Chapter 10 - The Mutability of Repair of DNA

Question 1

Explain the difference between transition and transversion mutations.

Answer 1

Transistions - pyridine-to-pyrimidine and purine-ro-purine substitutions (T to G and A to G).

Transversions - pyrimidine-to-purine and purine-to-pyrimidine substitutions.

Question 2

What are DNA microsatellites?

Answer 2

Mutation-prone sequences with di-, tri-, or tetra nucleotide sequences. Example: CA. Due to slippage of DNA polymerase.

CAG repeats cause polymorphism –> multiple extension causes polyglutamine stretches –> ex. Huntington’s disease.

Question 3

Which protein detects mismatches in E. coli? How is this proteins specificity determined?

Answer 3

MutS. DNA containing a mismatch is much more readily distorted than properly base-paired DNA.

Question 4

Explain how mismatch repair functions in E. coli.

Answer 4

MutS scans the DNA –> recognises a mismatch. The complex then recruits MutL. MutL activates MucH, an enzyme that causes an incision or nick on one of the strands. Nicking is followed by a helices (UvrD) and one of the three exonucleases. The helices unwinds the DNA and the exonucleases progressively digest the displaced single strand. The gap is filled in with DNA polymerase III.

Question 5

How does the E. coli mismatch repair system know which of the two mismatched strands to repair?

Answer 5

E. coli tags the parental strands by transient hemimethylation. Dam methylase methylates a residues on both strands of the sequence 5’-GATC-3’. For a few minutes, until Dam methylase catches up the replication fork –> only the daughter DNA duplex will be methylated.

Question 6

What are the proteins for mismatch repair called eukaryotic cells?

Answer 6

MutS –> MutS homologs (MSH)
MutL –> MutL homolog (MLH)
and PMS

Question 7

How does the mismatch repair system know which of the two mismatched strands to repair in eukaryotes?

Answer 7

Okazaki fragments. MHS interacts with the sliding-clamp and would thereby be recruited tot he site of discontinuous DNA synthesis on the lagging strand.

Question 8

What is the most common kind of hydrolytic damage?

Answer 8

Deamination of cytosine.

Question 9

What occurs when cytosine is deaminated?

Answer 9

Cytosine undergoes spontaneous deamination, generating uracil. Uracil preferentially pairs with adenine and thus introduces that base in the opposite strand upon replication.

Question 10

What occurs when adenine is deaminated?

Answer 10

Hypoxathine, which hydrogen bonds to cytosine rather than to thymine; guanine is converted to xanthine, which continues to pair with cytosine, although with only two hydrogen bonds.

Question 11

Explain what occurs when DNA undergoes depurination.

Answer 11

By spontaneous hydrolysis of the N-glycosyl linkage, and this produces an basic site.

Question 12

Explain what occurs when DNA undergoes alkylation?

Answer 12

Methyl and ethyl groups are transferred to reactive site on the base and phosphates int he DNA backbone.

Question 13

Explain how DNA is damaged bu ultraviolet light?

Answer 13

Radiation with wavelength 260 nm is strongly absorbed by the bases, one consequence is the photochemical fusion of two pyrimidines that occupy adjacent positions not he same polynucleotide chain.

Question 14

What does clastogenic mean?

Answer 14

Ionizing radiations and agents that cause DNA to break.

Question 15

What are intercalating agents? What how do these agents causes short insertion and deletions?

Answer 15

Flat molecules containing several polycyclic rings that bind to the equally flat purine and pyrimidine bases of DNA. They slip between the bases in the template strand and causes the DNA polymerase to insert an extra nucleotide opposite the intercalated molecule.

Question 16

What is excision repair?

Answer 16

The damaged nucleotide is note repaired but removed from the DNA. A new nucleotide is incorporated by DNA polymerase.

Question 17

What is recombinational repair?

Answer 17

Repair when both strands are damaged. Sequence information is retrieved from a second undamaged copy of the chromosome.

Question 18

What is photoreactivation?

Answer 18

Reverses the formation of pyrimidine dimers.

Question 19

Explain base excision repair and nucleotide excision repair.

Answer 19

In base excision repair, an enzyme called glycosylase recognises and removes the damaged base by hydrolysing the glycosidic bond. The resulting basic sugar is removed from he DNA backbone in a further endonucleolytic step. Endonucleolytic cleavage also removes apurinic and apyrimidinic sugars that arise by spontaneous hydrolysis. (Endonuclease cuts the DNA at the 5’ position of the AP site, leaving the 3’ OH, exonuclease cuts the 3’position of the AP site, leaving the a 5’-phosphate. The resulting gap is fulled in by DNA polymerase I)

In excision repair, the enzyme recognises distortion to the shape of the helix. ATP hydrolysis promotes dimer formation by UvrA, which formed a complex with a dimer UvrB. This complex scans the DNA to identify distortions. UvrA leaves the complex, and the remaining UvrB dimer melts DNA locally around the distortion. UvrC forms a complex with UvrB and creates nicks 3’to the lesion and 5’ lesions. DNA helices UvrD releases the single-strand fragment from the duplex and DNA pol ! and ligase repair and seal the gap.

Question 20

Transcription-coupled repair.

Answer 20

Rescuing RNA polymerase after being arrested by the presence of lesion in the transcribed strand of a gene.
Recruitment to the stalled RNA polymerase of nucleotides excision repair proteins.

Question 21

When is NHEJ used?

Answer 21

When an unreplicated chromosome suffers a break and there is no sister chromosome present to serve as a template in the recombination-based DSB repair pathway.

Question 22

Describe the mammalian pathway of NHEJ.

Answer 22

A heterodimer of Ku70 and Ku80 binds to the broken DNA and recruits the protein kinase DNA-PKcs. DNA PKcs in turn receipts Artemis, an enzyme having exonuclease and endonuclease activities, which processes the broken ends. Finally a complex of Ligase IV with XRCC4 and Cemunos-XLF joins the broken ends to each other.

Question 23

Explain translesion synthesis.

Answer 23

Fail-safe mechanism that lets the replication machinery bypass the site of damage or tolerate the DNA damage.

Question 24

Which family DNA polymerases catalyses translation synthesis?

Answer 24

In E. coli –> DNA Pol IV or DNA Pol V. DinB and UmuC are members of a distinct family of DNA polymerases found in many organisms known as the Y family of DNA polymerase.

Question 25

What are transposons?

Answer 25

So called jumping genes that insert themselves randomly in the DNA sequence.

Question 26

Name chemical agents that cause DNA damage.

Answer 26

For example Acrylamide, monosodium glutamate.

Question 27

Name the different types of mutation.

Answer 27

1. Point mutations –> substitution, insertion ot deletions.
2. Insertions and deletions (often caused by transposons)
3. Base substitution –> Transition or transversion.

Question 28

What is the result of deamination of purines?

Answer 28

Adenine –> Hypoxathine

Guanine –> Xanthine

Question 29

Name the 3 main DNA repair mechanisms.

Answer 29

1. Mismatch repair - by proofreading activity of DNA polymerase.
2. Direct repair - by photo reactivation of the pyrimidine dimers where the offending covalent bonds are reversed by DNA photolase.
3. Excision repair - involves endonuclease, helices, DNA polymerase enad ligase.

Question 30

What is the most common UV irradiation damage? How is it repaired?

Answer 30

Thymine dimers –> they causes distortion in the double helix. Usually repaired by excision repair or by photolase.

With nucleotide excision repair a multi enz. complex recognises bulky t-dimers. One cut is made on both sides of the lesion. About 12 bases are removed and DNA pol and ligase seal the gap

Question 31

How does DNA glycosylase mediate base excision?

Answer 31

The unusual bas flips out from the DNA, this causes a distension in the DNA helix-. Glycosylase recognises this distortion and removes the base.

Question 32

Explain the SOS response.

Answer 32

SOS response is triggered by a block in DNA replication and involves expression of cascades of repair enzymes. The signal is excess of single stranded DNA. It first activates recA protein. RecA removes suppression of the SOS response genes by cleaving LexA repressor. This induces translation repair by UmuC and UmuD. These polymerases start synthesis of DNA from the mismatch site.

Question 33

Explain the mechanism of DNA damage tolerance by translesion synthesis in E. coli.

Answer 33

DNA pol IV or DNA Pol V (a complex of the proteins UmuC and UmuD) performs translation synthesis. DinB and UmuC are members of a distinct family of DNA polymerase found in many organisms known as the Y family of DNA polymerases. Upon encountering a lesion in the template during replication, DNA pol III with its sliding clamp dissociates from the DNA and is replaced by the translesion DNA polymerase, which extends DNA synthesis across the thymine dimer not he template strand.Translesion polymerase is then replaced by the DNA polymerase III.