! www.clutchprep.com

!

GENETICS - CLUTCH

CH.17 MUTATION, REPAIR, AND RECOMBINATION

CONCEPT: TYPES OF MUTATIONS

● There are many different types of ______________________________________ □ The first way to classify mutations is to describe how they arise

- Spontaneous mutations are changes that randomly occur

- Induced mutations are changes caused via natural or artificial agents (ex: radiation, chemicals, etc…)

□ A second way to classify mutations is by where the mutation occurs

- Somatic cell mutations occur in somatic cells

- Germ cell mutations occur in germ cells

- Germ cell mutations are passed onto offspring

EXAMPLE:

□ A point mutation is a mutation that affects only a _________________ nucleotide

- Base substitutions changes on base for another

- Transitions replaces the base with one from a similar category (ex: purine to purine)

- Transversions replaces the base from a different category (ex: purine to pyrimidine)

- Base insertions add extra bases, and base deletions remove bases

- An indel mutation is when both an insertion and a deletion occur

- These can easily effect codons

Somatic Cell Germ Cell

Not passed ontooffspring

Passed onto offspring

GENETICS - CLUTCH

CH.17 MUTATION, REPAIR, AND RECOMBINATION

Page 2

EXAMPLE:

□ A third way to classify mutations is their effect on _________________________

- Synonymous (silent) mutations change a codon to another codon that codes for the same amino acid

- Missense mutations change a codon to another codon that codes for a different amino acid

- Conservative changes one codon to another chemically similar amino acid

- Nonconservative changes one codon to another chemically different amino acid

- Nonsense mutations change a codon to a stop codon

- Frameshift mutations alter the codon reading frame

EXAMPLE:

GENETICS - CLUTCH

CH.17 MUTATION, REPAIR, AND RECOMBINATION

Page 3

Base Distortions

● Base distortions disturbe the __________________________ structure of the bases □ Bases can lose part of their structure

- Apurinic sites are regions that have lost their purines

- Depurination is the loss of the purine

- Apyrmidinic sites are regions that have lost their pyrimidines

□ Deamination is the removal of an amino group from a base or molecule

- This process can change cytosine to uracil and adenine to hypoxanthine

□ Oxidative damage occurs when reactive oxygen species affect the ___________________

- O2-, H2O2, and OH target the DNA and chemically alter the bases

EXAMPLE: Deamination of cytosine to uracil

GENETICS - CLUTCH

CH.17 MUTATION, REPAIR, AND RECOMBINATION

Page 4

Mutations and Phenotypes

□ A fourth way to classify mutations is by their effect on the protein’s _________________ or organism phenotype

- Loss of function and gain of function mutants refer to the activity of the mutated gene

- Null mutations describe mutations that result in a complete loss of function

- Visible mutations alter the physical phenotype of the organism

- Nutritional mutations cause loss of an ability to synthesize an amino acid or vitamins

- Behavioral mutations cause changes in behavioral

- Conditional mutations are only detectable under certain conditions

- Ex: temperature sensitive mutants

- Lethal mutations cause death of the organism

- Neutral mutations have no observable affect on the organism

EXAMPLE:

□ A fifth way to classify mutations is by their effect on individual _____________________________

- Hypomorphic mutation is a loss of function mutation that produces a tiny amount of functional protein

- Haploinsufficiency describes when one WT allele doesn’t provide enough gene product

- Other allele has been mutated to null or a loss-of-function

- Dominant-negative describes when a mutant allele blocks the protein production produced by WT allele

- Hypermorphic mutations is a gain of function mutation that produces a more efficient protein than WT

- Neomorephic mutations generate a novel phenotype

+

+

+

m

+

m

Loss of function Gain of functionWild-Type

GENETICS - CLUTCH

CH.17 MUTATION, REPAIR, AND RECOMBINATION

Page 5

EXAMPLE:

□ A sixth way to classify mutations is by their __________________________________- activity

- Suppressor mutations are genetic changes that suppress the effect of another mutation

- Intragenic are mutations found in the same gene as the mutation being suppressed

- Intergenic are mutations found in a separate gene than the mutation being suppressed

100% Activity

WT alleles

0% Activity

30% Activity

160% Activity

Null Allele

Hypomorphic

Hypermorphic

GENETICS - CLUTCH

CH.17 MUTATION, REPAIR, AND RECOMBINATION

Page 6

PRACTICE:

1. A mutation occurred that changed the sequence 5’ AAGCTTGC 3’ to 5’ AAGCTTTGC 3’. What is the name for this type of mutation?

a. Substitution b. Transitions c. Transversion d. Insertion

2. Which of the following mutations changes one codon to a chemically different amino acid? a. Silent mutation b. Conservative missense mutation c. Nonconservative missense mutation d. Nonsense mutations

GENETICS - CLUTCH

CH.17 MUTATION, REPAIR, AND RECOMBINATION

Page 7

3. Which of the following point mutations changes a purine nucleotide to a pyrminidine nucleotide? a. Indel mutation b. Transversion c. Transitions d. Nonsense mutation

4. Changes in the codon reading frame can be caused from all but which of the following? a. Frameshift mutations b. Insertions c. Deletions d. Missense mutations

GENETICS - CLUTCH

CH.17 MUTATION, REPAIR, AND RECOMBINATION

Page 8

CONCEPT: SPONTANEOUS MUTATIONS

● The majority of DNA mutations are __________________________

□ Luria and Delbrünk fluctuation test proved that the majority of mutations are spontaneous

- Tested two bacterial cultures for their ability to develop resistance to a bacteriophage

1. 20 small cultures that were grown in the presence of the phage

2. One large culture that wasn’t grown in the presence of the phage

- A small amount of each culture was plated in the presence of the bacteriophage

- They found that resistance was not dependent on previous exposure to the phage

1. Each culture had a range of 1-107 resistant colonies per plate

2. The large culture had a range of 14-26 resistant colonies per plate

EXAMPLE:

GENETICS - CLUTCH

CH.17 MUTATION, REPAIR, AND RECOMBINATION

Page 9

● There are many different ________________________ that lead to spontaneous mutation

□ Errors in DNA replication can cause mutations

- Including: transitions, transversions, frameshift, indel, etc…

□ Naturally occurring DNA damage

- Including: depurination, deaminiation, oxidatively damaged bases

□ Tautomeric shifts occurs when different tautomers are used

- Tautomers are different forms of the bases which differ by positions of protons in the base

- Tautomers can form base pairings outside of A/T and C/G

EXAMPLE: Uracil tautomers

● Tri-nucleotide repeat diseases results from the addition of many nucleotide ______________________

□ Normal individuals contain a certain number of repeats, while diseased individuals contain many more

Ex: Fragile X: No disease = 6-54 CGG repeats, while diseased = 200-1300 CGG repeats

□ Strand slippage can cause insertions of DNA

- When the DNA is copied the nucleotides form a small loop by binding to repetitive regions

- This allows the DNA polymerase to replicate the looped regions twice

EXAMPLE:

Strand Slippage

GENETICS - CLUTCH

CH.17 MUTATION, REPAIR, AND RECOMBINATION

Page 10

PRACTICE:

1. Which of the following tests determined that most mutations are spontaneous? a. Luria and Delbrünk fluctuation test

b. Ames test c. Two-hybrid test d. Tautomer test

2. True or False: Thymine tautomers can base pair with cytosine. a. True b. False

GENETICS - CLUTCH

CH.17 MUTATION, REPAIR, AND RECOMBINATION

Page 11

3. Which of the following forms of DNA damage is NOT caused spontaneously? a. Errors in DNA replication b. Deamination c. Base Analogs d. Tautomeric shifts

GENETICS - CLUTCH

CH.17 MUTATION, REPAIR, AND RECOMBINATION

Page 12

CONCEPT: INDUCED MUTATIONS

● Mutagenesis is the process of creating mutations in a laboratory by exposing DNA to mutagens (substances that mutate)

□ Base analogs are chemicals that resemble bases and can induce mutations

□ Alkylating agents donate an alkyl group and alters proper base affinities (causes transition mutations)

□ Intercalating agents are chemicals that wedge between DNA bases and cause helix distortions

- Block DNA ______________________ and repair

□ Base damage is damage that prevents base pairings

- UV light can induce pyrimidine dimers

- Ionizing radiation (X-rays, gamma rays) causes DNA damage by creating free radicals

EXAMPLE:

● The Ames test is used to test chemicals for their ability to cause mutations

□ Bacteria are exposed to _____________________ and scientists examine mutation rates

□ For chemicals modified in the body, they are exposed to rat liver extracts

- The enzymes in the liver will breakdown the chemicals

- These processed chemicals are then tested for their ability to cause mutations

GENETICS - CLUTCH

CH.17 MUTATION, REPAIR, AND RECOMBINATION

Page 13

PRACTICE:

1. Which of the following mutagens wedges between DNA bases to disrupt the helix structure? a. Base analogs b. Alylating agents c. Intercalating agents d. UV light

2. Which of the following mutagens alters base affinities by adding an alkyl group? a. Base analogs b. Alylating agents c. Intercalating agents d. UV light

GENETICS - CLUTCH

CH.17 MUTATION, REPAIR, AND RECOMBINATION

Page 14

3. Which of the following mutations would have the least effect on an individual? a. 4 base pair insertion in an intron b. Deletion of an exon c. A nonsense mutation in the last exon of a gene d. A point mutation in the splice site of an intron

GENETICS - CLUTCH

CH.17 MUTATION, REPAIR, AND RECOMBINATION

Page 15

CONCEPT: DNA REPAIR

● DNA damage is repaired in a lot of different ___________________________-

□ DNA polymerase proofreading quickly fixes a mispaired base

□ Enzymes can reverse damaged DNA

- Many different enzymes exist to counteract damaged DNA

- Ex: CPD photolyase is an enzyme that repairs dimers caused from UV light

EXAMPLE:

GENETICS - CLUTCH

CH.17 MUTATION, REPAIR, AND RECOMBINATION

Page 16

Repair Pathways

□ Base-excision repair (BER) removes and replace damaged nucleotides

- DNA glycoslyases are enzyme that remove the damaged base from the sugar

- A deoxyribophosphodiesterase enzyme removes a stretch of neighboring DNA

- DNA polymerase fills the gap with complementary nucleotides

- DNA ligase seals the new nucleotides into the backbone

EXAMPLE:

□ Nucleotide-excision repair (NER) repairs damage that ______________________ the DNA helix

1. Global genomic NER repairs damage anywhere in the genome

2. Transcription-coupled NER repairs only transcribed regions of DNA

- Damaged base is recognized by proteins, which recruit more proteins

- A 30 nucleotide segment of DNA is removed from the damaged area

- DNA polymerase fills the gap, and DNA ligase seals the new nucleotides to the backbone

- Distortions in NER cause diseases like Xoderma pigmentosum and cockayne syndrome

- Causes light sensitivities

GENETICS - CLUTCH

CH.17 MUTATION, REPAIR, AND RECOMBINATION

Page 17

EXAMPLE:

□ Mismatch repair repairs damaged from insertions or deletions that occur immediately following replication

- First, the mismatched base is recognized, and proteins are recruited to the area

- The methylation status of the DNA strands is used to determine which is the mismatched base

- Methylated strand = old strand, unmethylated strand = new strand

- DNA polymerase fills the gap, and DNA ligase seals the new nucleotides to the backbone

EXAMPLE:

Methylated Old Strand

Unmethylated New Strand

Mismatch

GENETICS - CLUTCH

CH.17 MUTATION, REPAIR, AND RECOMBINATION

Page 18

Translesion Synthesis

□ Translesoin DNA synthesis is a last resort mechanism the cell uses to prevent mutations from causing death

- DNA damage causes DNA polymerase to _________________- while replicating

- Translesion (bypass) polymerases are recruited to the area

- These polymerases can overcome helical distortions and continue replicating

- But they have problems including: high error rate, no proofreading, and only add few nt at a time

- So translesion polymerases unblock stalled replication forks to allow for replication to continue

- They don’t fix the damage that caused the stall and they can miss mutations without proofreading

EXAMPLE:

DNAdamage

5'3'5'

Replicating Polymerase

5'3'5'

Stalling at damageTranslesion Polymerase

5'3'5'

5'3'5'

GENETICS - CLUTCH

CH.17 MUTATION, REPAIR, AND RECOMBINATION

Page 19

Double Strand Breaks

● Double strand breaks can be repaired in ______________________ ways

□ Nonhomologous end joining connects two broken ends together

- Proteins recognize DNA damage

- Other proteins are recruited which trim nucleotides off the damaged ends

- DNA ligase connects the two broken ends

- This type of repair occurs outside of S phase

□ Homologous recombination repairs double strand breaks directly after replication

- Synthesis-dependent strand annealing uses sister chromatids as a template to repair the broken strands

- Process is similar to crossing over, but uses sister chromatids instead of nonsister chromatids

EXAMPLE:

Nonhomologous end joining

Homologous Recombination

GENETICS - CLUTCH

CH.17 MUTATION, REPAIR, AND RECOMBINATION

Page 20

PRACTICE:

1. Which of the following repair pathways repairs damage that causes distortions in the double helix? a. Base Excision Repair b. Nucleotide Excision Repair c. Mismatch Repair d. Homologous Recombination

2. Which of the following repair pathways uses a methylated strand of DNA to correct DNA damage? a. Base Excision Repair b. Nucleotide Excision Repair c. Mismatch Repair d. Homologous Recombination

GENETICS - CLUTCH

CH.17 MUTATION, REPAIR, AND RECOMBINATION

Page 21

3. True or False: Translesion DNA synthesis is the first mechanism the cell uses to repair DNA damage? a. True b. False

4. Which of the following pathways is an error-free way to repair double-stranded breaks? a. Homologous recombination b. Non-homologous end joining c. Translesion synthesis d. Mismatch repair

GENETICS - CLUTCH

CH.17 MUTATION, REPAIR, AND RECOMBINATION

Page 22