Prokaryotes vs Eukaryotes and Microscopy
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Prokaryotes vs Eukaryotes and Microscopy

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Questions and Answers

What is a key difference between prokaryotic and eukaryotic cells?

  • Prokaryotes lack cell organelles. (correct)
  • Eukaryotes have cell walls.
  • Eukaryotes do not have DNA.
  • Prokaryotes have a nucleus.

    • Which structure is NOT present in prokaryotic cells?

  • Nucleus (correct)
  • Ribosomes
  • Cell wall
  • Plasma membrane

    • What is the purpose of the Gram stain in microbiology?

  • To classify bacteria into Gram-positive or Gram-negative. (correct)
  • To determine the size of bacteria.
  • To identify bacterial cell shape.
  • To visualize cellular organelles.

    • Which of the following is a characteristic of Gram-negative bacteria?

    <p>Contains an outer membrane.</p> Signup and view all the answers

    Which of the following structures is involved in the pathogenesis of bacterial infections?

    <p>Cell wall</p> Signup and view all the answers

    What enzyme is responsible for initiating transcription by binding to the promoter region?

    <p>RNA polymerase</p> Signup and view all the answers

    During bacterial translation, which molecule decodes the mRNA sequence?

    <p>Ribosome</p> Signup and view all the answers

    Which of the following enzymes adds nucleotides to the growing strand during DNA replication?

    <p>DNA polymerase</p> Signup and view all the answers

    What is an open-reading frame (ORF) most closely associated with?

    <p>Genetic information in DNA</p> Signup and view all the answers

    What is one function of plasmids in bacteria?

    <p>Conferring phenotypic advantages</p> Signup and view all the answers

    Which molecule plays a role in transporting amino acids to the ribosome during translation?

    <p>Transfer RNA</p> Signup and view all the answers

    What is one clinical significance of plasmids within hospital bacteria?

    <p>They enable multiple antibiotic resistance</p> Signup and view all the answers

    Which step in bacterial gene expression directly follows transcription?

    <p>Translation</p> Signup and view all the answers

    What is the primary method through which bacteria divide?

    <p>Binary Fission</p> Signup and view all the answers

    Which nutrient is NOT typically required for bacterial growth?

    <p>Zinc</p> Signup and view all the answers

    What is one of the main functions of catabolism in bacterial cells?

    <p>Generate energy</p> Signup and view all the answers

    What characteristic of Gram-negative bacteria contributes to its staining properties?

    <p>High lipid content in the cell envelope</p> Signup and view all the answers

    What structure carries the genetic information of bacteria?

    <p>Bacterial genome</p> Signup and view all the answers

    Which environmental factor is NOT directly related to bacterial growth requirements?

    <p>Color</p> Signup and view all the answers

    Which of the following bacteria would not stain using the Gram method?

    <p>Mycoplasmas</p> Signup and view all the answers

    What are the basic building blocks of DNA?

    <p>Nucleotides</p> Signup and view all the answers

    What is the function of the cytoplasmic membrane in bacteria?

    <p>Osmotic barrier and energy production</p> Signup and view all the answers

    How many genes are approximately present in a bacterial genome?

    <p>4000</p> Signup and view all the answers

    Which morphological shape does a bacillus represent?

    <p>Rod-shaped</p> Signup and view all the answers

    Which staining method is utilized for mycobacteria due to their cell envelope characteristics?

    <p>Ziehl-Neelsen stain</p> Signup and view all the answers

    Which factor contributes to the emergence of antibiotic-resistant bacteria?

    <p>Genetic variation processes</p> Signup and view all the answers

    What happens to the crystal violet-iodine complex in Gram-negative bacteria during alcohol treatment?

    <p>It diffuses out of the cell</p> Signup and view all the answers

    What content primarily composes the cytoplasmic membrane of bacteria?

    <p>Lipids and phospholipids</p> Signup and view all the answers

    What is a notable feature of Gram-positive bacteria compared to Gram-negative bacteria?

    <p>Presence of a thick peptidoglycan layer</p> Signup and view all the answers

    What is a common source of genetic variation?

    <p>Gene mutations</p> Signup and view all the answers

    What type of mutation is a substitution?

    <p>Changing one nucleotide to another</p> Signup and view all the answers

    What can result from a frameshift mutation?

    <p>Non-functional proteins due to truncation</p> Signup and view all the answers

    What is the expected mutation rate per cell division?

    <p>10-3 to 10-9</p> Signup and view all the answers

    How do deletions affect the amino acid sequence?

    <p>They can cause frameshift errors</p> Signup and view all the answers

    What is one potential effect of insertion mutations?

    <p>They can result in premature translation termination</p> Signup and view all the answers

    Which of the following is NOT associated with plasmid genes?

    <p>Ribosomal RNA genes</p> Signup and view all the answers

    Which type of mutation could be silent with no effect on protein function?

    <p>Substitution</p> Signup and view all the answers

    What is the primary function of the Ku protein in DNA repair?

    <p>To hold both strands of broken DNA</p> Signup and view all the answers

    Which process is characterized as error-prone and mutagenic?

    <p>NHEJ (Non-Homologous End Joining)</p> Signup and view all the answers

    What is a key difference between NHEJ and homologous recombination?

    <p>NHEJ does not use homologous chromosome information for repair</p> Signup and view all the answers

    What role do BRCA1 and BRCA2 proteins play in DNA repair?

    <p>They are involved in homologous recombination repair</p> Signup and view all the answers

    What is the result of mutations in BRCA1 and BRCA2 genes?

    <p>Significantly increased risk of certain cancers</p> Signup and view all the answers

    What is the primary function of DNA gyrase in bacteria?

    <p>To facilitate DNA replication by relieving supercoiling</p> Signup and view all the answers

    Which direction does DNA Polymerase read the DNA template?

    <p>3’ to 5’</p> Signup and view all the answers

    What is the role of the Proliferating Cell Nuclear Antigen (PCNA) in DNA replication?

    <p>To encircle the DNA template and stabilize DNA Polymerase</p> Signup and view all the answers

    How does DNA Polymerase prevent errors during DNA synthesis?

    <p>Through proof-reading activity that selects correct dNTPs</p> Signup and view all the answers

    What type of bond does DNA Polymerase catalyze during DNA synthesis?

    <p>Phosphodiester bonds between nucleotides</p> Signup and view all the answers

    What is the significance of the hydrolysis of pyrophosphate (PPi) during DNA synthesis?

    <p>It drives the synthesis reaction forward</p> Signup and view all the answers

    Which characteristic describes DNA Polymerase's processivity?

    <p>It can add nucleotides at a rate of up to 1000 bases/second</p> Signup and view all the answers

    What type of base pairing occurs during DNA replication facilitated by DNA Polymerase?

    <p>Watson-Crick base pairing</p> Signup and view all the answers

    What is the first step in the process of DNA replication in eukaryotic cells?

    <p>Unwinding of DNA strands</p> Signup and view all the answers

    What characterizes semi-conservative replication of DNA?

    <p>One strand is parental and one strand is newly synthesized</p> Signup and view all the answers

    What is the role of telomerase in eukaryotic DNA replication?

    <p>Extending telomeres</p> Signup and view all the answers

    Which of the following enzymes is crucial for unwinding the DNA double helix during replication?

    <p>Helicase</p> Signup and view all the answers

    How long does DNA replication take in cultured animal cells?

    <p>16-24 hours</p> Signup and view all the answers

    What is the significance of the random distribution of old and new histones in daughter strands during replication?

    <p>To maintain chromatin structure</p> Signup and view all the answers

    What is the primary reason for the interest in protein function relative to DNA replication?

    <p>Proteins are targets for drug design.</p> Signup and view all the answers

    During which phase do human cells typically replicate their DNA?

    <p>S phase</p> Signup and view all the answers

    Which component is essential for DNA replication to provide a template?

    <p>A single-stranded template</p> Signup and view all the answers

    What is the role of DNA helicase in the replisome?

    <p>To separate DNA strands in an ATP-dependent process</p> Signup and view all the answers

    Which enzyme is responsible for synthesizing the RNA primer during DNA replication?

    <p>Primase</p> Signup and view all the answers

    Where does the separation of DNA strands occur during replication?

    <p>At the origins of replication</p> Signup and view all the answers

    What role do Single-Strand Binding (SSB) proteins play during DNA replication?

    <p>They prevent DNA strands from re-associating</p> Signup and view all the answers

    What defines the origins of replication in eukaryotic cells?

    <p>Multiple sites with an AT-rich consensus sequence</p> Signup and view all the answers

    Which of the following is NOT a component required for DNA replication?

    <p>RNA polymerase</p> Signup and view all the answers

    What is the primary function of topoisomerase during DNA replication?

    <p>To prevent supercoiling of DNA</p> Signup and view all the answers

    What type of activity do eukaryotic DNA polymerases Pol e and Pol d possess?

    <p>3' to 5' exonuclease activity</p> Signup and view all the answers

    How is the lagging strand synthesized during DNA replication?

    <p>Piece by piece using Okazaki fragments</p> Signup and view all the answers

    Which DNA polymerase is primarily responsible for initiating DNA replication?

    <p>Pol a</p> Signup and view all the answers

    What is the main role of RNase H1 in DNA replication?

    <p>Remove RNA primers</p> Signup and view all the answers

    What characterizes the leading strand during DNA replication?

    <p>Synthesized continuously</p> Signup and view all the answers

    What happens after the removal of RNA primers during DNA replication?

    <p>Gaps are filled with DNA by DNA Pol d/e</p> Signup and view all the answers

    Which polymerase is primarily involved in the synthesis of mitochondrial DNA?

    <p>Pol g</p> Signup and view all the answers

    What is the primary function of flap endonuclease 1 (FEN1) during DNA replication?

    <p>Remove 5' ribonucleotide</p> Signup and view all the answers

    What limits the proof-reading ability of Pol a during DNA replication?

    <p>It lacks 5' to 3' exonuclease activity</p> Signup and view all the answers

    What determines the processivity of Pol e and Pol d during DNA replication?

    <p>Their association with PCNA</p> Signup and view all the answers

    What is the primary function of telomerase in eukaryotic DNA replication?

    <p>To extend the telomeres at the ends of chromosomes</p> Signup and view all the answers

    Which of the following correctly describes the semiconservative nature of DNA replication?

    <p>Each daughter strand contains one parental strand and one newly synthesized strand</p> Signup and view all the answers

    What is the role of DNA polymerase during DNA replication?

    <p>To synthesize new DNA strands by adding nucleotides</p> Signup and view all the answers

    How long does it generally take for DNA replication to occur in human cells?

    <p>8 hours to 100 days or permanent G0</p> Signup and view all the answers

    What is a primary function of the proteins involved in eukaryotic DNA replication?

    <p>To assist in unwinding the DNA and reassembling histones</p> Signup and view all the answers

    Which statement best describes the contributions of nucleosomes during DNA replication?

    <p>They disassemble and reassemble in daughter strands</p> Signup and view all the answers

    What do strict Watson-Crick base pairing ensure during DNA replication?

    <p>Accuracy in the synthesis of new DNA strands</p> Signup and view all the answers

    What is a likely consequence of errors during DNA replication in eukaryotic cells?

    <p>Potential mutations leading to disease</p> Signup and view all the answers

    What is the primary function of DNA glycosylase in base excision repair?

    <p>To recognize and remove damaged bases</p> Signup and view all the answers

    Which protein is primarily responsible for cutting the DNA backbone in base excision repair?

    <p>AP endonuclease</p> Signup and view all the answers

    What is a significant consequence of defects in the mismatch repair (MMR) system?

    <p>Higher mutation rates leading to cancer</p> Signup and view all the answers

    During mismatch repair, which protein carries the Proliferating Cell Nuclear Antigen (PCNA) to carry out the repair process?

    <p>MutL</p> Signup and view all the answers

    Which two genes are commonly mutated in Hereditary Non-Polyposis Cancer (HNPCC) leading to malfunction in the mismatch repair system?

    <p>MLH1 and MLH2</p> Signup and view all the answers

    What is the primary function of telomerase?

    <p>To synthesize DNA at the ends of chromosomes</p> Signup and view all the answers

    What does the RNA component of telomerase do?

    <p>Serves as a template for DNA synthesis</p> Signup and view all the answers

    Why can't DNA Polymerase add nucleotides at the extreme end of the lagging strand?

    <p>There are no available nucleotides to add</p> Signup and view all the answers

    What happens to telomerase function during cell differentiation?

    <p>It declines leading to telomere shortening</p> Signup and view all the answers

    What is the composition of telomeric DNA in humans?

    <p>Thousands of tandem repeats of TTAGGG</p> Signup and view all the answers

    In which type of cells is telomerase activity typically higher?

    <p>Rapidly dividing cells like unicellular eukaryotes</p> Signup and view all the answers

    What is the typical outcome after hundreds of cell divisions in relation to telomeres?

    <p>Telomeres gradually shorten</p> Signup and view all the answers

    What structural feature characterizes the ends of linear DNA strands?

    <p>Telomeric regions</p> Signup and view all the answers

    What is the primary function of the DNA helicase during DNA replication?

    <p>Separate the DNA strands in an ATP-dependent process</p> Signup and view all the answers

    Which component is necessary for the assembly of the replisome at the origin of replication?

    <p>Deoxyribonucleotide triphosphates (dNTPs)</p> Signup and view all the answers

    What role do single-strand binding (SSB) proteins serve during DNA replication?

    <p>Prevent the DNA strands from re-associating</p> Signup and view all the answers

    What initiates the separation of DNA strands at the origins of replication?

    <p>DNA helicase unwinding</p> Signup and view all the answers

    Which of the following statements about eukaryotic DNA replication is true?

    <p>Multiple replication origins exist across each chromosome</p> Signup and view all the answers

    What is the primary function of primase in DNA replication?

    <p>Synthesize RNA primers to initiate replication</p> Signup and view all the answers

    What is one of the key functions of topoisomerase during DNA replication?

    <p>Regulate DNA supercoiling and prevent tangling</p> Signup and view all the answers

    How do clusters of replicons benefit eukaryotic DNA replication?

    <p>They enhance the speed of genomic replication</p> Signup and view all the answers

    What activity allows DNA Polymerase to remove incorrectly matched nucleotides during replication?

    <p>3’ to 5’ exonuclease activity</p> Signup and view all the answers

    Which statement accurately describes the synthesis of the leading strand during DNA replication?

    <p>Synthesized continuously in the 5’ to 3’ direction</p> Signup and view all the answers

    How does the lagging strand differ from the leading strand during DNA replication?

    <p>It is synthesized discontinuously in segments</p> Signup and view all the answers

    What role does DNA Polymerase epsilon play during DNA replication?

    <p>Synthesizes the leading strand</p> Signup and view all the answers

    Which of the following enzymes is responsible for removing RNA primers during DNA replication?

    <p>Rnase H1</p> Signup and view all the answers

    What feature distinguishes DNA Polymerases delta and epsilon in eukaryotic DNA replication?

    <p>Both have 3’ to 5’ exonuclease activity</p> Signup and view all the answers

    What is the significance of Okazaki fragments during DNA synthesis?

    <p>They indicate the presence of RNA primers</p> Signup and view all the answers

    Which polymerase is primarily involved in initiating eukaryotic DNA replication?

    <p>DNA Polymerase alpha</p> Signup and view all the answers

    What is a crucial function of the Proliferating Cell Nuclear Antigen (PCNA) in DNA replication?

    <p>Increases the processivity of DNA Polymerases</p> Signup and view all the answers

    Why do eukaryotes lack defined termination sequences during DNA replication?

    <p>They exhibit no specific replication termination mechanism</p> Signup and view all the answers

    What is the primary disadvantage of non-homologous end joining (NHEJ) in DNA repair processes?

    <p>It can introduce mutations to the DNA.</p> Signup and view all the answers

    During homologous repair, what is the significance of the sister chromatid's proximity during S phase?

    <p>It serves as a template for genetic recombination.</p> Signup and view all the answers

    What is the consequence of mutations in the BRCA1 and BRCA2 genes?

    <p>Increased incidence of certain cancers.</p> Signup and view all the answers

    What process involves the alignment, trimming, and filling of broken DNA ends in preparation for ligation?

    <p>Non-homologous end joining.</p> Signup and view all the answers

    Which stage of homologous recombination involves the formation of a structure known as the Holiday junction?

    <p>Strand invasion.</p> Signup and view all the answers

    What is the role of DNA glycosylase in base excision repair?

    <p>Identifies and removes damaged base</p> Signup and view all the answers

    What defects in human MMR (mismatch repair) can lead to?

    <p>Higher cancer incidence, especially in HNPC</p> Signup and view all the answers

    Which enzyme is primarily responsible for joining the DNA backbone during repair processes?

    <p>DNA ligase</p> Signup and view all the answers

    How does AP endonuclease contribute to base excision repair?

    <p>Cuts the DNA backbone at apuric sites</p> Signup and view all the answers

    What happens to the DNA strand after Exonuclease 1 (Exo1) removes bases from the nick?

    <p>It is rebuilt by DNA polymerase</p> Signup and view all the answers

    What is the primary reaction product of DNA Polymerase during DNA synthesis?

    <p>Double-stranded DNA</p> Signup and view all the answers

    Which enzyme is responsible for organizing the replication process alongside DNA Polymerase?

    <p>Proliferating Cell Nuclear Antigen (PCNA)</p> Signup and view all the answers

    How does the proofreading activity of DNA Polymerase ensure fidelity during DNA replication?

    <p>By replacing incorrect bases with correct ones after polymerization</p> Signup and view all the answers

    What is the specific role of the sliding clamp in conjunction with DNA Polymerase?

    <p>To enhance the processivity of DNA polymerase</p> Signup and view all the answers

    Which statement accurately describes the mechanism of catalysis by DNA Polymerase?

    <p>It requires a distinct conformational change upon binding of the correct dNTP.</p> Signup and view all the answers

    What drive the reaction of nucleotide incorporation during DNA synthesis?

    <p>The hydrolysis of pyrophosphate (PPi)</p> Signup and view all the answers

    In which direction does DNA Polymerase assemble the new DNA strand during replication?

    <p>5’ to 3’</p> Signup and view all the answers

    Which attribute characterizes the processivity of DNA Polymerase?

    <p>The rapid addition of nucleotides exceeding 1000 bases/second</p> Signup and view all the answers

    What major mechanism is primarily responsible for repairing double-strand breaks in DNA?

    <p>Nonhomologous end-joining</p> Signup and view all the answers

    Which of the following conditions is primarily related to a deficiency in nucleotide excision repair?

    <p>Cockayne Syndrome</p> Signup and view all the answers

    What type of radiation is most commonly associated with causing thymine dimers in DNA?

    <p>Ultraviolet radiation</p> Signup and view all the answers

    Which protein acts as a broken DNA sensor in the process of nonhomologous end-joining?

    <p>Ku protein</p> Signup and view all the answers

    Which of the following symptoms is NOT associated with Xeroderma Pigmentosum?

    <p>Microcephaly</p> Signup and view all the answers

    Which of the following is a causative agent of DNA damage that nucleotide excision repair processes aim to correct?

    <p>Ionizing radiation</p> Signup and view all the answers

    What is the primary function of nucleotide excision repair in cells?

    <p>Repairing bulky DNA adducts</p> Signup and view all the answers

    In which disorder are mutations in nucleotide excision repair pathways notably linked to developmental delays and sensitivity to sunlight?

    <p>Xeroderma Pigmentosum</p> Signup and view all the answers

    What is the major function of the 3’ to 5’ exonuclease activity of eukaryotic DNA polymerases?

    <p>Removing mismatched nucleotides from the newly synthesized strand</p> Signup and view all the answers

    Which statement accurately describes the synthesis of the leading and lagging strands during DNA replication?

    <p>The leading strand is synthesized continuously while the lagging strand is synthesized in fragments</p> Signup and view all the answers

    What is the role of primase in DNA replication?

    <p>It creates an RNA primer to start DNA synthesis</p> Signup and view all the answers

    Which mechanism is primarily responsible for the removal of RNA primers in eukaryotic DNA replication?

    <p>Flap endonuclease 1 activity</p> Signup and view all the answers

    Which of the following describes the processivity of Pol e and Pol d during DNA replication?

    <p>They are highly processive when interacting with PCNA</p> Signup and view all the answers

    What characteristic differentiates Pol a from Pol e and Pol d in the context of DNA replication?

    <p>Pol a lacks exonuclease proofreading capability</p> Signup and view all the answers

    Which type of fragments are produced during the synthesis of the lagging strand?

    <p>Okazaki fragments</p> Signup and view all the answers

    What is the significance of the replication fork during DNA replication?

    <p>It is where the DNA unwinds and replication occurs</p> Signup and view all the answers

    How does the lack of termination sequences in eukaryotic DNA replication affect the process?

    <p>It allows replication to continue indefinitely</p> Signup and view all the answers

    Which enzyme is primarily involved in the extension of RNA primers into DNA during eukaryotic replication?

    <p>Pol a</p> Signup and view all the answers

    Study Notes

    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.

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    Description

    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.

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