Sept 25 DNA Repair and Recombination

Question 1

what are mutations and how can they occur?

Answer 1

permanent, transmissible changes to the genetic material of a cell (or organism). mutations can occur spontaneously, by transposable elements (segments of DNA that can move) and by errors during replication

Question 2

what are mutagens?

Answer 2

chemical compounds, UV radiation or ionising radiation that increase the frequency of mutations

Question 3

what happens if mutation are in somatic cells vs gametes?

Answer 3

if the mutation happens i the gametes, the mutation will be passed on to the offspring
if it is in a somatic cell, it will not be passed on but the somatic cell will be affected

Question 4

what are carcinogens?

Answer 4

agents that cause cancer, many carcinogens are mutagens

Question 5

what can defects in DNA repair systems cause?

Answer 5

some human genetic diseases and cancer susceptibility syndromes

Question 6

what is the accuracy of DNA polymerases without and with proofreading?

Answer 6

without proofreading: 1 error in every 10,000 nucleotides
with proofreading: 1 in every 1,000,000,000 (billion) nucleotides

Question 7

how does the polymerase proofreading mechanism work?

Answer 7

mistakes can be corrected as soon as they are made
DNA polymerases epsilon and delta (not alpha) have a 3’ to 5’ exonuclease, or “proofreading” activity
incorporation of an incorrect base causes the polymerase to pause
the 3’ end of the new strand is free to move to the 3’ –> 5’ exonuclease site and the mispaired base is removed

Question 8

what is exonuclease?

Answer 8

an enzyme that digests DNA

Question 9

what is the most common point mutation (single base change)?

Answer 9

from C to T
methylcytosine is deaminated (amino group is lost and replaced by a ketone group)
cytosine changes into uracil
this causes a wrong base pairing

Question 10

when must the repair occur to make sure that it is fixed?

Answer 10

before replication, because if that strand replicates there will be no wrong match

Question 11

how does the system recognise which base is the wrong one?

Answer 11

in a T-G mismatch, it almost always comes from deamination from C to U or 5mC to T, so T is wrong and should be replaced by C

Question 12

how does base excision repair (BER) work?

Answer 12

1. DNA glycosylase breaks the bond between T and the sugar phosphate backbone
2. APEI endonuclease recognises an abasic site and cuts the DNA strand on the sugar phosphate backbone where it is missing a base
3. AP lyase removes deoxyribose phosphate
4. DNA polymerase beta, a special DNA polymerase, inserts C using G as a template
5. ligase repairs the sugar phosphate backbone
   same thing happens when UG wrong base happens

Question 13

what are mismatch errors?

Answer 13

errors introduced during replication
base pair mismatches and insertions/deletions of one or a few nucleotides (repeat and skip over)

Question 14

how does the system recognise which strand is the wrong one in mismatch errors?

Answer 14

the newly synthesised strand is the wrong one, since the error is introduced during replication
every organism has a way to recognise which strand is the wrong one

Question 15

how does mismatch excision repair work?

Answer 15

happens after DNA replication
1. MSH2 and MSH6 recognise the mismatch and distinguish the newly synthesised daughter strand
2. this triggers the binding and the acitivity of MLH1 endonuclease (dimerised with PMS2)
MLH1 cuts the newly synthesised strand
3. DNA helicase unwinds the helix and DNA exonuclease digests several nucleotides of the daughter strand
4. DNA polymerase delta fills in the missing nucleotides using the other strand as a template and ligase repairs the sugar phosphate backbone

Question 16

what is the difference between endonuclease and exonuclease?

Answer 16

endonuclease cuts in the middle
exonuclease cuts from the ends

Question 17

what does nucleotide excision repair fix?

Answer 17

fixes DNA regions where chemically modified bases locally distort the double helix

Question 18

what is an example of bases distorting the helix?

Answer 18

with the influence of UV radiation, two thymine bases adjacent to each other can become covalently bound, and create a kink in the structure of the double helix

Question 19

how does nucleotide excision repair work?

Answer 19

1. XP-C and 23B proteins recognise the distorted double helix
2. TFIIH (helicase), XP-G and RPA unwind the helix to make a bubble of ~25 nucleotides
3. XP-F and XP-G (nucleases) cut the damaged strand
4. DNA polymerase fills in the missing nucleotides using the other strand as a template and ligase repairs the sugar phosphate backbone

Question 20

where do the names XPn derive from?

Answer 20

xeroderma pigmentosum
a genetic disease that causes a high disposition to UV induced cancer
mutations that affect XP proteins cause this disease

Question 21

what happens if a thymine-thymine dimer is not repaired and it enters a replication fork?

Answer 21

normal replicative DNA polymerases (delta and epsilon) stall when they reach a T-T dimer
polymerase eta can read through T-T dimer, but this polymerase does not have proofreading activity
therefore the region in the vicinity of the dimer will be likely to contain mutations caused by replication errors
eventually pol eta gets replaced by the normal polymerases

Question 22

what is a double strand break, what causes it and what happened if it is not fixed?

Answer 22

radiation (x-rays and gamma rays) and some anti cancer drugs (since they target DNA replication) cause double stranded breaks in DNA
if a double strand break is not repaired then the part of the chromosome distal to the break would be lost at the next cell division, causing lethality

Question 23

how does non homologous end joining (NHEJ) repair work?

Answer 23

Ku and DNA-PK bind the end of a double stranded break (DSB)
when two DBS ends bounds by these proteins come together, they recruit nucleases that remove several bases
the two double stranded molecules are then ligated together
this mechanism does not ensure that what is ligated together originated from adjacent parts of the same chromosome
this can produce chromosomal rearrangements

Question 24

what does double stranded break repair by homologous recombination do?

Answer 24

the damaged DNA is replaced with a copy of an undamaged copy of the same sequence on the homologous chromosome in diploid organisms
involves an exchange of strands between separate molecules, process of recombination

Question 25

how are BRCA1 and BRCA2 involved in recombination?

Answer 25

mutations in which are associated with increased susceptibility to breast cancer, encode proteins involved in homologous recombination repair

Question 26

what is an example of a damage that can be repaired through homologous recombination?

Answer 26

a collapsed replication fork

Question 27

how does double strand break repair by homologous recombination work?

Answer 27

look at notes on ipad (need pictures)