Sept 25 DNA Repair and Recombination Flashcards
what are mutations and how can they occur?
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
what are mutagens?
chemical compounds, UV radiation or ionising radiation that increase the frequency of mutations
what happens if mutation are in somatic cells vs gametes?
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
what are carcinogens?
agents that cause cancer, many carcinogens are mutagens
what can defects in DNA repair systems cause?
some human genetic diseases and cancer susceptibility syndromes
what is the accuracy of DNA polymerases without and with proofreading?
without proofreading: 1 error in every 10,000 nucleotides
with proofreading: 1 in every 1,000,000,000 (billion) nucleotides
how does the polymerase proofreading mechanism work?
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
what is exonuclease?
an enzyme that digests DNA
what is the most common point mutation (single base change)?
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
when must the repair occur to make sure that it is fixed?
before replication, because if that strand replicates there will be no wrong match
how does the system recognise which base is the wrong one?
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
how does base excision repair (BER) work?
- DNA glycosylase breaks the bond between T and the sugar phosphate backbone
- APEI endonuclease recognises an abasic site and cuts the DNA strand on the sugar phosphate backbone where it is missing a base
- AP lyase removes deoxyribose phosphate
- DNA polymerase beta, a special DNA polymerase, inserts C using G as a template
- ligase repairs the sugar phosphate backbone
same thing happens when UG wrong base happens
what are mismatch errors?
errors introduced during replication
base pair mismatches and insertions/deletions of one or a few nucleotides (repeat and skip over)
how does the system recognise which strand is the wrong one in mismatch errors?
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
how does mismatch excision repair work?
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
what is the difference between endonuclease and exonuclease?
endonuclease cuts in the middle
exonuclease cuts from the ends
what does nucleotide excision repair fix?
fixes DNA regions where chemically modified bases locally distort the double helix
what is an example of bases distorting the helix?
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
how does nucleotide excision repair work?
- XP-C and 23B proteins recognise the distorted double helix
- TFIIH (helicase), XP-G and RPA unwind the helix to make a bubble of ~25 nucleotides
- XP-F and XP-G (nucleases) cut the damaged strand
- DNA polymerase fills in the missing nucleotides using the other strand as a template and ligase repairs the sugar phosphate backbone
where do the names XPn derive from?
xeroderma pigmentosum
a genetic disease that causes a high disposition to UV induced cancer
mutations that affect XP proteins cause this disease
what happens if a thymine-thymine dimer is not repaired and it enters a replication fork?
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
what is a double strand break, what causes it and what happened if it is not fixed?
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
how does non homologous end joining (NHEJ) repair work?
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
what does double stranded break repair by homologous recombination do?
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
how are BRCA1 and BRCA2 involved in recombination?
mutations in which are associated with increased susceptibility to breast cancer, encode proteins involved in homologous recombination repair
what is an example of a damage that can be repaired through homologous recombination?
a collapsed replication fork
how does double strand break repair by homologous recombination work?
look at notes on ipad (need pictures)
