david ray 806-834-1677, esb 216 office hours m 1-3 pm class website // homework

• 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

• 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.

Yes, another one

http://www.davidraylab.com/genetics/

• 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 Assumptionsmy section of

>

• 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)

Objectives and Assumptions

• 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


• 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


intron 2

DNA Mutation and Repair• Gene structure• A typical prokaryotic gene

• A typical eukaryotic gene

coding regionoperatorpromoter

regulatory sequences

coding region

5’ UTR

promoter exon 1

3’ UTRintron 1 intron 3 intron 4

exon 2 exon 3 exon 4 exon 5

//

other regulatory sequences

DNA Mutation and RepairDifferent regions of a genome have different mutation rates

…and within genes themselves.


• 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

• Mutations• Indels – Insertions/deletions• via

• Chromosomal changes• Transposable element activity• Replication ‘slippage’


• 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


• The impact of point mutations• Varied impacts depending on what



• The impact of indel mutations• Varied impacts depending on what


• Inserting or deleting bases, even on a small scale can result in frameshift mutations• Change the reading frame of the

mRNA


• 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


• 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...


intron 25’ UTR

promoter exon 1



• 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


intron 25’ UTR

promoter exon 1



• 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)


• 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


• Spontaneous mutations• Strand slippage


• Spontaneous mutations• Strand slippage can result in trinucleotide repeat disorders• Increases beyond a certain length cause malformed proteins


• 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

• Spontaneous mutations• Hydrolytic depurination


• 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

a C


• 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


• 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


1

2

• 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

• Remember this?• Transitions vs. transversions

• Would you expect more transitions or transversions by chance?

• Transition bias


A G

C T

5’….CG……3’….GC……

m

5’….TG……3’….GC……

5’….TG……3’….AC……

• 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


• 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


• 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 TC mutation


• 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


• 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


• 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 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 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


• 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


• 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


• 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


• 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


• 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


• 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


• 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


• 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


• 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.)


• 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/

sites/9834092339/student_view0/chapter14/nucleotide_excision_repair.html


• 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


• 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


• 2015 Nobel Prize in Chemistry


Mismatch repairNucleotide excision repair

Base excision repair

• Repair Mechanisms• Direct Reversal

• Rare• Demethylation

• O6-methylguanine• Methyltransferase

• Photoreactivation• Thymine dimers• photolyase


• 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


• 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


• 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


• 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=31stiofJjYw


• Double-strand breaks are repaired via two mechanisms

• Synthesis dependent strand annealing• 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=86JCMM5kb2A


• 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


• A higher mutation rate in males vs. females?– JBS Haldane 1947


• 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


• 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)


• Are mutations good or bad?• Gene duplication and indels as adaptive

– 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


• Myth: Mutations cannot be good or add information to a genome• http://www.newscientist.com/article/dn13673-evolution-myths-

mutations-can-only-destroy-information.html


david ray 806-834-1677, esb 216 office hours m 1-3 pm class website // homework

Documents

mutation ratesdna mutation

different mutation ratesand

various sourcesdna mutation

working knowledge of

basic differences

dna sequence

chromosome structure

repairdifferent genes