Lecture 26 - Replication fidelity, DNA repair and recombination (pt 2) Flashcards
What nucleotide excision repair mechanism repairs
Chemical changes in DNA that affect normal helical structure
What induces chemical changes in DNA that affect normal helical structure
UV light
How chemical changes in DNA that affect normal helical structure are detected
Sensor proteins slide along for DNA looking for kinks in the helical structure
Example of chemical changes in DNA due to UV light that affect helical structure
Adjacent thymine bases covalent bonding
Protein that recognizes thymine dimers
XP-C/23B complex (dimer)
XP-C = big protein that surrounds it
23 B = protein that binds XP-C
First protein that XP-C/23B complex recruits and what it does
Transcription factor TFIIH (is a DNA helicase). Unwinds the DNA
2 proteins that further unwind the DNA after TFIIH and how what they exactly do
XP-G and RPA (replication fork protein A). Further unwind until a 25 nt bubble is formed
How TFIIH, XP-G and RPA are positionned together
TFIIH thymine dimer side (figure : top)
RPA on A A strand side (figure : bottom)
XP-G on right
2 proteins required/recruited after 25 nt bubble is formed and what they do
XP-G (already there) and XP-F = 2 ENDOnucleases
XP-G cuts at 3’ end of bubble (on T dimer strand)
XP-F cuts at 5’ end of bubble (on T dimer strand)
After endonucleases (XP-G and XP-F) action, what happens to 25nt strand that was cut away
is degraded
How 25nt strand that was cut away is replaced
DNAP and DNA ligase fill the gap
Name of cancer syndrome in families that lack genes for proteins in nucleotide excision repair
xeroderma pigmenosum (XP)
What induces double-strand DNA breaks
X-rays and gamma-rays
2 systems that repair double-strand DNA breaks
1) Homologous recombination
2) Nonhomologous end joining
Principle of homologous recombination and how perfect repair can be
Uses unbroken homologous chromosome as a repair template
Can make a perfect repair
Nonhomologous end joining difference with homologous recombination and how perfect repair can be
does not use unbroken homologous chromosome as repair template
error-prone repair (always errors)
Homologous recombination step 1
Generate 3’ ssDNA tails at the break site (on broken chromosome) by using 5’-exonucleases
Homologous recombination step 2
Use Rad-51 to invade (hybridize with) the template chromosome with one of the two tails (part of a strand on the template chromosome makes a bubble cause not coupled anymore)
What hybridization of 3’ ssDNA tail of break site to template chromosome allows
perfect alignement of the two chromosomes
Homologous recombination step 3
3’ end of invading tail is extended until DNA in ‘‘bubble’’ base-pairs with other 3’ ssDNA tail. (bubble is now bigger + one of two 3’ ssDNA tails is now repaired)
Homologous recombination step 4
Extension of the second (non-invading 3’ ssDNA tail) using the strand of the template chromosome that is not hybridized (that forms a bubble)
Homologous recombination step 5
DNA ligase ligates the repaired strands
Structure left after Homologous recombination + name
4-stranded DNA structure, all bases paired, but with a bubble of 2 strands mixing with other chromosome ->HOLLIDAY STRUCTURE = EACH CROSSING AT BOTH ENDS OF BUBBLE (so there are 2)
When nonhomologous end joining is used
when homologous chromosome that can act as a template is not nearby
First step of nonhomologous end joining/what happens at broken ends
A complex of Ku heterodimer + a DNA-dependent protein kinase binds each broken end of the chromosome AND FORM A SYNAPSE
Second step of nonhomologous end joining
Several nts are removed from broken end by nucleases
Third step of nonhomologous end joining
Ligation of the chromosome fragments together
2 reasons why nonhomologous end joining is an error-prone repair
1) Small deletions at junction point
2) Could join wrong chromosome fragments (if, for example, chrom 6 and 10 are being repaired at the same time)
What is metaphase
Step in mitosis where chromosomes aligned in the middle of the cell
What happens to chromosomes in cells grown in presence of drug that induces DNA double strand breaks
Due to the lot of homologous recombination occuring, the chromosomes’ strands are recombinated
What method allowed the detection of homologous recombination and what the method does
Bodycote and Wolff method : Differentially stains sister chromatids at metaphase
why genetic recombination leads to different looks within the population of humans
Because of alleles (gene variants)
consequence of homologous recombination if no gene variants
Homologous recombination would lead to identical chromosomes
Hypothesis for why sexual reproduction is prevalant (as opposed to asexual reproduction)
Allelic diversity generates phenotyping diversity
What happens during metaphase 1 of meiosis
strand breaks are induced
why strand breaks are induced during metaphase 1 of meiosis (WHICH OF THE TWO TYPES OF dsDNA BREAKAGE REPAIR OCCURS)
half of the time, they will result in HOMOLOGOUS RECOMBINATION so advantage for progeny diversity
How many of the gametes have a recombined version of the chromosome
2 of the 4
2 ways of solving a Holliday structure
1) Nicking one pair of strands restores original chromosomes
2) Nicking other pair of strands generates recombinant chromosomes
How many possibilities for solving the 2 Holliday structures after homologous recombination and how many lead to recombinant DNA
4 possibilities. 2 lead to recombinant DNA
