Lecture 3
what are the main proteins involved in TCR
CSA and CSB -which are activated when RNA polymerase is stalled -TFIIH and XPD/B are the early subunits of nucleotide excision repair pathway in human cells -recruitment to a highly transcribed region mediated by CSA/CSB at the stalled RNA polymerase site
TLS in E. coli
DNA polymerase III stalled -relies on alternative trans-lesion polymerases that are able to replicate through the lesion by substituting the canonical polymerase in bacteria, Pol IV or Pol V can do that job
double-strand breaks also form during
DNA replication (most common andogenous source)
consequence for humans if we have a mutation in pol-eta
Two A's not inserted correctly --> leads to less ability to correct TT dimers --> more sensitivity to the mutagens that create TT dimers, specifically UV light, which creates skin cancer
what are the main components in nucleotide excision repair in bacteria?
UvrA/UvrB UvrC UvrD helicase, polymerase, ligase
the gap-filling model works very well in
a lagging strand, where u already have formation of Okazaki fragments (already have gaps left behind) (unlikely to work this way in a leading strand where polymerase works highly processively until the end)
DS breaks form rarely but they are the most dangerous DAN lesions because
a single unrepaired DSM triggers cell death/genomic instability (an interruption fo the continuity of the DNA molecule that can lead to losing entire arms of chromosomes in mitosis, for example)
trans-lesion synthesis
allows damage tolerance, not repair -allows for replication, so you get 2 DNA molecules at the end, but lesion is not fixed so you still have problems at that site
trans-lesion synthesis TLS) replicates across a damage with
specific low-fidelity DNA polymerases
If RNA polymerase is producing a transcript and a sudden TT dimer is generated by exposure to UV light...
that stalled polymerase leads to recruitment of CSA/CSB which can facilitate recruitment of corresponding subunits for nucleotide excision repair to that site
nucleotide excision repair in bacteria does what (generally)
removes a long stretch of ssDNA
difference between the polymerase-switching and ga-filling model
-polymerase-switching occurs at the replication fork (will just continue progressing and just has a temporary substitution fo the polymerases) gap-filling - the trans-lesion polymerase can work behind the fork
difference between DSB that happen during replication from those that happen from a nuclear bomb or nuclear plant disaster
-the repair mechanisms have evolved to deal w/ spontaneous damages - occur during a normal cell cycle -but damages that occur in presence of nuclear accidents or atamoic bombs can occur in any phase of the cell cycle -so our cells are very well equipped w/ mechnaims to repair with spontaneous damage DS breaks, but not so much for those that occur outside of replication for instance, 160/cell is way too much for our cells to deal with and that's why the organism eventually declines and the cells simply die
so why does this mechanism exist?
-this is still better than having replication fork stalled for a long time, because that can lead to ds break (which are much more complex region to repair, and can lead to permanent damage) -so these pathways exist to alleviate risk for ds breaks during replication
explain the steps of nucleotide excision repair in E. coli.
1. UvrA/UvrB (tetrameric complex) scans the DNA for distortions -TT dimer recognized and they bind to it and associate with this region of the DNA 2. UvrA leaves and UvrB remains to keep the DNA helix open so the repair pathway can proceed 3. One of the two subunits of UvrB is released and another protein comes in (UvrC) UvrC is dumbbell-shaped and has nickase activity -introduces 2 nicks - one upstream (8 nucleotides) and one downstream (4-5 nucleotides) of the TT dimer 4. UvrD (helicase) comes in and separates this strand of DNA from the rest, creating a gap -gap is filled by polymerase and ligase to restore the info lost in the TT dimer
in nucleotide excision repair in humans, how many proteins are involved?
25 (XPA, C, D, F, G...)
how many trans-lesion polymerases are found in human cells?
5
transcript-coupled repair (TCR) uses:
TFIIH to couple transcription with nucleotide excision repair in eukaryotes
pol-eta
an exception in human cells, pol-eta correctly inserts two A's when it encounters a TT dimer (recruited specifically in the presence of unprepared TT dimers during replication and it inserts two A's in front of the dimer, inserting correct bases even though not based on base-pairing) (only able to insert A's)
why do these rearrangements happen?
because there were DS breaks in the cell that were not correctly processed and that lead to the fusion of 2 different chromosomes - extremely common in cancer cells and leads to chromosome gain, chromosome loss, additional copies of genes, loss of genes And if genes are onco supressors, you can imagine that leads to advancement of the cancer So the more DS breaks are repaired incorrectly, they can help the cancer become more aggressive over time Can replicate with these abnormalities
Xeroderma pigmentosum
caused by nucleotide excision repair mutation -extreme sensitivity to light -skin cancer
Zeroderma pigmentosum
caused by pol-eta mutations (a variant of XP) symptoms: -mild-to-severe UV sensitivity -late onset of skin cancer
Mutations in CSA/CBS lead to
cockayne syndrome
UV radiation causes
crosslinks between adjacent pyrimidine bases - blocks DNA replication (TT dimers)
what are the repair pathways for UV light?
damage reversal nucleotide excision repair
but, in general, even if repair pathways are constantly active to prevent damages from being around during replication, some will
escape repair
cockayne syndrome
from mutations in transcription-coupled repair defective DNA repair of transcribed regions --> a lot of transcript with be aborted or will incorporate the wrong sequences, but in addition, less transcription in regions that should be highly transcribed symptoms: -mental retardation -developmental defects -UV sensitivity, skin cancer -premature aging -patients usually die by age 6-7
majority of trans-lesion synthesis mechanisms are
highly mutagenic -introduce incorrect bases in front of DNA lesion by incorporating incorrect base or TT dimer
how can replication forks be a source of ds breaks with nicks/gaps?
if a fork arrives and has to go through a site that contains a nick or gap (that have not been repaired at the time when the fork arrives) that can lead to the replisome falling off (fork collapse) -one of the two filaments of DNA will not be replicated --> precocious end of polymerization, leading to termination of filament (incomplete replication)
explain the karyotype of a cancer cell
in a normal cell, u have 2 copies of each chromosome (one maternal, one paternal) here, abnormalities are shown for example, additional chromosome fused to one copy of chromosome 3, two additional copies of chromosome 5 - one is just partial, one is also subject to rearrangements
Double strand breaks are caused by _____. sources: (exogenous)
ionizing radiation (IR) exogenous sources: -radioactive materials (ex: uranium from the environment) -cosmic rays (more so when u fly) -medical x-ray imaging -cancer therapy -nuclear power plants -scientific research
how does pol-eta work?
its structure pol-eta's active site has a pocket that can only accommodate a TT dimer (whereas other polymerases have amino acids in that pockets that don't allow for the TT dimer to sit there) -can only incorporate dATP in front of the dimer --> only inserts 2 A's across from dimer
nucleotide excision repair is a versatile pathway
mostly for UV damage, but some of these distortions can come from additional types of damages - damaged bases that are not promptly recognized by base excision repair that remain in the cells as well as large adducts that don't have a DNA glycosilase that can deal with that specific lesion can activate nucleotide excision repair and generate single stranded DNA and repair the lesion
how can replication forks be a source of ds breaks? (two main ways)
nicks/gaps or lesions
how can replication forks be a source of ds breaks with lesions?
normally, if u have an unprepared lesion (such as an unprepared TT dimer), and the fork goes through, there are tolerance mechanisms to enable replication through the lesion and continue HOWEVER, there will be a few lesions that escape these additional mechanisms --> stalled fork in front of lesion if that persists for long enough, will eventually result in fork collapse (replisome falls off) -by base pairing, these two filaments will pair with each other, leading to a structure called a regressed foot or "chicken foot" -the chicken foot, at the left end, is read as a ds break and will be processed as a double stranded break -but also the the structure on the other end (part that looks like a four-way junction) can be recognized as a ds break repair intermediate, which can be cut by endonuclease, creating additional break at the fork
why is Zeroderma pigmentosum less severe than XP?
nucleotide repair is still there, as well as trans-lesion repair -so still able to repair the majority of UV damage -but this disease shows that pol-eta is critical for dealing with UV damage
what are the two models
polymerase-switching gap-filling
these proteins would eventually recognize TT dimers anyway, but CSA and CSB
promote recruitment -so cells dont stay for a long time with T T dimers at highly transcribed sites
gap-filling model
the translation polymerase can work behind the fork -replicative polymerase encounters lesion, but it cannot go. through the lesion, so it is released and a new polymerase is recruited downstream from the lesion, leaving a gap behind -the gap left behind promotes recruitment of a trans-lesion polymerase to replicate through the gap, random nucleotides are added, and than the trans-lesion polymerase is released
trans-lesion polymerases work differently than canonical bases because
they can incorporate bases independently of base pairing (consequence is that random bases can be incorporated, creating a mismatch situation) ---but this is better than have a stalled polymerase!
why is this important for our cells?
to assure abnormal base is not part of a transcript, but also to allow for cells to not have RNA polymerase stalling for a long time if RNA pol. cannot go through a gene that is normally highly transcribers, you will experience a much lower level of that transcript -not having a lot of that highly needed gene product for a long time = bad results
defective repair in DSB's results in
translocations in cancer cells
What triggers the switch to a trans-lesion polymerase in mammalian cells?
ubiquitination of PCNA
how does TLS work?
when DNA polymerase 3 encounters a lesion, it is released from the replication site then a Trans-lesion polymerase comes in and replicates through the region by incorporating some random nucleotides trans-lesion polymerases are not very prosessive, so they are quickly released from that site, so a normal polymerase can be re-recruited and replication can continue downstream from that lesion
