Bio3124
Lecture 9
Overview
Reading: Ch9
• The mosaic nature of genomes
● Mutations: Types and causes
● Mechanisms of DNA repair
● Mobile genetic elements
- Insertion sequences and transposons
How genomes evolve
Gene transfer: Transformation; conjugation;
and transduction
● Genetic recombination
●
Mosaic Nature of Genomes
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DNA sequence is not static
Mutations generated during replication
fixed by replication
Inherited vertically
Transmitted across species horizontally
How mutations arise?
Mutation
• Heritable change in DNA sequence
• Point mutations
– Change in one base pair (base substitution)
• Transition: purine  purine,
pyrimidine  pyrimidine
• Transversion: purine  pyrimidine
• Insertions and deletions
– 1 or 2 base, cause frameshift
• Larger mutations
– Insertion or deletion of a section of DNA in chromosome
– Inversion: flipping a portion of chromosome
Types of Mutations
• Silent: no amino acid change
• Missense mutation
– Change one codon to another
• Nonsense mutation
– Change a codon to Stop
• Frameshift mutation
CGA  CGG
Arginine  Arginine
CGA  CAA
Arginine  Glutamine
CGA  TGA
Arginine  STOP
AAA CGA CCC  AAA CTG ACC C
Lysine Arginine Proline  Lysine Arginine Threonine
– Insert or delete a single base
– Changes bases reading frame by ribosome
– Alters all codons downstream of mutation
Mutagenesis
1. Spontaneous mutagenesis
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Cytosine deamination
Depurination-> AP sites
Inherent error of polymerases
Tautomeric transition during replication
Mutagenesis
2. Induced mutagenesis
• Electromagnetic radiation
– X-rays, gamma rays
– UV light  Don’t go tanning!
• Chemicals
– Analogs of bases (5-BU)
– Base-modifying chemicals
• Nitrosoguanidine, nitrous acid
– Intercalators insert between
bases
• Cause frameshift mutations
Mutagenesis Test
• Ames Test
– Uses Salmonella strains auxotrophic
for histidine
• Has mutation in hisG gene
• Cannot grow unless histidine is supplied
– Place on medium + chemical
• Mutagen causes reversion
Revertant
colonies
– Changes mutation to normal form
» Rare mutation
– More colonies = stronger mutagen
• Most mutagens are carcinogens
– Cancer results from multiple
mutations
Mutagen on
paper disk
Surveillance: DNA Repair
• Collectively increase DNA replication
fidelity
• correcting misincorporated bases
• Minimize mutation bkg
• Error-free mechanisms
– MMR, NER, BER, PR and recombinational
repair
• Error-prone mechanisms
– SOS dependent translesion bypass
Error-free DNA Repair
Photoreactivating enzyme (PR)
DNA photo-adduct reversal (see A. Sancar)
• Thymidine dimers
– Induced by UV
Mismatch repair
• conserved function across prokaryotic and eukaryotic cells
• methyl directed MMR corrects base mismatches generated by
replisome
•
MutS, MutL, MutH, UvrD (helicase), SSB, Exo, RecJ and Pol III and
ligase are the major palyers
DNA Mismatch Repair
•MutS: dimeric MutS physically interacts with
MutS
replisome β-clamp
T
MutS
G
CH3
CH3
• probes base stacking geometry
ATP
ADP
• binds to mismatched bases
T
G
• conformational changes , recruit MutL
• flips out the mismatching base to minor groove
MutS
MutL
T
MutS
MutL
G
• α-loop formed in DNA , mismatching bases
MutS
MutS
MutL
move out and the MutS/L complex stays at the
MutL
MutH
MutH
bottom of the loop
Incision
• MutL: recruits MutH, a type II endonuclease, and
Exo
T
introduces nick at a hemimethylated GATC at either
side on the newly synthesized strand
• the gap is filled in by the concerted actions of
helicase, Exonucleases,Pol III and ligase
Exo
PIII
CH3
CH3
C
G
G
PIII Helicase
CH3
CH3
Animation: DNA Mismatch Repair
Erratum: the polymerizing enzyme is Pol lII not Pol I
Nucleotide Excision Repair
• NER: wide spectrum, photo
adducts to bulky base adduct,
cross linked strands etc
• UvrAB complex binds damaged
base
• Bending DNA and recruiting UvrC
• Two nicks in DNA (12-13 bases
flanking damaged base)
• UvrD helicase removes ~13 base
oligo including damaged base
• Gap filled by Pol I, ligase
Animation: Nucleotide Excision Repair
Base Excision Repair
• BER: deamination of C and A
produces uracil and hypoxanthine
• Special glycosylases cut base off
sugar
• AP sites formed
• AP endonuclease cut
phosphodiester bond 5’ to AP site
• PolI fills in the gap and removes
AP by its 5’->3’ exonuclease
activity
Animation: Base Excision Repair
Recombinational Repair
• Occurs just after strand has
replicated
• Replisome stalls at adduct
• Reassembles past adduct
• Undamaged strand is copied
– Replaces damaged strand
– Catalyzed by RecA recombinase
Error-prone DNA Repair
Miroslov Radman
• SOS dependent
translesion
bypass
• Regulon of ~60
genes
• Stress response
leads to
mutagenic bypass
of DNA adducts
• Relies on errorprone Pols II, IV
and V
Mobile Genetic Elements
Transposable elements move from one
DNA molecule to another,
- Exist in virtually all life-forms
- Can move within and between chromosomes
Insertion sequence (IS)
- Simple transposable elements containing a
transposase gene, flanked by short inverted
repeat sequences.
- Are targets for the transposase enzyme
Insertion Sequence elements
Mobile Genetic Elements
IS elements transfer by one of 2 mechanisms:
- Replicative or nonreplicative transposition
Mobile Genetic Elements
• Transposons are complex transposable
elements that carry additional genes (e.g.,
drug resistance).
Animation: Transposition
Horizontal Gene Transfer
• Movement of genes between cells
– Other than through cell division
• Transformation, conjugation, transduction
– Transposons carry genes into chromosome
– Plasmids carry genes between cells
• Without having to become part of chromosome
Effects of Gene Transfer
• Spreads genes among bacteria
– Antibiotic-resistance genes
• Spread wherever antibiotics are overused
– Hospitals, farms
– Pathogenicity islands
• Encode genes for cell to act as pathogen
– Difference between typical E. coli in gut
and pathogenic E. coli O157H7
– Genes to degrade special metabolites
• industrially important genes
Gene Transfer: Transformation
• DNA from environment
– Cells must be Competent
• DNA take up mediated by Translocasome
(transforasome)
• Population develops Competence
– G+ cells secrete
signal
– Comp. factor
(CF)
– Stress induces
competence
• Starvation
Gene Transfer: Conjugation
Transfer of DNA btw cells by physical
contact
• Type IV secretion system = F-pilus
• F factor (plasmid) codes for pilus
proteins
– F+ cells, donors
– F- cell, recipients
• replication origins on F plasmid
 oriV: used in nonconjugating cells
 oriT: used during DNA transfer
• F plasmid is mobilized entirely
– Recipient cells can form pilus and transfer
F plasmid
– Can intergrate to chromosome = Hfr
Gene Transfer: Conjugation
Gene Transfer: Conjugation
• F factor
lac genes
oriT
Flac
– F plasmid contains extra genes
– Engineered or acquired from chromosome
– Transfers extra genes to recipient
• Hfr
– F factor integrates to bacterial chromosome
– Can mobilize the entire chromosome
• Take about 100 minutes for E. coli
• Transfers genes in order
• Interrupted mating used for mapping genes on
chromosome
Gene Transfer: Transduction
• phages inject DNA into cell
• Package DNA into viral capsid
– Viral DNA
– Generalized transduction:
incorporate bacterial DNA
• Transfer DNA to new host
• Can bring new bacterial genes
to host
Recombination
• Incoming DNA replaces chromosomal DNA
– DNA enters via transformation, conjugation,
transduction
• Generalized recombination requires that the
two recombining molecules have a considerable
stretch of homologous DNA sequences.
– Replaces variable-sized section of DNA
– Also used to repair damaged DNA
– Requires specific recombination proteins
• RecA
• RecBCD
• RuvAB
Generalized Recombination
Animation: Recombination
Animation: Holliday junction
Summary
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All microbial genomes have a mosaic nature.
● Mutations
can be classified based on their effects
on DNA or the protein.
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Mutations can be spontaneous or induced.
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DNA repair pathways can be divided into 2 types:
- Error-proof pathways and error-prone pathways
● Transposable elements include insertion
sequences and transposons.
Summary
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There are three types of gene transfer in bacteria:
- Transformation: Free DNA from environment
- Conjugation: DNA transfer after cellular contact
- Transduction: DNA transfer via bacteriophages
● Recombination is the exchange of DNA sequences
between DNA molecules.
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Genomes evolve primarily by horizontal gene
transfer and by duplications followed by functional
divergence through mutation.