DNA Damage, Repair, and Genome Editing

Question 1

What are the two main outcomes of DNA damage if not repaired?

Answer 1

DNA damage can either lead to mutations (due to changes in base pairing) or double-strand breaks, which can cause genome instability. If not repaired, cells may undergo apoptosis (programmed cell death).

Question 2

List 6 common causes of DNA damage.

Answer 2

DNA damage can occur due to:

Cellular metabolism (oxidative damage)
Spontaneous deamination
Loss of base (abasic sites)
Replication errors
UV exposure (photo-cross-linking)
Chemical exposure (alkylation or methylation damage)

Question 3

What is the fidelity of DNA polymerase, and what 3 mechanisms contribute to this accuracy?

Answer 3

DNA polymerase has high fidelity with an error rate of ~10⁻⁹. This accuracy is maintained through:

Exonuclease proofreading activity
Shape discrimination
Mismatch repair pathways

Question 4

What are the consequences of DNA polymerase errors, and how often do they occur per cell division?

Answer 4

DNA polymerase errors lead to mutations. On average, there are about 60 mistakes per cell division across 6 billion base pairs. While many of these occur in non-coding regions, others can lead to lasting mutations, especially with age.

Question 5

What is spontaneous deamination, and how does it lead to mutations?

Answer 5

Spontaneous deamination is the non-catalyzed loss of an amino group, converting cytosine to uracil. Uracil, which can base pair with adenine, leads to A-T mutations if unrepaired.

Question 6

Why does DNA contain thymine instead of uracil?

Answer 6

DNA contains thymine instead of uracil because uracil is chemically similar to thymine and spontaneous deamination of cytosine frequently converts it into uracil. Having thymine in DNA helps the cell recognize and repair deamination damage.

Question 7

What is depurination, and what type of damage does it create?

Answer 7

Depurination is the hydrolysis of the Nβ-glycosyl bond between a base and the pentose sugar, leading to the loss of a purine (or sometimes a pyrimidine) and forming an abasic (AP) site. This creates a site lacking a nucleotide base, leaving the DNA unstable.

Question 8

What are reactive oxygen species (ROS), and how do they damage DNA?

Answer 8

Reactive oxygen species (ROS) such as hydrogen peroxide, hydroxyl radicals, and superoxide radicals cause oxidative damage to DNA. Hydroxyl radicals are particularly harmful, modifying guanine bases and increasing mutation rates through abnormal base pairing.

Question 9

What mutation is caused by 8-oxo-guanine formed from oxidative damage?

Answer 9

8-oxo-guanine, formed by oxidative damage, shifts the nucleotide’s conformation to syn, which results in it pairing with adenine instead of cytosine, causing G-C to A-T transversion mutations after replication.

Question 10

How does UV radiation damage DNA, and what kind of repair mechanism addresses this damage?

Answer 10

UV radiation promotes the formation of pyrimidine dimers, specifically cyclobutane pyrimidine dimers and 6-4 photoproducts. These lesions distort the DNA helix, stalling DNA polymerase. Translesion polymerases bypass these lesions, allowing replication to proceed, though the damage is not directly repaired by this mechanism.

Question 11

What are alkylation and methylation damages in DNA, and what mutation can O6-methylguanine cause?

Answer 11

Alkylation and methylation damages occur when alkyl groups are added to bases, such as O6-methylguanine. O6-methylguanine mispairs with thymine instead of cytosine, leading to G-C to A-T transitions.

Question 12

What are the two types of DNA damage, and how do they differ?

Answer 12

DNA damage can be:

Endogenous: caused by internal factors like spontaneous deamination, oxidative damage from metabolism, or replication errors.
Exogenous: caused by external factors like UV-induced photo-cross linking or chemical exposure (e.g., alkylation from burnt food or cigarette smoke).

Question 13

How do pyrimidine dimers formed by UV radiation affect DNA replication?

Answer 13

Pyrimidine dimers create significant distortions in the DNA double helix, preventing DNA polymerase from continuing replication. If unrepaired, they may cause polymerase stalling, leading to replication forks breaking and genome instability.

Question 14

What mechanisms exist in cells to defend against reactive oxygen species (ROS)?

Answer 14

Cells have an elaborate defense system that includes enzymes like superoxide dismutase, catalase, and glutathione peroxidase to neutralize ROS and prevent oxidative damage to DNA and other cellular components.

Question 15

Why is DNA the only biological molecule that undergoes repair, while other damaged molecules like proteins and RNA are degraded?

Answer 15

DNA stability is essential for encoding the proteins and RNA that organisms need, and because DNA is easily damaged, its repair is prioritized to maintain genetic integrity.

Question 16

What are the three types of mutations, and how do they differ?

Answer 16

Substitution mutation: Replacement of one base pair with another due to damaged DNA affecting hydrogen bonding.
Insertion mutation: The addition of 1 or more base pairs, changing the codon and altering the genetic code.
Deletion mutation: The deletion of 1 or more base pairs, also altering the codon and genetic sequence.

Question 17

What is a silent mutation, and can it still have an impact on gene function?

Answer 17

A silent mutation affects nonessential DNA or has a negligible effect on gene function by preserving amino acid identity. However, it may still impact regulatory or positional non-coding regions.

Question 18

What role do DNA glycosylases play in Base-Excision Repair (BER)?

Answer 18

DNA glycosylases recognize common DNA lesions, remove the affected base by cleaving the N-glycosyl bond, and generate an abasic site for further repair.

Question 19

What is an AP site, and how is it processed in Base-Excision Repair (BER)?

Answer 19

An AP site (apurinic or apyrimidinic) is created when a damaged base is removed. A nuclease cuts the backbone at the AP site, DNA polymerase fills in the missing nucleotides, and DNA ligase seals the strand.

Question 20

How does uracil DNA glycosylase contribute to BER, and why doesn’t it remove uracil residues from RNA?

Answer 20

Uracil DNA glycosylase specifically removes uracil from DNA resulting from spontaneous deamination of cytosine. It does not remove uracil from RNA or thymine residues from DNA.

Question 21

Describe the 4 steps of the Base-Excision Repair (BER) pathway.

Answer 21

A glycosylase recognizes and removes the damaged base, generating an abasic site.
An endonuclease cleaves the phosphodiester backbone at the abasic site.
DNA Pol I replaces the missing base and short extension.
DNA ligase seals the gap, completing the repair.

Question 22

What is the function of the nucleotide-excision system, and how does it repair large DNA distortions?

Answer 22

The nucleotide-excision system repairs large DNA lesions that distort the DNA helix. It recognizes these distortions, excises a large fragment of DNA using excinucleases, and DNA polymerase fills in the gap, followed by ligation.

Question 23

What is the function of O^6-methylguanine-DNA methyltransferase (MGMT) in direct repair of alkylation damage?

Answer 23

MGMT transfers the methyl group from O^6-methylguanine to its own Cys residue, restoring guanine’s normal structure. This enzyme is used up in the process, making it a “suicide” enzyme.

Question 24

Why is the direct repair of methylguanine by MGMT considered energetically expensive for the cell?

Answer 24

The repair consumes an entire MGMT protein per methyl transfer event, which is energetically costly because the enzyme is not regenerated, requiring the cell to synthesize new proteins for each repair event.

Question 25

What are the key differences between Base-Excision Repair (BER) and Nucleotide-Excision Repair (NER)?

Answer 25

BER: Removes a damaged base, creating an abasic site, and replaces a few nucleotides.
NER: Removes a large DNA fragment around a distortion and requires helicase and ATP for unwinding.

Question 26

What are the key points regarding single-stranded DNA repair mechanisms?

Answer 26

Damaged DNA is repaired through different mechanisms, including base-excision repair, which removes damaged bases, nucleotide-excision repair, which excises large DNA fragments, and direct repair pathways, which revert damaged bases to their original form.

Question 27

What are two programmed biological processes that cause double-strand breaks in DNA?

Answer 27

Double-strand breaks occur during meiosis (recombination) and V(D)J recombination as part of programmed cellular processes.

Question 28

How can incomplete NER or BER repair pathways lead to double-strand breaks in DNA?

Answer 28

If a nick in DNA is not repaired before DNA replication, the replication machinery may fail to read past the nick, resulting in a double-strand break.

Question 29

What types of environmental damage can cause double-strand breaks in DNA?

Answer 29

UV, ionizing radiation (IR), and other radiation types can cause extensive DNA damage, potentially leading to apoptosis if the damage is too sever

Question 30

What role does the histone variant γH2AX play in the response to DNA double-strand breaks?

Answer 30

γH2AX is recruited to the site of DNA damage, forming DNA repair foci, and is involved in the cellular decision-making process along with p53 on whether to repair the damage or trigger apoptosis.

Question 31

What are the two major pathways for repairing double-strand breaks in DNA, and how do they differ?

Answer 31

Non-homologous end-joining (NHEJ): Directly joins broken DNA ends, often leading to small insertions or deletions.
Homologous recombination (HR): Template-driven, accurate repair using the sister chromatid, primarily during the S and G2 phases of the cell cycle.

Question 32

How does the Ku70-Ku80 complex contribute to Non-Homologous End Joining (NHEJ)?

Answer 32

The Ku70-Ku80 complex binds to DNA ends, helping to recruit DNA-PKcs and Artemis, which process the break to allow ligation of the DNA strands.

Question 33

Why is Non-Homologous End Joining (NHEJ) considered error-prone?

Answer 33

NHEJ relies on microhomology between DNA ends, often leading to small deletions or insertions, making it error-prone.

Question 34

What is the role of Artemis in NHEJ?

Answer 34

Artemis is a nuclease that processes single-strand extensions or hairpins at the broken ends, ensuring the DNA ends can be ligated together.

Question 35

What is the function of RAD51 in homologous recombination?

Answer 35

RAD51 binds to single-stranded DNA overhangs, forming a nucleoprotein filament that invades homologous DNA to search for sequence complementarity.

Question 36

What is the role of BRCA1 and BRCA2 in homologous recombination (HR)?

Answer 36

BRCA2 mediates RAD51 filament formation, while BRCA1 (along with BARD1) facilitates strand invasion, enabling repair by homologous recombination.

Question 37

What is resection in the context of homologous recombination?

Answer 37

Resection is the process where nucleases degrade one strand of the broken DNA to create single-stranded overhangs, which are critical for strand invasion during homologous recombination.

Question 38

How is the DNA repair process through homologous recombination (HR) resolved?

Answer 38

After the strand invasion and polymerase filling of the gap, Holliday junctions are formed and later resolved by nucleases, followed by ligation of the nicks by DNA ligase.

Question 39

What is the commitment step in homologous recombination?

Answer 39

Resection of the DNA strands is the commitment step in homologous recombination, marking the point at which the HR pathway is activated.

Question 40

What is the primary difference in the outcome between NHEJ and HR in terms of fidelity and genetic impact?

Answer 40

NHEJ often results in small insertions or deletions, potentially disrupting genes.
HR repairs DNA with high fidelity, especially in S or G2 phase, without introducing errors into the genetic sequence.

Question 41

Why does NHEJ often lead to gene disruption, while HR generally preserves genetic integrity?

Answer 41

NHEJ can cause insertions or deletions at the break site, leading to gene disruption. HR, by using a homologous template, repairs the break without altering the genetic sequence, thus preserving genetic integrity.

Question 42

What are 6 reasons for engineering a genome?

Answer 42

Genome engineering is used to:

Analyze gene functions
Study genome rearrangements (e.g., cancer)
Engineer animal models
Create disease or insect resistance in crops, animals, or humans
Remove unwanted organisms
Treat severe genetic diseases

Question 43

How does the CRISPR-Cas9 system recognize and cleave target DNA?

Answer 43

CRISPR-Cas9 recognizes a PAM sequence in the target DNA and cleaves the phosphodiester backbone of both strands when the single guide RNA (sgRNA) is complementary to the target sequence.

Question 44

What happens when sgRNA binds to Cas9 in the CRISPR-Cas9 system?

Answer 44

The binding of sgRNA induces folding of the PAM recognition domain of Cas9, which allows it to recognize and bind the target DNA sequence.

Question 45

What conformational changes occur in Cas9 during DNA binding?

Answer 45

The HNH nuclease domain undergoes large-scale structural reorganization upon binding to sgRNA and the target DNA, allowing Cas9 to cleave both strands of the target DNA.

Question 46

How does Cas9 cleave the DNA once the guide RNA is aligned with the target sequence?

Answer 46

Cas9’s HNH domain cuts the 3’ strand, and the RuvC domain cuts the 5’ strand of the DNA, resulting in a double-strand break after proper alignment and unwinding.

Question 47

What are the two DNA repair pathways that can act after a CRISPR-Cas9-induced double-strand break?

Answer 47

Non-homologous end joining (NHEJ): Error-prone and often leads to insertions or deletions, disrupting the gene.
Homologous recombination (HR): High-fidelity repair, especially in S- or G2-phase, ensuring accurate repair when a repair template is provided.

Question 48

What outcome is expected when NHEJ repair is used after a CRISPR-induced DNA break?

Answer 48

NHEJ often results in small insertions or deletions, potentially disrupting the gene’s function, and preventing future targeting by Cas9 if the sequence is altered.

Question 49

How does Homologous Recombination (HR) differ from NHEJ in genome editing?

Answer 49

HR uses a repair template to accurately restore the DNA sequence and allows the insertion of new genetic material, while NHEJ introduces errors and disrupts the gene.

Question 50

What can be added to the DNA during Homologous Recombination repair?

Answer 50

Novel sequences, like red regions in diagrams, can be added to the DNA by providing a repair template that is identical to the sequences flanking the double-strand break.

Question 51

How can genome editing be used to treat or prevent HIV?

Answer 51

By editing the CCR5 gene to mimic a naturally occurring 32 bp deletion that prevents HIV from binding and entering T-cells, individuals could become immune to HIV.

Question 52

Why is NHEJ the favored repair pathway in humans?

Answer 52

NHEJ is quicker and more common in human cells. Without a repair template, it results in random gene disruptions, which is useful for knocking out genes.

Question 53

How does the repair template work in CRISPR-Cas9 genome editing during HR repair?

Answer 53

The repair template provides a perfect match for the regions flanking the break, allowing DNA polymerase to use it for accurate repair and the insertion of new sequences.

Question 54

hy does CRISPR-Cas9 require the presence of sgRNA and target DNA for cleavage?

Answer 54

Cas9’s nuclease domains undergo necessary conformational reorganizations only when bound to sgRNA and target DNA, ensuring specificity in DNA cleavage.