GENETICS 380

:
DNA Mutation and Repair

Mutations Are Heritable Changes
In The DNA Sequence
The most common (often called normal) sequence is the wild-type
sequence; all changes in the DNA base sequence are referred to as
mutations for the purposes of this unit
Mutations can be helpful, neutral or detrimental to the organism’s effort
to thrive in its environment, depending on the demands the
environment makes on the organism
Mutations are necessary for evolution
Mutations are one of the mechanisms whereby organisms acquire new
characteristics

Forward, Reverse And Suppressor Mutations
Reverse mutation, aka reversion, replaces the original (forward)
mutation and restores the wild-type genotype and phenotype
Suppressor mutation is at another site, but it compensates for the
mutation and restore the wildtype phenotype

Suppressor Mutations
Suppressor mutations can hide or suppress the effects of other mutations
The individual is a double mutant, but has a normal phenotype
Some suppressor mutations are intragenic—within the same gene’s
coding sequence

Suppressor Mutations Some suppressor mutations are intergenic— in a second gene’s coding sequence Ex. C  G nonsense mutation creates a UAG STOP codon where there was a UAC tyrosine codon .

Suppressor Mutations A mutation changes the anticodon of a tyrosinecarrying tRNA to AUC. which can bind to the STOP codon UAG and cause the ribosome to put tyrosine into the polypeptide Problem—The AUC tRNA is supposed to bring in the releasing factor that stops translation at UAG codons This will cause a failure to stop translation in other genes with the same STOP codon .

therefore present in every cell the individual has Somatic mutations—Arise after fertilization. therefore only present in a subset of the individual’s cells . during cell replication/division/differentiation/migration.Germline Versus Somatic Mutations Germline mutations—Present in either (or both) the sperm or the egg that made the individual.

Somatic Mutations Get Passed Down To One Fourth Of The Descendants Of The Original Cell One chromosome 2 DNA strands After mitosis. one daughter cell has the mutation on one strand Mutation arises during DNA replication in S phase After next S phase All the descendants of this cell have the mutation After mitosis .

this is a germline mutation—it was present in one of the gametes that made the child. it is a somatic mutation—it occurred after fertilization and only existed in a subset of the parent’s cells . and it will exist in every cell the child has From the parent’s perspective.Somatic Mutations Can Be Passed Down To Offspring If the mutation exists in the cells that make sperm (spermatogonia) or eggs (oogonia). the mutation can be packaged into a gamete From the offspring’s perspective.

Some Mutations Are Conditional Conditional mutation—Some mutations only cause consequences under certain conditions Ex. Many people who have a glucose-6-phosphate dehydrogenase deficiency (an X-linked recessive disease) will experience “flare-ups” (main symptom = hemolytic anemia) if they eat fava beans .

Some Mutations Involve Large Pieces Of One Or More Chromosomes Structural Abnormalities Deletions and duplications range in size from single nucleotides to pieces of the chromosome that are large enough to include many genes in them Inversions and translocations that rearrange large pieces of chromosomes Numerical Abnormalities Entire chromosomes can be added or deleted More on this when we discuss cytogenetics .

The Smallest Mutations Are Single-Nucleotide Point Mutations Point mutation = substitution. and truncates the protein . deletion or addition of a single nucleotide Transition = purine-purine substitution or pyrimidine-pyrimidine substitution Transversion = purine-pyrimidine substitution or vice versa Missense mutation—single-nucleotide substitution causes one amino acid to replace another Nonsense mutation—single-nucleotide substitution creates a STOP codon at the site of the mutation.

but has no functional consequences .Point Mutations Have Variable Effects On The Organism’s Phenotype Not all mutations change the activity of a protein--Those that do not are often called benign polymorphisms Some mutations are silent mutations—they do not change the organism’s phenotype Synonymous mutation—does not change the amino acid content of the protein Neutral mutation—changes the amino acid content of the protein.

by a small or large amount If the amino acid substitution. deletion or addition changes the protein’s 3D shape or ability to move as it works. it will disrupt the protein’s function The degree to which it alters the protein’s ability to bend and move determines how severely it affects protein activity .Point Mutations Have Variable Effects On The Organism’s Phenotype Those that alter protein activity may increase or decrease it.

histidine and lysine are positively charged Glutamic acid and aspartic acid are negatively charged Some of the neutral ones are polar.Amino Acids Differ In Size. some are nonpolar . Polarity. Bendability And Electrical Charge Arginine.

A Protein’s 3D Shape Is Determined By Interactions Among Amino Acids .

Some Amino Acid Substitutions Will Disrupt The Protein's Function. Others Will Not Alanine replaces glycine will probably be tolerated Arginine replaces glycine will probably not be tolerated Small uncharged Small uncharged Small uncharged Large positive charge .

these are called synonymous mutations .Some Mutations Are Synonymous The genetic code is partially redundant (often referred to as degenerate) Some base substitutions do not change the amino acid content of the protein.

a C T mutation changes a GC that lies in an exon to a GT).Synonymous And Intronic Mutations May Change The Pattern Of RNA Splicing Synonymous mutations may change the splicing of the mRNA (intronic mutations may also) Recall that almost all human introns begin with GT and end with AG If a mutation creates a new splice site (ex. the spliceosome will splice the pre-mRNA abnormally This will either add intronic nucleotides to the mRNA or delete exonic nucleotides from the mRNA Either way. this changes the instructions the ribosome gets. and it will incorporate the wrong amino acids into the polypeptide—this almost always abolishes the activity of the protein .

Deletion of a codon results in loss of one amino acid Normal sequence TAC TGG CTA GCT CCU tyr trp leu ala pro 3 bp deletion TAC TGG GCT CCU tyr trp ala pro The amino acid sequence is normal before and after the deletion .Insertions And Deletions May Shift The Ribosome’s Reading Frame The ribosome reads the mRNA in 3-base codons An insertion or deletion in the mRNA involving a multiple of three nucleotides will not shift the ribosome’s reading frame—the amino acid sequence will be normal before and after the mutation Ex.

until it hits a STOP codon (usually fairly soon) Normal sequence TAC TGG CAT TCT CCT tyr trp his ser pro 1 bp deletion TAC TGG ATT CTC CT tyr trp ile leu leu All the amino acids after the deletion are abnormal .Insertions And Deletions May Shift The Ribosome’s Reading Frame If the insertion or deletion does not involve a multiple of three bases. the ribosome’s reading frame gets shifted The ribosome will incorporate the wrong amino acids.

i.Mutations In The Regulatory Sequences Affect The Rate Of Protein Production Coding sequence mutations can change the activity of each molecule of the protein Mutations in the promoter and other regulatory sequences affect the rate at which the gene makes its protein. the number of protein molecules in the body .e.

but sometimes the repeat can contract Whether it expands or contracts often depends on the parent of origin— more likely to contract during spermatogenesis than oogenesis . the more it disrupts gene function Expansions are most common. with different people often having a different number of repeated units in the string Trinucleotide repeats are found in both coding regions and regulatory sequences These trinucleotide repeats can expand during meiosis The larger the repeated string gets.Trinucleotide Repeats Can Expand And Disrupt A Gene’s Activity There are some places in the genome where three bases get repeated (trinucleotide repeats).

Trinucleotide Repeats Can Cause Strand Slippage In this case. both strands end up having the expanded repeat . the newly synthesized strand forms a hairpin loop This causes DNA polymerase to replicate that portion of the template strand again When the newly synthesized strand is used as a template itself.

Fragile X Syndrome Is Due To An Expanding CGG Repeat In The X Chromosome (Xq28) Chromosomes from cells that are grown in culture will show fragile sites. where they look stretched out as if the chromosome was about to break There are common fragile sites all over the genome. Fragile X mental retardation syndrome—fragile site depicted here . in everyone’s chromosomes—these are not associated with disease There are also rare fragile sites that are associated with diseases—ex.

which silences the gene . the CGG repeat and the FMR1 gene promoter get methylated.Fragile X Syndrome Is Due To An Expanding CGG Repeat In The X Chromosome (Xq28) The Fragile X Syndrome CGG repeat is located in the 5' untranslated region of the FMR1 gene Unaffected individuals have 5-44 CGGs in the string 45-54 repeats is a grey area—sometimes these can expand Those with 55+ CGGs often see the repeat expand during meiosis When there are greater than 200 repeats.

the repeat will be big enough to affect the children . but will pass on an expanded repeat to some of their children.Fragile X Syndrome Is Due To An Expanding CGG Repeat In The X Chromosome (Xq28) The FraX trinucleotide repeat can also contract during meiosis The repeat is more likely to contract during Dad’s spermatogenesis than Mom’s oogenesis There are asymptomatic male carriers who pass a repeat down to their daughters that is smaller than the repeat they have in their genome The daughters may not be affected.

Fragile X Syndrome .

subsequent generations are affected earlier and more severely than previous generations At the clinical level.Trinucleotide Repeat Expansion Causes Anticipation At The Clinical Level The larger a repeat gets. this is known as genetic anticipation . the more severely it disrupts gene activity When the repeat expands through meiosis.

Anticipation In Three Generations With Myotonic Dystrophy .

Transposable Elements Can Affect Gene Activity Transposable elements (aka transposons) can move from one place to another within the same cell’s DNA They can “cut and paste. or they can make a copy of themselves and “copy and paste” (replicative transposition) themselves into another location Approx. 45% of the human genome has come from transposons .” (nonreplicative transposition) and leave their original place to insert elsewhere.

All Organisms’ Genomes Contain Transposable Elements Most transposable elements have inverted terminal repeats at their ends (shown in green in the figure below) ex 5’-ACAGTTCAG…CTGAACTGT-3’ 3’-TGTCAAGTC…GACTTGACA-5’ This enables them to form loops that help them break out .

Inverted Repeats Help Transposable Elements Break Out Of Their Places .

The Enzyme Transposase Makes Staggered Cuts In The DNA This creates short single-stranded overhangs that are complementary to each other The transposon inserts between the ends of the cut .

Transposable Elements Generate Direct Flanking Repeats When They Insert The inverted terminal repeat is part of the transposon The direct flanking repeat is not part of the transposon—it is created as a result of the transposition process .

Transposable Elements Generate Direct Flanking Repeats When They Insert If you were searching through a genome for transposons. you would see the terminal inverted repeat flanked by the direct repeat .

Retrotransposons Use Reverse Transcription

Transposable Elements Can Disrupt Genes

Transposable
Elements Can
Disrupt Genes
In this case, the transposable
element is inserted in the gene’s
regulatory sequences
This shuts the gene down,
causing the grapes to stop
producing the anthocyanin
pigments that make them look
black, making white grapes
Later, a portion of the
transposable element leaves,
partially restoring gene function
and allowing the grapes to
produce enough anthocyanins to
look red

Transposable Elements Can Jump Back
Out And Restore Gene Function
In corn, there is a transposable element that contains an Ac element and
a Ds element
The Ac element produces transposase, while the Ds element does not
By producing transposase, the Ac element can cause the Ds element to
transpose into the pigment-making gene’s sequence, disrupting pigment
formation and causing the kernels to be yellow

Transposable Elements Can Jump Back Out And Restore Gene Function .

as that cell continues to undergo mitosis in the developing kernel. thereby restoring the function of the pigment gene and allowing the cell to produce purple pigment again This happens one cell at a time.Transposable Elements Can Jump Back Out And Restore Gene Function The Ds element can transpose back out again. when there were still many rounds of cell replication and division to go before the kernel was completely developed Smaller speckles represent cells that have descended from a cell in which the Ds element transposed out of the pigment gene at a later time . this causes purple speckles in the kernel The larger speckles represent a group of cells that have descended from a cell in which the Ds element transposed out of the pigment gene early in the development of the kernel. the cells it gives rise to also produce purple pigment.

Transposable Elements Can Jump Back Out And Restore Gene Function .

Transposable Elements Can Also Activate Genes Inactive gene PRO PRO Transposable element has sequence that is capable of acting as a promoter Activated gene RNA .

Transposable Elements Can Cause Chromosome Rearrangements That Disrupt The Activity Of Genes .

Transposable Elements Can Cause Chromosome Rearrangements That Disrupt The Activity Of Genes .

Transposable Elements Can Cause Chromosome Rearrangements .

the cell uses interfering RNAs to interrupt transposase production .Regulating The Activity Of Transposons The transposable elements produces the enzyme transposase. the cell methylates the DNA in the region of the transposon. which is what enables transposition In many cases. which inhibits production of transposase In some cases.

Spontaneous And Induced Mutations Spontaneous Mutations Errors during DNA replication Hydrolytic and other reactions can cause base changes in DNA after replication Induced Mutations Environmental agents such as UV light or radon Chemical exposures Metabolic byproducts such as the superoxide ion O2Agents that induce mutations are called mutagens .

A Gene’s Mutation Rate Is Influenced By Factors That Vary By Gene And Species Spontaneous DNA mutation rate (varies from gene to gene) Exposure to dietary. environmental and lifestyle factors Capacity to repair mutations--DNA polymerase has 3’-5’ exonuclease activity so it can remove an incorrect base Some mutations convey advantages in some environments Stressful environment may increase mutation rate .

Bases Exist In Protonated And Nonprotonated Forms Shifting the proton changes the number and position of hydrogen bonds the base makes This causes the base to pair with a different base than it usually pairs with .

Incorporation Errors Arise During DNA Replication The ability to pair with an atypical base because of shifts in the arrangement of hydrogen bonds is sometimes referred to as “wobble” or “non Watson-Crick basepairing” .

the strands with the mismatched bases serve as templates for DNA replication Replication proceeds properly. resulting in one of the two new chromatids having the wrong base on both strands of its DNA .Incorporation Errors Lead To Replication Errors During the next cell cycle.

Depurination: Hydrolysis Removes The Purine Ring From Adenines And Guanines The N-glycosidic bond that holds the base to the sugar is cleaved—the base is removed but the sugar/phosphate remains Usually corrected quickly. because the base that belongs there is either a T or a C . but sometimes an A gets incorporated across from the depurinated site during the next round of replication—this results in a mutation.

U and T) can be deaminated by hydrolysis of the NH 2 group. they get deaminated to yield T’s Unmethylated C’s get deaminated to yield U’s . the reaction doesn’t require an enzyme Some C’s exist in methylated form.Deamination Converts Between Pyrimidines Pyrimidines (C.

Deamination Followed By Replication A T G C T A C G 1ST round A T replication A T T A G C U G Deamination converts C to U T A G C U A + A T G C T A C G 2nd round replication A T G U T A C A + A T G T T A C A .

Many Chemical Mutagens Change The Number Of Hydrogen Bonds The Base Makes .

5-Bromouracil Replaces A Thymine But Bonds With A Guanine .

superoxide ion O2. superoxide ion O2-) oxidize guanine to 8oxoguanine.Reactive Oxygen Species Also Change Base Pairing Reactive oxygen species (ex. which pairs with A. rather than C.) are created by normal metabolism . as G should Reactive oxygen species (ex.

ethidium bromide . acridine orange. ex. confusing the DNA polymerase and causing insertions and deletions in the DNA Includes some common DNAvisualizing agents.Intercalating Agents Insert Themselves Between Nucleotides Intercalating agents increase the distance between neighboring basepairs.

Ultraviolet (UV) And Ionizing Radiation Break The DNA Strands Both UV radiation (from the sun) and ionizing radiation (from X rays or radon in homes) cause your cells to form highly reactive free radicals These free radicals can cause breaks in the DNA The attempt to fix the damage may result in mutations arising .

this will lead to mutation after replication . causing the cell to go into apoptosis This is why UV light kills bacteria and is a good water sterilizer Eukaryotes have a special DNA polymerase eta that puts AA across from the pyrimidine dimer and restores the DNA If the dimer is not a T-T dimer.The Sun’s UV Rays Cause Pyrimidine Dimers Bonds form between neighboring Cs or Ts in the same DNA strand This brings the two neighboring nucleotides closer together. which prevents the cell from going through the cell cycle. leaving the bases unable to bond with the bases from the other DNA strand ACTTGC | | | | | | T G AAC G UV rays A C T= T G C | | | | T GA AC G This prevents DNA replication.

.

000 nucleotides (104 – 105) After the repair systems fix what they can. which are recognized by the repair systems At the bubble. the cell must recognize which of the two strands is the one that needs to be taken out After replication. while the template DNA is methylated . but there’s a delay. 1 in 1 billion (109) nucleotides Mismatched bases and loops such as those that lead to deletions and duplications form bubbles in the DNA double helix.Base Mismatch Repair DNA polymerase III proofreads itself. 10. but still makes an error every approx. the frequency of errant nucleotides is only approx. the newly synthesized DNA is unmethylated. so immediately after replication.000-100. the A’s in GATC’s get methylated.

Methylation Differentiates The Old DNA Strand From The New One .

pyrimidine dimers. photolyase uses light energy to break the bonds in pyrimidine dimers. can be repaired directly by chemical reactions that restore the original base without removing anything Ex.Direct Repair Some mutations. the enzyme 06-methylguanine DNA methyltransferase removes the methyl group . ex. which enables the bases to make their proper bonds with their complementary bases again If guanine gets methylated to O6-methyl guanine.

Deaminations Are Repaired By Base Excision .

Deaminations Are Repaired By Base Excision .

Deaminations Are Repaired By Base Excision .

Deaminations Are Repaired By Base Excision .

10 new mutations uncorrected per day Sometimes the error leaves DNA ligase unable to seal the gap The AP endonuclease will return and excise the nucleotide. allowing DNA polymerase beta another chance to put the right nucleotide in . DNA polymerase beta.Base Excision Repair Is Error-Prone In eukaryotes. which has no proofreading ability. fills in the gap DNA polymerase beta’s error rate is high enough to leave approx.

Nucleotide Excision Repair Sometimes the cell removes a short string of nucleotides Same steps as base excision: 1. Seal with DNA ligase endonuclease . Replace with proper nucleotide(s) 4. Remove bad nucleotide(s) 3. Recognize problem 2.

then resumes after a short time on the other side of the gap .DNA Damage Bypass Replication stalls briefly at the gap.

DNA Damage Bypass The corresponding portion of the other DNA strand is excised and inserted into the gap This fixes the original gap but leaves a new gap DNA polymerase fills in the gap that was created by the excision The newly synthesized strand is available as a template .

there are three ways in which it can be resolved: Nonhomologous end joining—The ends simply join back together This results in there being a deletion in the chromosome There are two specific mechanisms for double-strand gap repair In both cases.Repairing Double-Stranded Breaks When there is a double-stranded break. the homologous chromosome is used as a template to fill in the gap on one of the broken strands The gap in the other strand may be filled using either the newly repaired strand or the homologous chromosome as the template .

The Newly Synthesized Strand May Be Used To Fill The Gap In The Other Strand The light blue strand uses a red strand as a template to fill its gap The light blue strand then acts as a template to fill the gap in the dark blue strand .

The Homolog May Be Used To Fill The Gap In The Other Strand .

The Homolog May Be Used To Fill The Gap In The Other Strand .

.

9. 36. 16 Application Questions—34. 10. 12. 42 .Good Practice Questions From Chapter 18 Comprehension Questions—1-7. 15.

Answers Not Given By The Textbook Comprehension Questions 1. neutral changes the amino acid sequence but not the activity of the protein . 3. nonsense creates a STOP codon at the site of the mutation. than previous generations. 2. Silent doesn’t change the amino acid sequence of the protein. Missense = one amino acid substituted for another. Transition = purine to purine. as does the effect the repeat has on gene function. The size of the repeat increases from one generation to the next. transversion = purine to pyrimidine or pyrimidine to purine. This causes subsequent generations to be more severely affected. and at younger ages. or pyrimidine to pyrimidine.

hydroxylamine add an OH group to cytosine. nitrous acid deaminates cytososine. a frame-shifting insertion or deletion could be followed by a deletion or insertion (respectively) that restores the reading frame 5. All cause the base to pair with a base other than the one it is supposed to pair with . Strand slippage. then pair during DNA replication with the wrong nucleotide. 7. unequal crossovers due to repeated sequences causing misalignment of chromosomes 6.Answers Not Given By The Textbook Comprehension Questions 4. They get incorporated into the DNA. A second mutation in the codon could restore the original amino acid. Alkylating agents add methyl or ethyl groups.

The Ac element produces transposase. This causes the Ds element to insert itself into the pigment-producing gene. preventing pigment production and causing the kernels to be yellow instead of purple. Replication and division create more pigment-producing cells from those that have lost the Ds element. the Ac element can cause the Ds element to transpose back out of the gene in certain cells. then reverse transcription creates a DNA copy of the sequence. . allowing the gene to resume making pigment in those cells only. Flanking direct repeats around it after it has inserted into a new location. The earlier in the development of the kernel the transposon jumps out. Transcription produces RNA. 12. the more opportunity for replication and division to produce descendants of that cell. Later. 10. leading to larger pigmented spots around that cell. Terminal inverted repeats and the transposase or reverse transcriptase gene as part of the transposon. which then translocates into a new spot in the genome.Answers Not Given By The Textbook Comprehension Questions 9.

DNA polymerase replaces the nucleotides. DNA ligase knits it all together . Base excision repair—glycosylase removes the damaged base. DNA ligase knits it all together Nucleotide excision repair—System recognizes the mismatch. DNA polymerase replaces the nucleotide. nucleases and helicases remove a portion of the new (unmethylated) strand. Mismatch repair—Mismatched bases on the new (unmethylated) strand are removed and replaced by DNA polymerase. Direct repair—Original base and bonds are restored by enzymes such as photolyase or O6-methylguanine DNA methyltransferase. AP endonuclease removes the rest of the nucleotide.Answers Not Given By The Textbook Comprehension Questions 15.

Homologous recombination requires a sister chromatid and a complex sequence of invasion. . Nonhomologous end joining simply joins the broken ends. restoring pigment production.Answers Not Given By The Textbook Comprehension Questions 16. displacement and synthesis of new DNA. The white eyes are due to a transposable element that interrupts the function of the gene that makes the red pigment. Application Questions 34. Red spots represent the cells that have descended from cells that have had the transposon transpose back out.

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