DNA damage and Repair

Question 1

How many errors does DNA polymerase make?

Answer 1

10^4 to 10^5 nucleotides

Question 2

How many errors are left after repair mechanisms?

Answer 2

1 in 10^9 nucleotides are errors.

Question 3

Which types of abnormalities need repair?

Answer 3

Base mismatches

Damage to the structure of the DNA itself (break in the chromosome or pyrimidine dimers)

Question 4

What does mismatch repair do?

Answer 4

repairs replication errors: mispaired bases and strand slippage.

Question 5

What does direct repair do?

Answer 5

repairs pyrimidine dimers and other specific types of alterations

Question 6

What does base excision do?

Answer 6

repairs abnormal bases, modified bases and pyrimidine dimers.

Question 7

What does nucleotide excision do?

Answer 7

Repairs DNA damage that distorts the double helix, including abnormal bases, modified bases and abnormal dimers.

Question 8

Which enzyme repairs nicks in the DNA strand?

Answer 8

DNA ligase

Question 9

How are chemically modified bases repaired?

Answer 9

Base excision repair: removal of base-specific DNA glycolase.

Question 10

How are mismatched bases repaired?

Answer 10

Corrected by mismatch repair: excision and resynthesis.

Question 11

What does the AP endonuclease repair system do?

Answer 11

Fixes apurining or apyrimidinic sites.

Question 12

How is a damaged region of DNA fixed?

Answer 12

Nucleotide excision repair (excision and resynthesis across partner strand)

Question 13

How are pyrimidine dimers fixed?

Answer 13

enzymatically reversed.

Question 14

Describe direct repair in detail.

Answer 14

Damaged nucleotide, chemically damaged and abnormal: it is not chemically stable in relation to the rest of the DNA molecule. Not a standard ATCG. Direct repair would be to chemically modify the damaged nucleotide (little evidence of occurring substantially). For example, is deamination of thymidine occurs, forming uracil, one cannot just go back and add an amine groupe.

Question 15

Describe excision repair in detail.

Answer 15

A damaged nucleotide not forming the standard ATCG can excise a region: the sugar phosphate backbone is removed and the missense strand is used as a template by DNA polymerase. DNA ligase then reforms the backbone.

Question 16

Describe mismatch repair in detail.

Answer 16

Occurs when either a missing base has occurred or a mis-matching of DNA bases. A segment of DNA is excised and the other strand is used as a template to add newly synthesised nucleotides whilst reforming the sugar phosphate backbone. Can be as much as 500 bases excised.

Question 17

Describe recombination repair.

Answer 17

Double strand break: hard to repair as there are no sticky ends. Recombination repair involves two molecules (via the other chromosomes) to direct the process of joining these two broken DNA strands.

Question 18

What is required for DNA ligase to operate?

Answer 18

Requires a function 5’-phosphate and 3’hydroxyl group.

Question 19

How are alkyl groups repaired?

Answer 19

This is a highly specific removal of chemical groups: O6-methylguanine-DNA methyltransferase (MGMT)

Question 20

How does photoreactivation occur?

Answer 20

DNA photolyase is stimulated by 300-500 nm light. It is not present in humans but is found in plants.

Question 21

How is base-excision repair performed?

Answer 21

Specific DNA glycosylases cut between the base and the sugar backbone (or cut out the whole backbone or remove th bases that reside within the DNA molecule, glycosylases will further remove the bases, leaving the backbone intact. This creates and AP site, AP endonucleases cut the backbone and resynthesis by DNA polymerase, with the damaged strand acting as a primer and original strand as a template.

Question 22

How does nucleotide-excision repair differ from base excision repair?

Answer 22

They are very similar, however NER does not require initial removal of damaged base. There is recognition of damaged region, followed by excision of up to 12 nucleotides. Excised region is replaced by DNA polymerase/DNA ligase. Regions up to 2kb may be excised and repaired.

Question 23

Why is mismatch repair difficult?

Answer 23

It is difficult to tell which was the original base and which needs to be retained. Mismatch repair specifically repairs errors of replication where the bases are normal. Excision of daughter strand followed by new polymerisation.

Question 24

What is replication slippage?

Answer 24

It is expansions of repeats (mostly microsatellites: AAA homopolymer or GCGCGCGCG; the simpler they are the more difficult they are to replicate.
Expansions are common but sometimes retractions can occur. Expansion/contraction may depend on the parent of origin. The larger the repeat gets, the more they expand from one generation to the next.

Question 25

What are replication loops?

Answer 25

There is constant pairing/unpairing in DNA, that can cause loops where the 3’ end of the growing strand detaches from the template and reanneals to the template at a point upstream from its original location. Continued replication duplicates the region between the points of detachment and reannealing. Mismatch repair of the shorter strand creates a duplex with a trinucleotide expansion. Results in two strands of different lengths.

Question 26

Describe in detail microsatellites.

Answer 26

Microsatellites can vary in number of repeat units between individuals. They differ from the usual bi-allelic nature of SNPs: there can be 30-40 different alleles of 1 locus. They are very rarely found in coding sequences: the exception is trinucleotide repeats and changes in reading frames. They are quite easily genotyped. They generally do not have much function but can be exemplary of anticipation (Huntington’s) which breaks the usual mendelian rules of inheritance.

Question 27

How can CNVs come about?

Answer 27

Through insertions and deletions (Indels). Can be individual exons to whole genes or chromosomes.

Question 28

What are transitions?

Answer 28

A transition replaces a pyrimidine with another pyrimidine or a purine with another purine

Question 29

What are transversions?

Answer 29

A transversion replaces a pyrimidine with a purine or a purine with a pyrimidine.

Question 30

What are the effects of mutations?

Answer 30

Loss of function
Gain of function
Germline mutations
Somatic mutations
Transitions
Transversions. 
Missense mutations
nonsense mutations
Neutral mutation changes
Silent mutations (can still cause disease)
Frameshift mutations
Splice mutations.

Question 31

What are the effects of SNPs on function?

Answer 31

Functional consequences vary: subtle but completely depend on context. 
Junk DNA (no effect)
Promoter regions (mRNA levels)
UTRs (tissue distribution, mRNA stability) 
Coding regions (synonymous and nonsynonymous) 
Splice sites (altered splice sites)
Introns (splicing enhancers/ silencers)
Premature stop codons (truncated proteins) 
Single nucleotide variations that alter protein activity may increase or decrease it by a small or large degree.
Silent mutations may change the splicing of the mRNA, as can intronic mutations.