DNA repair in terminally differentiated cells
This page summarizes research interests
in the Nouspikel lab, and outlines current and future directions of research
Click on any picture to enlarge it.
DNA repair is a daunting task
Even though DNA is pretty stable, we have so much of it that its decay
pauses quite a challenge: due to spontaneous depurination we loose
about 3000 purines per cell per day. In addition, because of byproducts
of the oxygen metabolism within each cell, several hundreds of
pyrimidines become oxidized every day. As if this were not enough, DNA
duffers multiple aggressions from the environment: from UV rays in
sunlight, to chemical carcinogens in food, cigarette smoke, or polluted
To cope with this damage load, we have evolved a whole set of DNA
Maintaining that many systems in working order does not come cheap: NER
alone involves over 30 polypeptides that constantly scan our genome. To
which one should add the energy cost of carrying the repair
operation itself. The energy expense is critically important, though,
as illustrated by the variety of
horrible diseases (generally cancer prone)
caused by defects in a
given repair system.
- A few specialized enzymes can directly reverse a given lesion
(e.g. methylation or UV-induced damage).
- Nucleotide excision repair (NER) takes care of UV-induced
lesions, chemical adducts, DNA crosslinks, etc.
- Base excision repair (BER) deals mainly with small modifications
of a base: oxidations, methylations, etc.
- Mismatch repair corrects mismatched nucleotide pairs
- Stand break repair tackles double-strand breaks, such as those
caused by ionizing radiation.
Instability and decay of the primary
structure of DNA Lindahl,T.
Quality control by DNA repair
& Wood,R.D. Science
The "parsimony" hypothesis
Terminally differentiated cells never replicate their DNA, so why
should they bother repairing it? Think of all the energy they could
save by dispensing with producing DNA repair enzymes, not to mention
performing the actual repair.
Of course cells need to maintain the
few genes that they are currently using, but this can be done by a
mechanism called Transcription-Coupled Repair (TCR). It is generally
believed that TCR
is achieved by RNA polymerase II, which calls repair enzymes for help
when it encounters a blocking DNA lesion.
Subpathways of nucleotide excision
repair and their regulation
Oncogene 21:8949-8956 (2002)
Repair phenotype of human neurons
This is precisely the phenotype that we have observed in human neurons,
perhaps the epitome of differentiated cells. Because it is known to be
TCR, we initially focused on Nucleotide Excision
neurons show a marked drop in NER at the global genome level. By
contrast, transcribed genes are still proficiently repaired on both
DNA strands. The latter is quite a surprise: TCR only works
on the transcribed strand, as RNA polymerase II is not affected by
lesions on the non-transcribed strand. The non-transcribed strand is
normally repaired by NER like the bulk of the genome, but in neurons
the bulk of the genome is barely repaired at all. So what is repairing
the non-transcribed strand in neurons? We decided to call it
Differentiation-Associated Repair (DAR) and elucidating its mechanism
is one major goal of the lab.
But of course, DAR makes a lot of sense retrospectively. The way TCR
(and global NER) work is by removing a ~30-nucleotide piece of DNA
around the lesion, then filling the gap using the other strand as a
template. If neurons were to let the non-transcribed strand fall in
dereliction, sooner or later TCR would run into problems, when
presented with a damaged template (click on picture for details).
Terminally differentiated human
neurons repair transcribed genes but
display attenuated global DNA repair Nouspikel
& Hanawalt, Mol.
(2000) (download pdf file 138 K)
Answers from macrophages
At this point, there were two main questions that need to be addressed:
Because neurons are difficult to work with (you can get so few of
switched to another type of terminally differentiated cells:
These have essentially the same repair phenotype as mature neurons: low
global NER, proficient TCR, and presence of DAR. But the advantage is
that the precursor cells can be grown to the billions, before being
differentiated into macrophage. This allowed us to set up an in-vitro DNA
repair assay that would not have been possible with neurons.
- What's the mechanism for DAR?
- What's the mechanism for shutting of global NER in neurons?
Using this assay, we purified a protein that could complement the NER
deficit in macrophage extracts. Mass spectroscopy identified it as the
ubiquitin-activating enzyme E1, i.e. the first step on the
ubiquitination pathway (it passes ubiquitin to an E2 enzyme, then an E3
enzyme transfers it to the target protein).
As usual, answering one
question raises even more questions. In this case:
- What's wrong with E1 in differentiated cells?
- How does E1 affect NER?
1) It is known that E1 is phosphorylated on at least 4 different
serines. We have evidence that one of these sites (we don't know which
one yet) is
de-phosphorylated in differentiated cells. The assumption is that this
prevents proper interaction with the E2 used by NER (we don't know yet
which E2 it is), without affecting the E2s used by other pathways.
2) Ubiquitination is best known for its ability to tag a protein for
degradation, but it is by no means its only function. Depending on the
number of ubiquitin moieties attached to the target, and on the type of
they form, it is possible to control the activity of the target
protein, positively or negatively. An obvious possibility is thus that
one of the NER enzymes is activated by ubiquitination. We have
indications as to which one it is, but still need a formal proof...
The catalytic mechanisms of protein
Barford, Biochem. J. 379:513-525 (2004)
Mechanisms of DAR
Pei-hsin Hsu, a very good student who worked with me at Stanford,
studied the NER phenotype of four different leukemia cell lines, at
various stages of differentiation. His goal was to find out whether
there was a progressive loss of NER, correlating with the degree of
differentiation. The answer was no, but Pei confirmed that DAR exists
in these cells, and most importantly that it can exist in proliferating
cells also. Which means that we may have to change the name "DAR".
since it's not unique to differentiated cells...
As for the mechanisms of DAR, we found out that it happens in areas
transcribed gene that RNA polymerase II never reaches (click here for details). In addition, it
also happens when RNA polymerase II is stalled half-way through the
gene by transcription
inhibitors. We thus believe that DAR is nothing more than a subset of
NER, concentrated in the nuclear sub-compartments where transcription
takes place. Bringing a gene into these "transcription factories"
it preferential access to NER enzymes, whether it is actually
transcribed or not. Knocking down NER enzymes with siRNA confirmed that
DAR depends on the same enzymes than global NER, but not on those
specific for TCR.
Directions of research
Filling in the blanks
There are many questions that remain to be answered, besides those
- Which phosphorylation site(s) on E1 affect(s) its interaction
with E2 enzymes?
- What are the E2 and the E3 involved in controlling NER by
- What is the final target, i.e. the NER enzyme controlled?
- Is the pathway identical in neurons?
- Would DAR be apparent in a silent gene located near an active
I would like to know how universal these mechanisms are. In particular:
Generalization of the model
- Do all
differentiated cells show a lack in NER and the presence of DAR?
- And what about cells like hepatocytes, that are
quiescent but must retain the ability to proliferate when needed?
- Is this only true for NER, or does differentiation produce
changes in BER, double-strand break repair, mismatch
What we have here is a natural control mechanism that shuts down DNA
repair at the global genome level, while preserving it in transcribed
genes. If we could learn how to trigger it in the whole organism,
rather than just in differentiated cells, it could be a tremendous help
for cancer therapy.
Potential for cancer therapy
For instance, by combining it with anti-cancer drugs
like cisplatin, which work by damaging the DNA. It would allow for
lower doses of the drug, hence reducing the nasty side effects. It
may also prevent relapses due to the emergence of a resistant clone of
cancer cells overexpressing NER enzymes.
Potential for neuro-regeneration
It has been observed in many neurodegenerative diseases, among which
Alzheimer's disease, that neurons attempt to re-enter the cell cycle.
They even manage to replicate their DNA, but die shortly afterwards.
This is good news and bad news: it's good to know that there is a
molecular switch allowing to trigger neuro-regeneration, and that this
switch must be fairly accessible for so many diseases to trigger it.
The bad news is, of course, that it doesn't work because neurons die
and we don't know why.
My (so far totally unproved) hypothesis is that they die because they
have neglected DNA repair over decades and are now attempting to
replicate and transcribe a crippled genome. If this were true, and if
we could learn how to control NER via ubiquitination, then we might be
able to trigger a bout of DNA repair in neurons, prior to causing them
to replicate. This would open the gate to neuro-regeneration approaches!
When parsimony backfires Nouspikel
& Hanawalt: BioEssays
25:168-173 (download pdf file 169K)
E1 as a master switch
As far as I know, this is the first time that control of ubiquitination
has been observed at the level of the E1 enzyme. Because there is only
one E1, versus dozens of E2s and hundreds of E3s, it has always been
assumed that control mechanisms would operate on the E3s. But if we are
right that differential phosphorylation of E1 affects the way it
interacts with the various E2, this may constitute a control mechanism
affecting many systems other than NER. It may even be a "master switch"
used to trigger all at once many of the phenotypic changes required
I am very interested in identifying other ubiquitination pathways
regulated at the E1 level, and I believe that we have a very good
hand, macrophage extracts, to undertake this task.
Techniques used in the lab
Thierry Nouspikel's CV