Yes, another one
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David Ray  806-834-1677, ESB 216
Office Hours  M 1-3 pm
Class website  http://www.davidraylab.com/genetics/
Homework  Still trying to figure it out
REEF  Think I’ve got it working but will need to experiment
Ask questions IN CLASS
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Makes things more interesting for me
Others probably have the same question
You’re paying, get your money’s worth
Interaction with other humans tends to wake people up
Office hours!!!!!!!!!! I have them. Take advantage.
Objectives and Assumptions
• Objectives: By the end of this course you should…
• have a working knowledge of how mutations occur,
impact and are repaired by/in cells
• be able to describe chromosomal aberrations and their
and impacts
• describe the basic mechanisms of bacterial gene
regulation
• describe the basic mechanisms of bacterial gene
regulation
• solve problems related to basic population genetic
scenarios using Hardy-Weinberg principle
Objectives and Assumptions
• I am working on the assumption that you…
• have a working knowledge of Mendelian genetics (Chapter 2)
• have a working knowledge of DNA, RNA and protein structure
(Biology 1403)
• understand the basic differences between eukaryotes and
prokaryotes (Biology 1403)
• have a basic understanding of cell division and chromosome structure
(Chapter 3, Chapter 11)
• have a working knowledge of transcription and translation (Biology
1403, Chapter 8)
• give a rat’s behind about learning this stuff (Tuition)
DNA Mutation and Repair
• Faithful replication of the genome is necessary for life
• Mutation rates are generally low in all genomes and vary from
organism to organism
• Most mutations are either neutral or deleterious
• But without the occasional mistake natural selection has nothing on
which to act
• Genetically homogeneous species experience great peril
• Cells must
• Act to replicate DNA faithfully
• Identify mistakes
• Distinguish old (accurate) strand from new (possibly mutated)
strand
• Act to repair mistakes from various sources
DNA Mutation and Repair
• Mutation rate is measured as the number of times a mutation alters
the DNA sequence at a particular locus per replication cycle or
generation
• Some factors that impact mutation rates
• genome size
• environment
• effectiveness of molecular repair mechanisms
• life cycle
DNA Mutation and Repair
• Different genes in a genome can have different mutation rates
• Factors such as the gene/locus size, local conditions in the nucleus,
whether or not the gene/locus is ‘important’ can impact mutation
rates
DNA Mutation and Repair
• Gene structure
• A typical prokaryotic gene
operator
promoter
coding region
regulatory sequences
• A typical eukaryotic gene
coding region
other regulatory
sequences
5’ UTR
intron 1 intron 2
intron 3
3’ UTR
intron 4
//
promoter exon 1
exon 2
exon 3
exon 4
exon 5
DNA Mutation and Repair
Different regions of a genome have different mutation rates
DNA Mutation and Repair
…and within genes themselves.
DNA Mutation and Repair
• Mutations
• Point mutations
• Transitions vs. transversions
• Would you expect more transitions or
transversions by chance?
• Transition bias
• Can be permanent if not repaired immediately
A
G
C
T
DNA Mutation and Repair
• Mutations
• Indels – Insertions/deletions
• via
• Chromosomal changes
• Transposable element activity
• Replication ‘slippage’
DNA Mutation and Repair
• The impact of point mutations
• Varied impacts depending on what
changes and where the changes
occur
Silent mutation: a base-pair change
that does not alter the resulting amino
acid due to the redundancy of the
genetic code
Missense mutation: a base-pair
change that results in an amino acid
change in the protein
Nonsense mutation: a base-pair
change that creates a stop codon in
place of a codon specifying an amino
acid
DNA Mutation and Repair
• The impact of point mutations
• Varied impacts depending on what
changes and where the changes
occur
Silent mutation: a base-pair change
that does not alter the resulting amino
acid due to the redundancy of the
genetic code
Missense mutation: a base-pair
change that results in an amino acid
change in the protein
Nonsense mutation: a base-pair
change that creates a stop codon in
place of a codon specifying an amino
acid
DNA Mutation and Repair
• The impact of point mutations
• Varied impacts depending on what
changes and where the changes
occur
DNA Mutation and Repair
• The impact of indel mutations
• Varied impacts depending on what
changes and where the changes
occur
• Inserting or deleting bases, even on
a small scale can result in
frameshift mutations
• Change the reading frame of the
mRNA
DNA Mutation and Repair
• The impact of mutations
• Both point and indel mutations can alter the amount of protein product
from a gene
• Regulatory mutations
• Occur in:
• Promoters
• 5’ UTRs
• 3’ UTRs
• Interfere with the regulation of transcription and/or translation
DNA Mutation and Repair
• The impact of mutations
• Remember those introns?
• Both point and indel mutations can alter whether or not introns are
spliced out correctly
• Knock out splice sites or...
5’ UTR
intron 1 intron 2
promoter exon 1
exon 2
intron 3
exon 3
3’ UTR
intron 4
exon 4
exon 5
DNA Mutation and Repair
• The impact of mutations
• Remember those introns?
• Both point and indel mutations can alter whether or not introns are
spliced out correctly
• …introduce new splice sites
5’ UTR
intron 1 intron 2
promoter exon 1
exon 2
intron 3
exon 3
3’ UTR
intron 4
exon 4
exon 5
DNA Mutation and Repair
• Mutations
• Sources of mutation
• Radiation
• Chemical modification
• Endogenous/spontaneous mutations
• DNA replication and repair errors
• ~1017 replications in normal human lifespan
• Each cell division = copy 6 x 109 bases
• Average replication error rate = ~10-10/nucleotide
• Any given gene may be the site of ~109 mutations when
considering all cells and cell divisions
• Human heterozygosity - measure of allelic difference within
an individual
• ~0.08% (~1/1250 bp)
DNA Mutation and Repair
• Spontaneous mutations
• DNA polymerase has a proofreading activity that normally keeps
mutation rates low but accidents happen and
• Some types of mutation are invisible to the polymerase
• Strand slippage is common in repetitive regions of the genome
• DNA forms a temporary hairpin
• DNA slips along it’s length but no mismatch exists
• Replication proceed but there is an increase or decrease in
repetitive units
DNA Mutation and Repair
• Spontaneous mutations
• Strand slippage
DNA Mutation and Repair
• Spontaneous mutations
• Strand slippage can result in trinucleotide repeat disorders
• Increases beyond a certain length cause malformed proteins
DNA Mutation and Repair
• Spontaneous mutations
• Strand slippage
– Repeat expansion and human
pathogenicity
• Cystatin B gene and epilepsy
– protects against the proteases leaking
from lysosomes
– “the majority of [eplilipsy associated]
alleles contain expansions of a 12-mer
repeat located about 70 nucleotides
upstream of the transcription start site
… of the CSTB gene. Normal alleles
contain 2 or 3 copies of this repeat
whereas mutant alleles contain more
than 60 such repeats.” (Lalioti et al.
1997)
Note instability from one
generation to the next
• Sorry – 206 ESB
• Sorry – Homework has been re-opened
• REEF questions
DNA Mutation and Repair
• Spontaneous mutations
• Hydrolytic depurination
DNA Mutation and Repair
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Spontaneous mutations
Hydrolytic deamination of cytosine
Loss of an amino (NH2) group from a nucleotide
When cytosine is deaminated, the amino is most often replaced
with oxygen atom, making it uracil
• There is a repair system in place to remove the U and replace with
aC
DNA Mutation and Repair
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Spontaneous mutations
Hydrolytic deamination of 5-methylcytosine
There are many chemically modified cytosines in many genomes
5-methylcytosine
When 5-methylcytosine is deaminated, it becomes a thymine
DNA Mutation and Repair
• Spontaneous mutations
• Hydrolytic deamination of 5-methylcytosine
• Three scenarios can play out
• 1. repair system changes T back to a C  true repair
• 2. repair system changes the G to an A  mutation
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1
DNA Mutation and Repair
• Spontaneous mutations
• Hydrolytic deamination of 5-methylcytosine
• Three scenarios can play out
• 1. repair system changes T back to a C  true repair
• 2. repair system changes the G to an A  mutation
• 3. no repair occurs and a second allele is formed during
replication
3
DNA Mutation and Repair
• Remember this?
• Transitions vs. transversions
• Would you expect more transitions or
transversions by chance?
• Transition bias
A
G
C
T
m
5’….CG……
3’….GC……
5’….TG……
3’….GC……
5’….TG……
3’….AC……
DNA Mutation and Repair
• Induced mutations
• Radiation, chemicals and other exogenous agents causing
mutations are called mutagens
• The type of mutation depends on the mutagen
• Chemical mutagens are classified by the changes they make:
• 1. nucleotide base analogs
• 2. deaminating agents
• 3. alkylating agents
• 4. oxidizing agents
• 5. hydroxylating agents
• 6. intercalating agents
DNA Mutation and Repair
• Induced mutations
• Base analogs are chemicals that are structurally similar to normal
DNA bases
• Can replace normal nucleotides without being recognized as
incorrect by polymerase
• 5-bromodeoxyuridine
DNA Mutation and Repair
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Induced mutations
Deaminating agents are chemicals that remove amino groups
Already discussed 5-methylcytosine
Adenine deamination with nitrous acid produces hypoxanthine,
which can mispair with cytosine, inducing a TC mutation
DNA Mutation and Repair
• Induced mutations
• Alkylating agents are chemicals that add bulky chemical groups
like methyl (CH3) and ethyl (CH2-CH3) chains to bases
• Ethyl methansulfonate (EMS) is such an alkylating agent
• The addition of ethyl group distorts the helix and leads to incorrect
base pairing
DNA Mutation and Repair
• Induced mutations
• Oxidizing agents either add an oxygen atom or remove a
hydrogen atom
• Bleach or hydrogen peroxide
• Oxidized guanine mispairs with adenine  transversion mutation
DNA Mutation and Repair
• Induced mutations
• Intercalating agents insert themselves between the rungs of the
DNA ladder, distorting the helix and cause stress, leading to breaks
that are not efficiently repaired  frameshift mutations
DNA Mutation and Repair
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DNA damage
Radiation
Photochemical fusion of pyrimidines
DNA repair systems exist in most
organisms to repair the problem
• If not repaired dimers stall DNA and
RNA polymerases, producing
replication gaps
• Gaps are eventually filled by an error
prone process known as translesion
synthesis
• The polymerases involved in
translesion synthesis lack proofreading
activity and are error-prone
DNA Mutation and Repair
• DNA damage
• Radiation
• Higher energy radiation (X-rays, etc.) can break the DNA backbone
resulting in single- or double-stranded breaks
• These will interfere with replication/transcription and must be
repaired
DNA Mutation and Repair
• Multiple distinct repair systems exist in prokaryotes and
eukaryotes
• The first line of defense is DNA polymerase proofreading
• Works by ensuring the proper geometry of base pairs
DNA Mutation and Repair
• If proofreading fails, mechanisms exist to identify and repair the
mismatch
• Mismatch repair
• Works on mismatched bases
• Two challenges
• Identify mismatches
• Identify the strand with the mistake (the new strand)
• E. coli players:
• MutS – dimer; scans DNA, recognizes mismatch
• MutL – recruited by MutS, recruits MutH
• MutH – nicks DNA upstream of lesion
• Exonucleases and helicase
• DNA pol III
• Ligase
DNA Mutation and Repair
• Mismatch repair
• Works on mismatched bases to increase
fidelity of DNA synthesis
• E. coli players:
• MutS – dimer; scans DNA, recognizes
mismatch
• MutL – recruited by MutS, recruits MutH
• MutH – nicks DNA upstream of lesion
• Exonucleases and helicase
• DNA pol III
• Ligase
DNA Mutation and Repair
• Mismatch repair
• Works on mismatched bases to increase
fidelity of DNA synthesis
• E. coli players:
• MutS – dimer; scans DNA, recognizes
mismatch
• MutL – recruited by MutS, recruits MutH
• MutH – nicks DNA upstream of lesion
• Exonucleases and helicase
• DNA pol III
• Ligase
DNA Mutation and Repair
• Mismatch repair
• Works on mismatched bases to
increase fidelity of DNA synthesis
• Two challenges
• Identify mismatches
• Identify the strand with the
mistake (the new strand)
• Dam methylase - methylates A in
‘GATC’ on both strands (note error in
figure)
• Frequency of GATC ~ 1/256 bp
(1/44)
• Replication results in transient
(a few minutes)
hemimethylation
DNA Mutation and Repair
• Mismatch repair
• Works on mismatched bases to increase fidelity of DNA synthesis
• Two challenges
• Identify mismatches
• Identify the strand with the mistake (the new strand)
• Dam methylase - methylates A in ‘GATC’ on both strands
• Frequency of GATC ~ 1/256 bp (1/44)
• Replication results in transient (a few minutes) hemimethylation
• http://highered.mheducation.com/sites/9834092339/student_view0/
chapter14/methyl-directed_mismatch_repair.html
DNA Mutation and Repair
• Mismatch repair
• Eukaryotes utilize homologs to
each prokaryotic protein
• MSH proteins – MutS homologs
• MLH proteins – MutL homologs
• No MutH homolog – recognition
via nicks in lagging strand
DNA Mutation and Repair
• Human DNA repair
deficiencies
– Hereditary nonpolyposis colon
cancer (HNPCC)
• Deficient mismatch repair
leads to instability
• “The majority (70%) of
HNPCC patients have a
germline mutation in either
hMSH2 (human mutS
homolog-2) or hMLH
(human mutL homolog),
giving a lifetime risk of about
80% for colorectal cancer.”
Wheeler et al. 2000
• REEF
DNA Mutation and Repair
• Excision repair mechanisms
• common
• Base excision repair
• Used to replace chemically modified bases
• Four step process
• Glycosylase – removes altered base (not the entire nucleotide)
• Endonuclease – removes the remainder of the nucleotide
• Polymerization – fills empty spot
• Ligation – seals the nick in the DNA backbone
DNA Mutation and Repair
DNA Mutation and Repair
• Nucleotide excision repair
• Recognizes general distortions
caused by bulky chemical adducts
• In E. coli – UvrA, B, C, & D
• UvrA/B – scan for lesions
• UvrB – melts dsDNA
• UvrC – nicks DNA
• UvrD – helicase
• DNA pol I and ligase
• In eukaryotes – 25+ enzymes
• Seven XP genes (A, C, D, G etc.)
DNA Mutation and Repair
• Nucleotide excision repair
• Recognizes general distortions
caused by bulky chemical adducts
• In E. coli – UvrA, B, C, & D
• UvrA/B – scan for lesions
• UvrB – melts dsDNA
• UvrC – nicks DNA
• UvrD – helicase
• DNA pol I and ligase
• In eukaryotes – 25+ enzymes
• Seven XP genes (A, C, D, G etc.)
• http://highered.mheducation.com/si
tes/9834092339/student_view0/ch
apter14/nucleotide_excision_repair
.html
DNA Mutation and Repair
• Repair Mechanisms
• Nucleotide excision repair
• Trascription-coupled repair
• Prioritizes the ‘most important’
genes
• RNA pol stalled at lesions
• Initiates NER pathway because
XPA and XPD are part of TFIIH
complex
DNA Mutation and Repair
• Human DNA repair
deficiencies
– Xeroderma pigmentosa (XP)
• 7 distinct types, all caused by
deficient NER system
• Extreme sensitivity to sunlight,
high incidence of skin cancer
• DNA repair enzyme containing
creams help
DNA Mutation and Repair
• 2015 Nobel Prize in
Chemistry
Nucleotide excision repair
Mismatch repair
Base excision repair
DNA Mutation and Repair
• Repair Mechanisms
• Direct Reversal
• Rare
• Demethylation
• O6-methylguanine
• Methyltransferase
• Photoreactivation
• Thymine dimers
• photolyase
DNA Mutation and Repair
• What if repairs are not made before replication?
• All of these damage scenarios have the potential to lead to
mutation if not fixed
• Some of them can prevent replication from occurring so
mechanisms have evolved to allow replication in spite of the
problems
• Most of these mechanisms are ‘last resort’ type processes and
are very error prone
• The idea is to just get the cell through replication because if
replication stalls for too long, the cell will die
• Typically are used when there is widespread DNA damage
• The SOS repair system
DNA Mutation and Repair
• The SOS repair system
• Typically utilizes translesion
polymerases
• Translesion polymerases usually
not expressed in bacteria but are
expressed under conditions of
widespread damage
• Repressed by LexA protein
• DNA damage (ssDNA & ATP)
induces expression of RecA
• RecA modifies LexA
• LexA autocatalytic cleavage
• [RecA] increases  target genes
activated
• DNA damage repaired  [RecA]
decreases  LexA represses
genes
DNA Mutation and Repair
• Double-strand breaks are repaired
via two mechanisms
• Nonhomologous end-joining (NHEJ)
• Mutagenic in itself b/c nucleotides
are lost or gained via the repair
• Three step repair
• 1. Break is recognized by PKcs,
Ku70 and Ku80, which bind to
broken ends
• 2. The protein complex trims the
ends of the breaks
• 3. The blunted ends are ligated
DNA Mutation and Repair
• Double-strand breaks
• Nonhomologous end-joining (NHEJ)
• Mutagenic in itself b/c nucleotides
are lost or gained via the repair
• Three step repair
• 1. Break is recognized by PKcs,
Ku70 and Ku80, which bind to
broken ends
• 2. The protein complex trims the
ends of the breaks
• 3. The blunted ends are ligated
• Note the loss of information
• https://www.youtube.com/watch?v=31s
tiofJjYw
DNA Mutation and Repair
• Double-strand breaks are repaired via
two mechanisms
• Synthesis dependent strand
annealing
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Uses the information in the
homologous chromosome to direct the
repair
1. broken ends are trimmed and
coated by Rad51
2. Rad51 initiates a process known as
strand invasion, where the
homologous chromatid pairs with the
broken one
3. The sister chromatid directs
synthesis across the break
4. Strands dissociated and the ends
are ligated
• Note the potential loss of alleles due to
homogenization of chromomsomes
• https://www.youtube.com/watch?v=86J
CMM5kb2A
DNA Mutation and Repair
• Controlled double-strand breaks
• In many organisms homologous
recombination is a mechanism to
induce the mixing of genes to
increase potentially beneficial
diversity in the genome
• Occurs during meiosis in
eukaryotes
• Had planned to cover mechanisms
of recombination but time doesn’t
permit
DNA Mutation and Repair
• A higher mutation rate in males vs. females?
– JBS Haldane 1947
DNA Mutation and Repair
• Are mutations good or bad?
• Not all mutations are bad. Some can be
beneficial
• CCR5 Δ32
• Chemokine receptor 5 – a receptor molecule on T-cells
that allows HIV (and other pathogens) to enter cells
• 32-bp deletion that inactivates the receptor
• Homozygotes are immune to HIV, heterozygotes exhibit
resistance
• Likely evolved in northeast Europe and has spread via
long-range dispersal and strong selection in European
populations
DNA Mutation and Repair
• CCR5 Δ32
• The Viking hypothesis – a single
origin ~700-2000 years ago
• Increased prevalence associated
with the Black Death (1348-1350)
• The short duration of the Black
Death is probably not responsible
for continued positive selection
• Smallpox? (Galvani and Novembre
2005)
DNA Mutation and Repair
• Are mutations good or bad?
• Gene duplication and indels as adaptive
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The nylon-eating bacterium (http://www.nmsr.org/nylon.htm)
Nylon was invented in 1935 – no advantage for any organism to digest it before then
1974 – Bacterium discovered that digests nylon (Flavobacterium)
Examination of the Flavobacterium revealed two mutations compared to other
species
• 1. gene duplication - provides something for evolution to play with
• 2. frameshift mutation in duplicated gene – provides novel amino acid sequence
• Ohno 1984 – Proceedings of the National Academy of Sciences 81 2421-2425
– Both of these are simple and common occurrences
– 2006 study identified 470 such events in the human genome (108 in mouse)
• Okamura et al. 2006 – Genomics 88 (6) 690-697
DNA Mutation and Repair
• Myth: Mutations cannot be good or add information to a genome
• http://www.newscientist.com/article/dn13673-evolution-mythsmutations-can-only-destroy-information.html