topic 16

Question 1

why is it more important to repair DNA damage, compared to RNA damage?

Answer 1

RNA is continually being synthesized and degraded. DNA is permanent. thus, it’s more important to repair DNA damage which could lead to mutations

Question 2

why do organisms strive to preserve the DNA sequence given to them by their ancestors?

Answer 2

DNA damage is the unintended physical/chemical change in DNA. It could lead to reduced cell function, cell death, or cancer.

Question 3

what are the types of DNA damage? (6)

Answer 3

copying mistakes

depurination

deamination

pyrimidine dimers

strand breaks

other base modification

Question 4

what are copying mistakes? what are the types? (3)

Answer 4

a type of DNA damage made by DNA polymerase during replication. the resulting DNA double helix will have a distorted structure. change in sequence could have functional consequences.

types: mismatch, insertion, deletion

Question 5

what are the types of mutations in protein-coding sequences? (4) describe them

Answer 5

silent: the genetic code is redundant so some mutations will not change the amino acid a codon is coding for

missense: other mutations will change the amino acid at affected codon

nonsense: some mutations will introduce a stop codon which will cause premature termination of translation of mRNA.

frameshift: insertion of base(s) which causes the reading frame of the gene to shift, affecting all the codons after the site of mutation. frameshift mutations can be either insertion or deletion. insertion/deletion of a multiple of 3 bases is said to be in frame since it doesn’t change the reading frame.

Question 6

what is depurination? what problem arises and how is it resolved?

Answer 6

a type of DNA damage. it is the loss of A or G bases from the spontaneous hydrolysis of the A/G base from the deoxyribose sugar. this creates an abasic site (site with no base) on the DNA strand. the sugar phosphate backbone remains intact.

when DNA polymerase encounters an abasic site during replication, the enzyme stalls and can not go further. to solve this, the replicative DNA polymerase dissociates and something called a translesion DNA polymerase is recruited to synthesize DNA past the site of damage (not a repair process).

the drawback is that translesion polymerases make mistakes much more often than normal DNA polymerases. at an abasic site, translesion polymerases will either skip a site entirely, resulting in a deletion in the new strand, or they will put a random base in the new strand, opposite to the abasic site. so, translesion polymerases are more likely to result in new mutations in the DNA

Question 7

what is deamination?

Answer 7

a type of DNA damage. it is the conversion of an amine to a carbonyl caused by the spontaneous chemical change which often occurs at cytosine residues to produce uracil. this doesn’t hinder DNA replication but could affect the sequence of a protein or regulation of gene expression.

Question 8

what are pyrimidine dimers? how are they resolved?

Answer 8

a type of DNA damage. they are ring formations of a 4 membered carbon ring between adjacent pyrimidines (usually thymines) caused by UV light. it blocks DNA replication which can be overcome with the use of translesion polymerases (which could lead to mutations). mutations arising from pyrimidine dimers explain why exposure to sunlight increases the risk of skin cancer.

Question 9

what are strand breaks?

Answer 9

type of DNA damage. can be caused by ionizing radiation or mechanical stress. both types of strand break will prevent complete transcription/replication, but double stranded breaks are more dangerous because it could lead to serious chromosomal abnormalities such as rearrangements that’re likely to cause a disease state.

Question 10

what leads to base modifications (DNA damage)?

Answer 10

can arise from exposure of ionizing radiation and chemical mutagens such as oxidizing agents (e.g. mutagen is the superoxide radical, O2 - ; the first intermediate formed during reduction of oxygen to water by Complex IV) and polycyclic aromatic hydrocarbons. results in chemical changes in the base(s).

a large number of different reactive compounds can modify bases to alter the structure and function of DNA.

Question 11

describe the steps in the mismatch repair process

Answer 11

1. when a mismatch is found by MutS, the proteins in the repair machinery scan the DNA in either direction, looking for a site that’s methylated on one strand but not on the other.
2. once the new strand is determined, an endonuclease cuts the strand at the site of methylation.
3. then, an exonuclease digests the DNA from the site of methylation to the site of mismatch.
4. a DNA polymerase then fills in the gap, using the intact strand as a template for synthesis. the DNA ligase will seal the backbone to create the intact double-stranded DNA.

Question 12

describe mismatch repair. when is it active? when does it occur? how does it determine which base to correct (in bacteria? eukaryotes?)? what are the down sides of this mechanism?

Answer 12

it is active during replication. it happens when an incorrectly incorporated base escapes proofreading by DNA polymerase.

in bacteria, the base to be corrected is determined by the presence of methylation. most bacteria methylate certain bases on their DNA after the DNA has been synthesized. mature DNA in these organisms are methylated on strands, but it takes some time for the modifying enzymes to come along and add the methyl group to newly synthesized DNA. thus, there exists a time window after a replication fork has passed, during which the template strand of the DNA is methylated.

in eukaryotes, the method of detection remains unknown.

this process is energetically costly for the cell and the site of methylation can be hundreds of nucleotides away from the mismatch.

Question 13

what is direct repair? what types of damage does it repair?

Answer 13

it is the repair of specific modified bases, carried out by enzymes which specialize in repairing specific types of damage.

Question 14

what are types of repair mechanisms?

Answer 14

proofreading

mismatch repair

direct repair

base excision repair

nucleotide excision repair

non-homologous end-joining

homologous recombination

Question 15

what is base excision repair (BER)? what types of damage does it repair? what are the types of BER?

Answer 15

it is the repair of modified bases (e.g. cytosine deamination), abasic sites (e.g. depurination), and single-stranded breaks. it fixes damage that’s fairly localized to the DNA (generally affecting only a single base).

there are 2 types: short patch and long patch. short patch is the regular/default pathway.

Question 16

describe the steps in the process of base excision repair’s short patch

Answer 16

1. DNA glycosylase breaks the N-glycosidic bond joining the base to the deoxyribose, removing the damaged base from the DNA, creating an abasic site. (if the damage was depurination, an abasic site already exists, so no need for glycosylase to act)
2. then, an endonuclease will cut the sugar phosphate backbone. if the damage is a single stranded break, then this step isn’t necessary.
3. cutting the backbone creates a free 3’ hydroxyl to which a repair DNA polymerase can add a new base at this site using the undamaged strand as the template. after a base has been added, DNA ligase will seal up the backbone, restoring an intact double helix. short patch repair requires synthesis of only a single base to replace the damaged base.

Question 17

when would base excision repair’s long patch be used? describe how it works

Answer 17

if the original damage affected the sugar phosphate backbone, it won’t be possible for DNA ligase to seal the backbone properly.

in such a case, a longer stretch of DNA will replaced, moving the nick further away from the site of damage, allowing DNA ligase to do its job. long patch replacement involves replacement of no more than 10 bases on the damaged strand.

Question 18

what is nucleotide excision repair (NER)? what types of damage does it repair?

Answer 18

it is the repair of pyrimidine dimers or has a bulky nature and disrupts the double helix structure.

Question 19

describe the steps in the process of nucleotide excision repair

Answer 19

1. helicase is involved which unwinds the strands around the site of damage.
2. an endonuclease cuts the damaged strand in 2 places, about 30 bases apart, which results in in the removal of that stretch of nucleotides.
3. a repair DNA polymerase uses the 3’ end on one side of the damage to synthesize DNA to replace the missing piece. the DNA ligase seals the backbone.

Question 20

what is xeroderma pigmentosum? what causes this?

Answer 20

people with a defect in NER suffer from xeroderma pigmentosum.

in this condition, exposure to UV light causes pyrimidine dimers to accumulate, greatly increasing the risk of skin lesions and skin cancer.

Question 21

what is non-homologous end-joining (NHEJ)? what types of damage does it repair? what is the advantage of using this method? the disadvantage?

Answer 21

it’s goal is to join the 2 ends of DNA back together by whatever means necessary without worrying about preserving the DNA sequence or recovering segments that might’ve been lost when damage occurred.

it repairs double stranded breaks

non-homologous end-joining has the advantage that no other DNA is required and so it doesn’t take a lot of time to be performed.

the disadvantage is that the resulting sequence is almost never restored to what was before the break occurred. so, some mutations, insertions, and/or deletions are likely to happen. however, it is still preferable over the broken state.

Question 22

describe the steps in the process of non-homologous end-joining

Answer 22

1. protein Ku binds to both ends of the broken DNA and aligns both portions of the double strand
2. protein Ku recruits a nuclease and polymerase. the ends of the DNA are usually trimmed back. regions of complementarity are found or created and the 2 strands are made to anneal to each other. polymerase fills in any gaps
3. DNA ligase joins the backbones on both strands.

Question 23

what is homologous recombination? what types of damage does it repair? what is the advantage of using this method? the disadvantage?

Answer 23

it is a method that requires a 2nd double helix with a homologous/identical sequence to the one that was broken.

it repairs double stranded breaks

homologous recombination has the advantage in the way that it’s more likely to restore the original sequence of before the double-strand break.

the disadvantage is that the cell must be able to find the appropriate section of double stranded DNA to copy from. this process is most convenient shortly after replication, when another copy of the chromosome is nearby. it is used less often than non-homologous end-joining.

Question 24

describe the steps in the process of homologous recombination

Answer 24

1. an endonuclease trims the ends of the broken DNA
2. then the overhang invades the intact copy, base pairing to the complementary sequence.
3. for both strands, the intact DNA is used as a template for new DNA synthesis.
4. the sequence in the entire area is copied and the crossed over strands are resolved to give 2 complete double helices.