for repair.
• Non-mutagenic, as it accurately copies the undamaged sister chromatid.
• BRCA1 and BRCA2:
  • Genes that are involved in homologous recombination repair.
  • Mutations in BRCA1 and BRCA2 are associated with increased risk of breast, ovarian, prostate, and pancreatic cancers.
  • Mutations in these genes can lead to an 80% lifetime risk of developing cancer.

Eukaryotic DNA Replication

• Complex process involving many proteins.
• Essential for cell division and life cycle.
• Has medical relevance, with proteins serving as targets for drug design in areas like antibiotics and cancer therapy.

Eukaryotic DNA Replication Specifics

• Large amounts of DNA need to be replicated.
• Chromosomes undergo structural changes, including the disassembly and reassembly of nucleosomes.
• Involves a variety of proteins and enzymes, and takes a significant amount of time.
• Replication occurs in the S-phase of the cell cycle, with varying timeframes for different cell types.
• Human cells can replicate for varying durations, ranging from 8 hours to 100 days, or enter a permanent G0 phase.

Semiconservative Replication

• Parental DNA strands are unwound.
• Exposed bases facilitate strict Watson-Crick base pairing with new nucleotides.
• A template strand is used to create a new complementary strand.
• Each daughter strand contains one parental and one newly synthesized strand.

DNA Replication Requirements

• Single-stranded DNA template.
• Deoxyribonucleotide Triphosphates (dNTPs) – A, G, C, T, and Mg2+ are necessary.
• Replisome: a nucleoprotein complex coordinating replication activities. It comprises various enzymes and proteins.
• Primer with a free 3’ hydroxyl group.

Initiation

• Initiation occurs at specific points on DNA called "origins of replication".
• These origins consist of short AT-rich consensus sequences.
• Eukaryotes have multiple origins of replication.
• Replication forks move in opposite directions from each origin.
• The replisome assembles at the origin to initiate replication.
• Each chromosome has multiple replication origins, spaced every 3-300 kb.
• This allows for rapid replication of the genome.

Replisome Proteins

• DNA Helicase: unwinds the DNA strands using ATP.
• Single-Strand Binding (SSB) Proteins: prevent the separated strands from re-associating.
• Topoisomerase: regulates DNA twisting by controlling supercoiling. It has nuclease and ligase activity.
• Primase: synthesizes short RNA primers that provide a starting point for DNA polymerase.
• DNA Polymerase: replicates DNA by extending primers in a 5’ to 3’ direction. It possesses proofreading capabilities with 3’ to 5’ exonuclease activity, ensuring accuracy.

Semi-Discontinuous Replication

• Both strands are replicated simultaneously at the replication fork.
• However, DNA polymerase can only synthesize DNA in a 5’ to 3’ direction.
• Leading Strand: synthesized continuously by DNA polymerase, moving alongside the replication fork.
• Lagging Strand: synthesized discontinuously due to the 5’ to 3’ polymerase constraint.

Lagging Strand Synthesis

• Synthesized in pieces called Okazaki fragments.
• Primase creates new primers at regular intervals on the lagging strand.
• DNA polymerase replicates the template using each primer, producing a new strand in the 5’ to 3’ direction.
• DNA polymerase is blocked by the proximity of the next primer, resulting in individual Okazaki fragments (approximately 1,000 bp long).

Eukaryotic DNA Polymerases

• Multiple DNA polymerases are present in humans.
• Three main enzymes participate in eukaryotic replication:
  • Pol α: initiates replication and is tightly associated with primase, forming a Pol α/primase complex. Synthesizes a short primer (7-10nt RNA + 15 dNTPs) and extends the primer 5' to 3'. No exonuclease activity for proofreading. Moderately processive.
  • Pol ε: Involved in leading strand synthesis. Highly processive and interacts with PCNA. 3’ to 5’ exonuclease activity for proofreading.
  • Pol δ: Responsible for lagging strand synthesis. Highly processive and interacts with PCNA. 3’ to 5’ exonuclease activity for proofreading.
• Pol β: Involved in DNA repair.
• Pol γ: replicates mitochondrial DNA.

Primer Removal

• RNA primers need to be removed for continuous DNA synthesis.
• Rnase H1: Removes most of the RNA primer, leaving a single 5' ribonucleotide adjacent to the DNA.
• Flap Endonuclease 1 (FEN1): Removes the remaining 5' ribonucleotide. It possesses endonuclease activity, removing up to 15 bp from the 5' end of annealed DNA.
• Pol δ or Pol ε: Fill the remaining gaps.

Replication Termination in Eukaryotes

• Eukaryotes lack specific termination sequences.
• Replication continues until replication forks collide with adjacent replicons.
• Challenges arise in replicating the ends of linear DNA strands (telomeres).

Telomeres

• Located at the 3' ends of each chromosome.
• Consist of thousands of tandem repeats (TTAGGG in humans).
• Synthesized and maintained by telomerase.
• Telomerase is a ribonucleoprotein, containing RNA and protein components.
• The RNA acts as a template for DNA synthesis, adding tandem repeats to the 3' end.
• This provides a new template for primase and lagging strand synthesis.

Telomerase Activity

• High in rapidly dividing cells (e.g., unicellular eukaryotes).
• Present in gamete cell production (sperm) and germline cells.
• Declines during development as cells differentiate, leading to telomere shortening.

Mismatch Repair

• Differentiates parental and daughter strands.
• Uses methylation differences to identify the daughter strand.
• Exo1 (exonuclease) removes bases from the nick on the daughter strand.
• DNA polymerase rebuilds the copy strand.
• DNA ligase rejoins the backbone.
• Defects in mismatch repair increase cancer incidence.

Base Excision Repair

• Replaces bases damaged by chemical processes like depurination or deamination.
• DNA glycosylase: identifies and removes the damaged base, creating an apurinic or apyrimidinic site.
• AP endonuclease: recognizes and cuts the backbone.
• Deoxyribose phosphate lyase: removes the sugar phosphate residue.

DNA Polymerase

• Uses single-stranded DNA as a template
• Reads the template from 3' to 5'
• Synthesizes new DNA from 5' to 3'
• Aligns and adds nucleotides along the template strand based on Watson-Crick base pairing
• Catalyzes the formation of phosphodiester bonds
• Reaction is driven by the hydrolysis of pyrophosphate (PPi)
• Highly processive, capable of adding up to 1,000 bases per second

Proliferating Cell Nuclear Antigen (PCNA)

• Encircles DNA template and keeps DNA polymerase closely associated with the template
• Involved in replication, repair synthesis, methylation, chromatin assembly, and remodeling
• Coordinates DNA metabolism with cell cycle progression by interacting with cyclins, cyclin-dependent kinases (CDK), and CDK inhibitors

DNA Polymerase Proofreading

• Prevents errors during DNA replication.
• Two mechanisms: substrate specificity and proofreading activity
• Substrate specificity:
  • The enzyme's active site can bind all four dNTP types
  • Catalysis only occurs when the correct dNTP is bound
  • A conformational change in the enzyme occurs when the correct Watson-Crick base pair is formed
• Proofreading:
  • 3' to 5' exonuclease activity (reverse direction)
  • Removes mismatched nucleotides from the 3' end of the new strand

Semi-Discontinuous Replication

• Both strands of duplex DNA are replicated simultaneously at the replication fork
• DNA polymerase can only synthesize DNA from 5' to 3'
• Leading strand: Synthesized continuously by DNA polymerase traveling with the replication fork
• Lagging strand: Synthesized discontinuously, piece by piece

Lagging Strand Synthesis

• Primase makes a new primer at regular intervals
• DNA polymerase replicates the template from the RNA primer, producing a new strand in the 5' to 3' direction
• DNA polymerase is blocked by the proximity of the next primer
• Result: a DNA strand of approximately 1,000 bp called an Okazaki fragment
• RNA primers are removed and gaps are filled by DNA polymerase
• Backbone is joined by DNA ligase

Eukaryotic DNA Polymerases

• Pol α: Involved in initiating replication; associates with primase to form a complex
  • Synthesizes 7-10 nt RNA + 15 dNTPs
  • Extends primer 5' to 3'
  • No exonuclease activity
  • Moderately processive
• Pol ε: Involved in leading strand synthesis; highly processive with PCNA
  • Extends primer 5' to 3'
  • Has 3' to 5' exonuclease activity
• Pol δ: Involved in lagging strand synthesis; highly processive with PCNA
  • Extends primer 5' to 3'
  • Has 3' to 5' exonuclease activity
• Pol β: Involved in DNA repair
• Pol γ: Replicates mitochondrial DNA

Primer Removal

• Requires two enzymes: RNase H1 and flap endonuclease 1 (FEN1)
• RNase H1: Removes most of the RNA primer, leaving a single 5' ribonucleotide adjacent to DNA
• FEN1: Removes the 5' ribonucleotide
  • Has endonuclease activity
  • Can remove up to 15 bp from the 5' end of annealed DNA
• DNA Pol δ or ε fills the gaps

Replication Termination in Eukaryotes

• No specific termination sequences
• Methylation (CH3 added to GATC) differentiates the parental and daughter strands
• DNA Polymerase and Exo1 remove bases from the nick past the mutation
• DNA Ligase rejoins the backbone
• Defects in mismatch repair (MMR) result in a high cancer incidence

Base Excision Repair

• Replaces bases lost through chemical damage (depurination or deamination)
• DNA glycosylase: Identifies and removes damaged base, leaving an apurinic or apyrimidinic site
• AP endonuclease: Recognizes and cuts the backbone
• Deoxyribose phosphate lyase: Removes the sugar phosphate residue
• DNA polymerase fills the gap
• DNA ligase seals the nick

E. coli Nucleotide Excision Repair (NER)

• Responds to helix distortion, such as pyrimidine dimers (T-T)
• Cleaves DNA on both sides of the damaged region
• Replaces up to 30 bases
• Protects against carcinogens like tobacco smoke and sunlight

Human NER Disorders

• Mutations affecting different proteins in the NER pathway can lead to two disorders:
  • Cockayne syndrome: Microcephaly, premature aging, sensitivity to sunlight, developmental delays, shortened lifespan
  • Xeroderma pigmentosum (XP): Rare autosomal recessive skin disease; deficiency in NER. Lack of enzymes for repair of DNA damage by UV radiation (thymine dimers). Symptoms: extreme sensitivity to light, skin cancer, frequent secondary tumors, high cancer-related death before age 30

Repair of Double Strand Breaks

• Two main mechanisms: Nonhomologous end joining (NHEJ) and recombination or homologous repair
• Causative agents: Ionizing radiation, chemotherapeutic agents, oxidative free radicals

Nonhomologous End Joining (NHEJ)

• Ku protein recognizes and holds both strands of broken DNA
• Nucleases trim and fill gaps
• Strands are ligated
• Error-prone and mutagenic
• Associated with cancer predisposition and immunodeficiency syndromes

Recombination or Homologous Repair

• Utilizes enzymes and proteins involved in genetic recombination during meiosis
• Uses homologous chromosome as a template for repair
• Non-mutagenic
• During S phase, the sister chromatid is close, providing a homology donor for repair
• Defects in proteins BRCA1 and BRCA2 are associated with an increased incidence of breast, ovarian, prostate, and pancreatic cancers.
• Mutation in genes coding for BRCA1 and BRCA2 can lead to an 80% lifetime risk of certain cancers.

Explore the differences between prokaryotic and eukaryotic cells, focusing on their structural characteristics. Learn about microscopy techniques and the Gram Stain method essential in microbiology for identifying bacterial types based on cell structure. This quiz covers fundamental concepts of bacterial cell structure and function.