Bacterial Classification, Anatomy, Nutrition, Growth, Metabolism and Genetics

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Bacterial Classification, Anatomy, Nutrition,
Growth, Metabolism and Genetics
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Classification Systems in the Prokaryotes

• Macroscopic morphology
• Colony appearance color
• Texture size
• Microscopic morphology
• Cell shape, size
• Staining
• Physiological / biochemical characteristics
• Enzymes
• Chemical analysis
• Chemical compound of cell wall
• Serological analysis
• Ag/ Ab binding
• Genetic and molecular analysis
• G C base composition
• Nucleic acid sequencing and rRNA analysis

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G C base composition

• Low GC Gram-Positive Bacteria
• Clostridia
• Mycoplasmas
• High GC Gram-Positive Bacteria
• Corynebacterium
• Mycobacterium

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Bacterial Taxonomy Based on Bergeys Manual

• Bergeys Manual of Determinative Bacteriology
  five volume resource covering all known
  procaryotes
• classification based on genetic information
  phylogenetic
• two domains Archaea and Bacteria
• five major subgroups with 25 different phyla

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Major Taxonomic Groups of Bacteria

• Vol 1A Domain Archaea
• primitive, adapted to extreme habitats and modes
  of nutrition
• Vol 1B Domain Bacteria
• Vol 2-5
• 2 - Phylum Proteobacteria Gram-negative cell
  walls
• 3 - Phylum Firmicutes mainly Gram-positive with
  low G C content
• 4 - Phylum Actinobacteria Gram-positive with
  high G C content
• 5 Loose assemblage of phyla All gram negative

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Species and Subspecies

• Species
• bacterial cells which share overall similar
  pattern of traits
• Subspecies
• Strain or variety
• culture derived from a single parent that differs
  in structure or metabolism from other cultures of
  that species
• E. coli O157H7
• Type
• subspecies that can show differences

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Bacterial Shapes, Arrangements, and Sizes

• Typically described by one of three basic shapes
• coccus
• Spherical
• bacillus
• Rod
• coccobacillus
• vibrio
• spirillum
• Helical, twisted rod,
• Spirochete

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Bacterial Shapes, Arrangements, and Sizes

• Arrangement of cells dependent on pattern of
  division and how cells remain attached after
  division
• cocci
• singles
• diplococci
• tetrads
• chains
• irregular clusters
• cubical packets
• bacilli
• chains
• palisades

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Bacilli
Cocci
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Bacterial anatomy
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Generalized structure of a prokaryotic cell
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Appendages Cell Extensions? Flagella

• 3 parts
• filament
• long, thin, helical structure composed of
  proteins
• Hook
• curved sheath
• basal body
• stack of rings firmly anchored in cell wall
• rotates 360o
• 1-2 or many distributed over entire cell

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Flagellar Arrangements

• monotrichous
• single flagellum at one end
• lophotrichous
• small bunches arising from one end of cell
• amphitrichous
• flagella at both ends of cell
• peritrichous
• flagella dispersed over surface of cell, slowest

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Movement by flagella
Fig. 4.4

• Polar
• Rotates counterclockwise
• Cell swims forward in runs
• Reverse will stop it
• Peritrichous
• All flagella sweep towards one end

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Chemotaxis
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Internal Flagella ? Axial Filaments

• aka Periplasmic
• Endoflagella
• Spirochetes
• enclosed between cell wall and cell membrane of
  spirochetes

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Appendages for Attachment ? Fimbrae

• fine hairlike bristles from the cell surface
• function in adhesion to other cells and surfaces

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Appendages for Mating? Pili

• rigid tubular structure
• made of pilin protein
• found only in Gram negative cells
• Functions
• joins bacterial cells for DNA transfer
  (conjugation)
• Adhesion
• to form biofilms and microcolonies

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The Cell Envelope

• External covering outside the cytoplasm
• Composed of few basic layers
• glycocalyx
• cell wall
• cell membrane
• Maintains cell integrity

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The Cell Membrane

• fluid layer of phospholipid and protein
• phospholipid molecules are arranged in a bilayer
• Hydrophobic fatty acid chains in the
  phospholipids form a permeability barrier

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The Bacterial Surface Coating? Glycocalyx

• Coating of molecules external to the cell wall
• Made of sugars and/or proteins
• functions
• attachment
• inhibits killing by white blood cells
• receptor

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The Bacterial Surface Coating? Glycocalyx

• 2 types
• slime layer - loosely organized and attached
• capsule - highly organized, tightly attached

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Cell Wall

• Four Groups Based on Cell Wall Composition
• Gram positive cells
• Gram negative cells
• Bacteria without cell walls
• Bacteria with chemically unique cell walls

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Structure of the Cell Wall? Peptidoglycan

• macromolecule composed of a repeating framework
  of long glycan chains
• cross-linked by short peptide fragments
• provides strong, flexible support
• keep bacteria from bursting or collapsing because
  of changes in osmotic pressure

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Gram Positive Cell Wall (1)

• Consists of
• a thick, homogenous sheath of peptidoglycan
• tightly bound acidic polysaccharides
• teichoic acid and lipoteichoic acid
• Periplasmic space
• cell membrane

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Gram Negative Cell Wall (2)

• Consists of
• an outer membrane containing lipopolysaccharide
  (LPS)
• periplasmic space
• thin shell of peptidoglycan
• periplasmic space
• cell membrane
• Protective structure while providing some
  flexibility and sensitivity to lysis

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Gram Negative Cell Wall

• LPS
• endotoxin that may become toxic when released
  during infections
• may function as receptors and blocking immune
  response
• contains porin proteins in upper layer
• Regulates molecules entering and leaving cell

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The Gram Stain

• Important basis of bacterial classification and
  identification
• Practical aid in diagnosing infection and guiding
  drug treatment
• Differential stain
• Gram-negative
• lose crystal violet and stain red from safranin
  counterstain
• Gram-positive
• retain crystal violet and stain purple

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Atypical Cell Walls

• Some bacterial groups lack typical cell wall
  structure
• Mycobacterium and Nocardia
• Gram-positive cell wall structure with lipid
  mycolic acid
• pathogenicity
• high degree of resistance to certain chemicals
  and dyes
• basis for acid-fast stain
• Some have no cell wall
• Mycoplasma
• cell wall is stabilized by sterols
• pleomorphic

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Chromosome

• single, circular, double-stranded DNA molecule
• contains all the genetic information required by
  a cell
• DNA is tightly coiled around a protein
• dense area called the nucleoid
• central subcompartment in the cytoplasm where DNA
  aggregates

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Plasmids

• small circular, double-stranded DNA
• stable extrachromosomal DNA elements that carry
  nonessential genetic information
• duplicated and passed on to offspring
• replicate independently from the chromosome

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Plasmids

• may encode antibiotic resistance, tolerance to
  toxic metals, enzymes toxins
• used in genetic engineering
• readily manipulated transferred from cell to
  cell
• F plasmids allow genetic material to be
  transferred from a donor cell to a recipient
• R plasmids carry genes for resistance to
  antibiotics

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Storage Bodies? Inclusions Granules

• intracellular storage bodies
• vary in size, number content
• Examples
• Glycogen
• poly-b-hydroxybutyrate
• gas vesicles for floating
• sulfur

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Endospores

• resting, dormant cells
• produced by some G genera
• Clostridium, Bacillus Sporosarcina
• resistance linked to high levels of calcium
  certain acids
• longevity verges on immortality
• 25 to 250 million years
• pressurized steam at 120oC for 20-30 minutes will
  destroy

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Endospores

• have a 2-phase life cycle
• vegetative cell
• endospore
• sporulation
• formation of endospores
• Germination
• return to vegetative growth
• withstand extremes in heat, drying, freezing,
  radiation chemicals

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Endospores

• stressed cell
• undergoes asymmetrical cell division
• creating small prespore and larger mother cell
• prespore contains
• Cytoplasm
• DNA
• dipicolinic acid
• mother cell matures the prespore into an
  endospore
• then disintegrates
• environmental conditions are again favorable
• protective layers break down
• spore germinates into a vegetative cell

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Microbial nutrition, growth, and metabolism
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Obtaining Carbon

• Heterotroph
• organism that obtains carbon in an organic form
  made by other living organisms
• proteins, carbohydrates, lipids and nucleic acids
• Autotroph
• an organism that uses CO2 (an inorganic gas) as
  its carbon source
• not dependent on other living things

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Growth Factors
Carbon source Energy source
photoautotrophs CO2 sunlight
chemoautotrophs CO2 Simple inorganic chemicals
photoheterotrophs organic sunlight
chemoheterotrophs organic Metabolizing organic cmpds

• organic compounds that cannot be synthesized by
  an organism must be provided as a nutrient
• essential amino acids, vitamins
• Nutritional types
• Chemo-
• Chemical compounds
• Photo-
• light

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Types of Heterotrophs

• Saprobes
• Parasites / pathogens
• Obligate

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Nutritional Movement

• Osmosis
• Facilitated diffusion
• Active transport
• Endocytosis
• Phagocytosis
• Pinocytosis

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Extracellular Digestion

• digestion of complex nutrient material into
  simple, absorbable nutrients
• accomplished through the secretion of enzymes
  (exoenzymes) into the extracellular environment

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Environmental Influences on Microbial Growth

• 1. temperature
• 2. oxygen requirements
• 3. pH
• 4. Osmotic pressure
• 5. UV light
• 6. Barophiles

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1. Temperatures

• Minimum temperature
• lowest temperature that permits a microbes
  growth and metabolism
• Maximum temperature
• highest temperature that permits a microbes
  growth and metabolism
• Optimum temperature
• promotes the fastest rate of growth and metabolism

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Temperature Adaptation Groups

• Psychrophiles
• optimum temperature 15oC
• capable of growth at 0 - 20oC
• Mesophiles
• optimum temperature 40oC
• Range 10o - 40oC (45)
• most human pathogens
• Thermophiles
• optimum temperature 60oC
• capable of growth at 40 - 70oC
• Hyperthermophiles
• Archaea that grow optimally above 80C
• found in seafloor hot-water vents

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2. Oxygen Requirements

• Aerobe
• requires oxygen
• Obligate aerobe
• cannot grow without
• oxygen
• Anaerobe
• does not require oxygen
• Obligate anaerobe
• Facultative anaerobe and aerobe
• capable of growth in the absence OR presence of
  oxygen

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• Fluid thioglycollate media can be used to test an
  organisms oxygen sensitivity
• Gas chamber

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3. pH

• The pH Scale
• Ranges from 0 - 14
• pH below 7 is acidic
• H gt OH-
• pH above 7 is alkaline
• OH- gt H
• pH of 7 is neutral
• H OH-

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3. pH

• Acidophiles
• optimum pH is relatively to highly acidic
• Neutrophiles
• optimum pH ranges about pH 7 (plus or minus)
• Alkaphiles
• optimum pH is relatively to highly basic

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4. Osmotic Pressure

• Bacteria 80 water
• Require water to grow
• Sufficiently hypertonic media at concentrations
  greater than those inside the cell cause water
  loss from the cell
• Osmosis
• Fluid leaves the bacteria causing the cell to
  contract
• Causes the cell membrane to separate
• Plasmolysis
• Cell shrinkage
• extreme or obligate halophiles
• Adapted to and require high salt concentrations

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5. UV Light

• Great for killing bacteria
• Damages the DNA (making little breaks)
• in sufficient quantity can kill the organisms
• in a lower range causes mutagenisis
• Endospores tend to be resistant
• can survive much longer exposures

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6. Barophiles

• Bacteria that grow at moderately high hydrostatic
  pressures
• Oceans
• membranes and enzymes depend on pressure to
  maintain their three-dimensional, functional
  shape
• Barotolerants
• Grows at pressures from 100-500 Atm
• Barophilic
• 400-500
• Extreme barophilic
• Higher than 500

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Microbial Associations

• Symbiotic
• organisms live in close nutritional
  relationships
• Mutualism
• Obligatory
• Dependent
• Both members benefit
• Commensalism
• One member benefits
• Other member not harmed
• Parasitism
• Parasite is dependent and benefits
• Host is harmed

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Microbial Associations

• Non-symbiotic
• organisms are free-living
• relationships not required for survival
• Synergism
• members cooperate and share nutrients
• Antagonism
• some member are inhibited or destroyed by others

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Microbial Associations

• Biofilms
• Complex relationships among numerous
  microorganisms
• Develop an extracellular matrix
• Adheres cells to one another
• Allows attachment to a substrate
• Sequesters nutrients
• May protect individuals in the biofilm

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Microbial Growth in Bacteria

• Binary fission
• Prokaryotes reproduce asexually
• one cell becomes two
• basis for population growth
• Process
• parent cell enlarges
• duplicates its chromosome
• forms a central septum
• divides the cell into two daughter cells

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Population Growth

• Generation / doubling time
• time required for a complete fission cycle
• Length of the generation time is a measure of the
  growth rate of an organism
• Some populations can grow from a small number of
  cells to several million in only a few hours!!

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Prokaryotic Growth

• Bacterial Growth Curve
• lag phase
• no cell division occurs while bacteria adapt to
  their new environment
• logarithmic (log) phase
• Exponential growth of the population occurs
• Human disease symptoms usually develop
• stationary phase
• When reproductive and death rates equalize
• decline (exponential death) phase
• accumulation of waste products and scarcity of
  resources

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Other Methods of Analyzing Population Growth

• Turbidity
• Direct microscopic count
• Coulter counting

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Turbidity
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Direct Microscopic Count
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Electronic Counting
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Microbial genetics
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Genomes
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Prokaryotic Genomes

• Prokaryotic chromosomes
• Main portion of DNA, along with associated
  proteins and RNA
• Prokaryotic cells are haploid (single chromosome
  copy)
• Typical chromosome is circular molecule of DNA in
  nucleoid

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DNA Replication in Prokaryotes
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Genetic Recombination in Prokaryotes

• Genetic recombination
• occurs when an organism acquires and expresses
  genes that originated in another organism
• Genetic information in prokaryotes can be
  transferred vertically and horizontally
• Vertical gene transfer (VGT)
• transfer of genetic material from parent cell to
  daughter cell
• Horizontal gene transfer (HGT)
• transfer of DNA from a donor cell to a recipient
  cell
• Three types
• Bacterial conjugation
• Transformation
• Transduction

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DNA Recombination Events

• 3 means for exogenous genetic recombination in
  bacteria
• Conjugation
• Transformation
• Transduction

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Transmission of Exogenous Genetic Material in
Bacteria
conjugation requires the attachment of two related species formation of a bridge that can transport DNA
transformation transfer of naked DNA
transduction DNA transfer mediated by bacterial virus
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1. Conjugation

• transfer of a plasmid or chromosomal fragment
  from a donor cell to a recipient cell via direct
  connection
• Gram-negative
• cell donor has a fertility plasmid
• (F plasmid, F' factor)
• allows the synthesis of a conjugation (sex) pilus
• recipient cell is a related species or genus
  without a fertility plasmid
• donor transfers fertility plasmid to recipient
  through pilus
• F and F-

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Physical Conjugation
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2. Transformation

• chromosome fragments from a lysed cell are
  accepted by a recipient cell
• genetic code of DNA fragment is acquired by
  recipient
• Donor and recipient cells can be unrelated
• Useful tool in recombinant DNA technology

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Transformation of Insulin Gene

• human insulin gene isolated and cut from its
  location on the human chromosome
• using a restriction enzyme
• plasmid is cut using the same restriction enzyme
• desired DNA (insulin gene) and plasmid DNA can be
  joined using DNA ligase
• plasmid now contains the genetic instructions on
  how to produce the protein insulin
• Bacteria can be artificially induced to take up
  the recombinant DNA plasmids and be transformed
• successfully transformed bacteria will contain
  the desired insulin gene
• transformed bacteria containing the insulin gene
  can be isolated and grown
• As transformed bacteria grow they will produce
  the insulin proteins coded for the recombinant
  DNA
• Insulin harvested and used to treat diabetes

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3. Transduction

• DNA is transferred from one bacterium to another
  by a virus
• Bacteriophages
• Virus that infects bacteria
• consist of an outer protein capsid enclosing
  genetic material
• serves as a carrier of DNA from a donor cell to a
  recipient cell

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Other ways genetics can change

• Transposons
• Mutations

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Transposons

• Special DNA segments that have the capability of
  moving from one location in the genome to another
  
• jumping genes
• Can move from
• one chromosome site to anotherr
• chromosome to a plasmid
• plasmid to a chromosome
• May be beneficial or harmful
• Changes in traits
• Replacement of damaged DNA
• Transfer of drug resistance

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Mutations

• Result of natural processes or induced
• Spontaneous mutations
• heritable changes to the base sequence in DNA
• result from natural phenomena such as radiation
  or uncorrected errors in replication
• UV light is a physical mutagen that creates a
  dimer that cannot be transcribed properly

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• Nitrous acid is a chemical mutagen that converts
  adenine bases to hypoxanthine
• Hypoxanthine base pairs with cytosine instead of
  thymine
• Base analogs bear a close resemblance to
  nitrogenous bases and can cause replication errors

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Point Mutation

• Result of spontaneous or induced mutations
• affects just one base pair in a gene
• Base-pair substitutions
• result in an incorrect base in transcribed mRNA
  codons
• Base-pair deletion or insertion
• results in an incorrect number of bases

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Repair Mechanisms

• Attempt to correct mistakes or damage in the DNA
• Mismatch repair involves DNA polymerase
• proofreading the new strand
• removing mismatched nucleotides

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• Excision repair
• involves cutting out damaged DNA
• replacing it with correct nucleotides