Mutation and Repair

Question 1

Phenotype vs genotype

Answer 1

Phenotype: observable properties of an organism
Genotype: Sequence of DNA

Question 2

What is a mutation?

Answer 2

Inheritable change in the DNA sequence
(damage to DNA can’t be inherited so not mutation)

Question 3

What are point mutations? what are the two kinds?

Answer 3

when a basepair in DNA is changed to a different basepair

Transition: a transition mutation is where a purine base (A & G) changes to another purine base and a pyrimidine base (C & T) changes to another pyrimidine base

Transversions: a transversion mutation is where a purine base (A & G) changes to a pyrimidine base (C & T) and vice versa

Question 4

Missense mutations

Answer 4

point mutations in the coding region of a gene can cause a change in the protein where one amino acid is replaced by another

Question 5

Nonsense mutations

Answer 5

a point mutation in the coding region of a gene can change a codon into one of the 3 nonsense codons that specify a STOP to translation (UAA, UAG, or UGA) causing production of a truncated, and usually, an inactive version of the gene product

Question 6

Frameshift mutation

Answer 6

a high percentage of all spontaneous mutations are frameshift mutations – this kind of mutation arises when small insertions or deletions of one or a few basepairs occurs – when this happens in the coding region of a gene the reading frame of the gene is disrupted downstream of the frameshift mutation

Because the reading frame is disrupted the gene product will be truncated, usually within 20 amino acids, downstream of the frameshift

Frameshift mutations usually inactivate the gene product

Question 7

What is triplet expansion

Answer 7

a disease caused by the insertion of many additional copies of a repeated codon triplet into a gene due to template slippage in the DNA replication process.

Question 8

what is fragile X syndrome

Answer 8

triplet expansion disease

Question 9

What is a deamination

Answer 9

removal of amino groups from biomolecules such as nucleotides
Deamination of nucleotide bases by spontaneous hydrolysis
A hydrolysis reaction in which water is added to cytosine, resulting in deamination to uracil and ammonia

Question 10

What is the origin of point mutations

Answer 10

oxidative damage

Question 11

what is oxidative damage

Answer 11

bacteria with aerobic metabolism and pathogens exposed to an inflammatory response triggered by the innate immune system are exposed to reactive oxygen species (ROS) that can damage DNA by chemically modifying the bases of the DNA

A common oxidative modification occurs to G to produce 8-oxo-G which can sometimes mispair with A instead of C inducing GC to TA and CG to AT transversions

Question 12

How is nitrous acid a source of mutation

Answer 12

Deamination by nitrous acid.
Nitrous acid can deaminate C and A residues, causing them to base-pair with the wrong nucleotide during replication
C after deamination turns into U which pairs with A instead of G, a CG basepair becomes a TA (a transition mutation). A after deamination turns into hypoxanthine which pairs with C instead of T, an AT basepair becomes a GC basepair (a transition mutation).

Question 13

how can chemicals produce mutations

Answer 13

The two-step process of a point mutation (b) The parental DNA contains a damaged adenosine base (X). The damaged A more readily pairs with a C than a T, so replication over the damaged base results in a mismatched nucleotide in one of the first-generation cells. Further replication can result in a permanent mutation in a cell of the second generation.
DNA repair processes that act to remove the damage, damaged base or the resulting mismatch lower the mutation rate that would arise from these damaged bases.

Question 14

how can sunlight cause DNA damage

Answer 14

One type of reaction caused by UV light results in a cyclobutane ring, which involves C-5 and C-6 of adjacent pyrimidine (in this case, thymine) bases

An alternative reaction results in a 6-4 photoproduct that links C-6 and C-4 of adjacent pyrimidines.
T’s stacked on each other in the same strand can become crosslinked by excitation afforded by UV spectrum light.

Question 15

How can DNA damaging agents be used to treat cancer? What are some agents?

Answer 15

Chemotherapeutic DNA-damaging agents. Chemotherapeutic agents act by preferentially damaging the DNA of fast-dividing tumor cells

Cisplatin, bleomycin, doxorubicin

Question 16

how does cisplatin and bleomycin work

Answer 16

Cisplatin is a cross-linking reagent that reacts with N-7 of two G residues to form intrastrand or interstrand cross-links.

Bleomycin binds to DNA and forms reactive oxygen species that cause strand breaks. Iron normally binds bleomycin, but cobalt was used to prevent DNA modification in the crystal structure shown here

Question 17

how does doxorubicin work

Answer 17

Doxorubicin is a DNA intercalator, inserting between adjacent nucleotide residues. Intercalators can cause frameshift mutations during replication, or they can block replication forks, making the forks susceptible to nucleases that cause strand breaks.

Question 18

Inversion mutation? Translocation mutation?

Answer 18

inversion mutation: a mutation that results from the inversion of a large segment of DNA in a chromosome.
translocation mutation: a mutation that results from the exchange of large segments of DNA between nonhomologous chromosomes

Question 19

transversion mutation

Answer 19

a point mutation resulting in the exchange of a purine-pyrimidine base pair for a pyrimidine-purine pair, or vice versa

Question 20

How can radiation damage DNA

Answer 20

Radiation can produce chromosome breaks during replication.

Question 21

Nicks in two strands vs Nicks in one strand

Answer 21

One:
When a replication fork encounters a single-strand break (nick), one daughter chromosome is broken, while the other daughter chromosome remains intact. The intact chromosome can become a template for repair of the broken one through homologous recombination.

Two:
When the break occurs in both strands, both daughter chromosomes are broken, and neither daughter chromosome is completed.

Question 22

mismatch repair (first 3 steps)

Answer 22

In E. coli, MutS2MutL2 binds a mismatch and scans the DNA for a GATC site. MutH nicks the DNA at the nearest unmethylated GATC site, facilitating repair of the mismatch on the newly synthesized strand by excision, filling in of the gap, and ligation.
Recall, once DNA has just been copied the newly synthesized strands (nascent strands) lack dam methylation for a while. This allows the MMR system in E. coli to distinguish between the parental strand and the nascent strand. Since mismatches from errant replication are more likely to occur on nascent strand, repair is directed towards the nascent strand.

Question 23

Mismatch repair (steps 4-7)

Answer 23

Question 24

What does MutS do

Answer 24

Homo dimer
Binds to a mismatch in DNA

Question 25

Direct repair of pyrimidine dimer mechanism

Answer 25

E. coli photolyase has two chromophores (light-absorbing groups) that work in sequence to use the energy of light to repair a pyrimidine dimer. Photoreactivation was discovered in the study of UV damaged phages. It was discovered that prior exposure to sunlight allowed ‘reactivation’ of some phages inactivated by exposure to UV light. Phage infected cells kept in the dark prior to UV inactivation did not undergo this reactivation after UV exposure

Question 26

Base excision repair (bacterial

Answer 26

In bacteria, a glycosylase excises a damaged nucleotide base, then an AP endonuclease nicks the backbone at the a basic site. Nick translation by Pol I excises the 5′ deoxyribose phosphate (5′-dRP) and some dNMPs, and synthesizes a new strand. Ligase seals the gap

Question 27

Base excision repair (eukaryotes)

Answer 27

Eukaryotic BER, after the first two steps (similar to those in bacteria), can take either of two paths. In long patch repair (left), a DNA polymerase extends the DNA strand from the 3′ terminus, displacing the 5′ single-stranded DNA; this is followed by cleavage by a flap endonuclease and ligation. In short patch repair (right), only one nucleotide is inserted (by Pol β) prior to ligation.

Question 28

When does BER tend to act?

Answer 28

BER tends to act on damaged baes with small chemical modifications. (deaminations, oxidative damage etc)

Question 29

UvrA, UvrB, UvrC, UvrD, Pol I, DNA ligase roles in NER

Answer 29

Question 30

# Uvr NER mechanism in E. coli

Answer 30

The NER pathway uses several proteins, including UvrA (red), UvrB (purple), and UvrC (green), that recognize the lesion and make incisions on either side, allowing UvrD (helicase II) to displace a section of lesion-containing DNA. The single-strand gap is filled in by Pol I, and the DNA is sealed by ligase. A transcription-coupled repair (TCR) path can also be taken, in which RNA polymerase stalls at the lesion on the coding strand (not shown). After the RNA polymerase is displaced, the reaction proceeds as shown here, using UvrA–D, Pol I, and ligase.

Question 31

Translesion synthesis

Answer 31

Translesion synthesis by TLS DNA polymerases. When the high-fidelity E. coli replicase, Pol III (yellow), encounters a leading-strand lesion (red X), it stalls, and a TLS polymerase (TLS Pol, gray) takes over the β sliding clamp (purple) to extend the leading strand across the lesion. After lesion bypass, Pol III resumes its function with the β clamp Pol III stalls when it finds lesion, TLS polymerase takes over and extends strand across the lesions, After bypass pol III resumes

Question 32

Which 3 E. Coli polymerases are capable of translesion synthesis? What family are they in

Answer 32

Question 33

Double-strand break repair mechanism? What does it require?

Answer 33

Accurate repair of a DSB requires an undamaged source of duplicate genetic information—that is, homologous double-stranded DNA. The broken ends are first processed to generate 3′ single-stranded extensions, and both ends are used for strand invasion of the homologous double-stranded DNA to form D-loops. The invading 3′ ends are extended by DNA polymerases.

Question 34

What does RecA do (structure and function)

Answer 34

protein filament Segment of a RecA filament with four helical turns (24 RecA subunits). Notice the bound double-stranded DNA in the center. The core domain of RecA is structurally related to the domains in helicases a bacterial recombinase that binds single-stranded DNA and promotes homologous recombination. RecA protein also has co-protease activity in the autocatalytic cleavage of some transcription repressors (LexA).

Question 35

RecA mechanism

Answer 35

forms filaments on ssDNA RecA and other recombinases in this class function as filaments of nucleoprotein. (c) Filament formation proceeds in discrete nucleation and extension steps. Extension occurs by adding RecA subunits so that the filament grows in the 5′→3′ direction. When disassembly occurs, subunits are subtracted from the trailing end.

Question 36

what does RecA promote

Answer 36

strand invasion

Question 37

Example of damage repair and notable enzyme for each of the following processes Mismatch repair Base excision repair Photoreactivation Nucleotide excision repair Double-strand break repair Translesion synthesis

Answer 37

Question 38

Duplication mutation

Answer 38

the duplication of a large tract of DNA, leading to an increased dosage of genes in the affected area.

Question 39

Dam methylase

Answer 39

an enzyme of E. coli that methylates adenine residues in the palindromic sequence GATC on both strands of the DNA. Transient hemimethylation of a DNA duplex following replication distinguishes the parental strand from the daughter strand

Question 40

DNA photolyase

Answer 40

a flavoprotein enzyme that becomes an electron donor when activated by visible light. DNA photolyases can repair pyrimidine dimers and other lesions caused by ultraviolet light.

Question 41

DNA glycosylase

Answer 41

DNA glycosylase: an enzyme that hydrolyzes the N-β-glycosyl bond between a nucleotide base and pentose, creating an abasic site in the DNA.

Question 42

AP endonuclease

Answer 42

AP endonucleases: enzymes that cleave the DNA backbone at an AP (apurinic or apyrimidinic; abasic) site as part of the base excision repair pathway.

Question 43

Excinuclease

Answer 43

excinuclease: an enzyme that cleaves a phosphodiester bond in the DNA on either side of a bulky lesion in DNA. Also called an excision endonuclease.

Question 44

what is DNA strand invasion

Answer 44

the pairing of a single-stranded extension of a DNA molecule with a homologous region of another DNA molecule, with displacement of one strand of the recipient molecule by the invading strand