DNA repair

Question 1

mismatch repair

Answer 1

1) Dam methylase - methylates n6 of adenine in sequence 5’-GATC-3’ (Newly synthesized DNA is unmethylated because it takes time to scan the genome)
2) MutS and MutL (with ATP hydrolysis) bind together at the bulge caused by mismatched bases
3) MutH binds to GATC sequences and the MutS:MutL complex pulls the dna towards it forming a loop
4) Once MutH (exonucleade) binds to MutL, MutH cleaves the strand opposite to the methylated base.
5) Helicase II works towards mismatch to unwind dna followed by an exonuclease that digests the DNA.
6) ssb binds the single stranded dna to stabilize it prior to reconstruction
7) polymerase fills the gap and ligase seals the left over nick

Question 2

photolyases (direct repair; no replacement of lesion)

Answer 2

1) chromophore of photolyase enzyme absorbs a photon of visible light and passes the excitation energy to FADH-
2) FADH- donates the electron (radical) to catalyze bond rearrangements in the pyrimidine dimer
3) radical rearrangement restores the pyramidines and the electron is transferred back to FADH to regenerate FADH-

Question 3

dealkylation (methyltransferase; direct)

Answer 3

accept methyl groups using a cysteine residue

Question 4

AlkB (enzyme)

Answer 4

oxidizes the methyl group into formaldehyde by a Fe(II) dependent mechanism

1-methyladenine and 3-methylcytosine

vitamin C is a necessary cofactor to recycle the iron

• AlkB, Fe(II), alpha-Ketoglutarate and oxygen for rxn

-alpha-Ketoglutarate turns to succinate (loses co2) then

Question 5

base excision repair (prokaryotic)

Answer 5

• for bases that have been chemically modified that result in small, local distortions
  -glycosylases recognize damaged bases by flipping it into their active site. Every different type of damage is recognized by a different enzyme

1) DNA glycosylase recognizes the damaged base and cleaves the glycosidic bond between base and sugar forming an AP/abasic site. AP sites can also occur spontaneously and this is fixed by BER as well.

2)AP endo nuclease cleaves the DNA containing the AP site.

3)DNA polymerase uses nick translation (prokaryotic). With the 5’ to 3’ exonuclease activity, it removes a short segment of DNA starting with AP site and replaces it with new DNA.

4) DNA ligase seals remaining nick

Question 6

Long patch repair (eukaryotic)

Answer 6

mechanistically similar to prokaryotic BER. Pol III fills in the remaining segment and the overhanging single stranded nuclei’s acid generated by strand displacement is removed by a flap endonuclease.

Question 7

short patch eukaryotic repair

Answer 7

The single a basic site is replaced by a new nucleotide using PolB and the gap sealed by DNA ligase.

Question 8

Nucleotide Excision Repair

Answer 8

-No specific mode of recognition between protein and damage. Dominant active system of repairing DNA
-UVR proteins

1) Initial complex (UVRA2:UVRB) slides down the DNA, looking for damage. When it hits damage, it separates the DNA, forming a bubble.

2) UVRA leaves DNA. UVRB still bound to the lesion. UVRC (exinuclease; cuts twice on a single strand) binds to UVRB and cuts 8 nucleotides on the 5’ side of lesion and 5 nucleotides on the 3’ side of lesion.

3) UVRD (helicase) frees the nicked strand

4) Pol 1 fills in gap

5) ligase seals

Question 9

NHEJ (non homologous end joining)

Answer 9

• double stranded break
• no error free template

The ends of the break are digested and modified for relocation. This induces mutations and loss of fidelity

Question 10

translesion polymerases

Answer 10

happens when strands separate

translesion polymerase skips over and continues

Question 11

Deamination

Answer 11

Cytosine —-> Uracil (base pairs with T)
Adenine —-> Hypoxanthine (bp cytosine)
Guanine —> Xanthine (bp to cytosine)
5-methylcytosine —> Thymine (bp to Adenine)