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DNA Damage and Its Repair

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Rachel Aronoff

on 20 March 2016

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Transcript of DNA Damage and Its Repair


byproducts of cellular metabolism can damage DNA.

Even
oxygen
(usually as Reactive Oxygen Species, ROS) can cause varied chemical damage to nucleotide bases.

Ordinary


water
contributes to base hydrolysis.

DNA damage
Biomarkers of DNA damage
include:
assays either directly for DNA breaks or induced repair processes
(by, for instance, 'comet' assays or probing for components of the DNA Damage Response, DDR, in cell nuclei);
new sequencing strategies for direct ID of DNA lesions;
assays for excreted metabolites of repair (8oxodG)

Comet Assays
a single cell electrophoresis technique
designed and used first by
Östling & Johansson in 1984

standardization difficult, but data
promising in many cases...

new commercial ventures &
more publications coming out!
Detection of Excreted
8oxodG
fraught also with uncertainty

though simple ELISA assay ...

other metabolic byproducts can contribute to its abundance, it is asserted

53bp1
DDR foci component,
has RNA binding domain, and interacts with p53 (key for choice between apoptosis and repair)

makes bright spots at dsDNA breaks
(with appropriate probing protocols)
Positive Controls??
Need to see what happens with lots of
ordinary folk, for seeing how reliable baseline levels are between individuals, and assess their responses to predicted DNA damaging compounds we use every day...

positive control protocols
join us! AGiR!!
Measures of DNA Damage
the simplest
in summary, can be triggered by:
Spontaneous reactions (base isomers)
Replication errors (misincorporation)
Oxygen radicals (ROS)
Energy from parts of the electromagnetic spectrum (UV, Ionizing radiation) and various
Chemicals (benzene, formaldehyde)
out in the cell and organism these ripple effects are felt
only as growth goes out of control
or disease occurs...

TAKE CARE!!
DDR components
form
detectable
foci in nuclei!
RNA dependent!
Alcohol metabolized to aldehydes directly attacks DNA, with connections to stress and impact on disease
on the way to quantitative and reliable measure of DNA damage
for assessment of risks to individuals
bacterial pathogens, additionally,
can directly attack DNA!
symptoms of
typhoid fever
are due to a
DNAseI related
toxin subunit!
our own gut
microbes can
produce similar proteins
or other unrelated molecules
that directly target
and attack
host DNA to
contribute to cancer &
chronic disease
infectious
processes
can indirectly
produce ROS
to damage DNA too
by products of growing colonies' metabolism
Genomic integrity
active maintenance of all* the genetic elements in cells of an organism for proper dynamic function.
(aka health).
antioxidants
can antagonise such
processes.
altogether accounts for the correlation
of alcohol consumption with cancer incidence...

* including DNA/RNA/epigenetic determinants and appropriate developmental gene expression
Is the RNA from the site making sure the DNA seq is ok??
Creative Commons Images:
Dog hip bone X-ray by Joel Mills, Wikimedia.
DNA by Christoph Bock, Max Planck Institute for Informatics.
Chemicals image: Courtesy of Milosz1
thymine dimer: http://commons.wikimedia.org/wiki/File:DNA_UV_mutation.gif
one turn helix (DNA structure): Zephyrus
Cobra snake: http://www.mediahex.com/Cobra_(snake)
Carcinogen symbol:http://www.unece.org/trans/danger/publi/ghs/pictograms.html
Cosmetics image: http://humaneeducation.org/blog/2013/12/12/6-resources/

cobra venom: contains active nucleases!
UV crosslinks certain adjacent
nucleotide bases, forming
'thymine dimers'

cigarette smoke not only contains compounds that damage DNA, but also those* which inhibit DNA repair!

*for instance, heavy metals like cadmium
X-rays induce breaks in DNA
even personal care products can contain known mutagens and other toxins!
some argue there is no
safe dose for ionizing radiation
This UN pictogram is from their
'global harmonized system' to
depict carcinogenesis.
Repair of DNA
damage
100s of genes repair DNA, and not only their protein products but also active RNAs and RNA transcription at the site of repair may be central to the process!

Mutation in genes that mediate DNA repair or recognize DNA damage is a key risk factor for several cancers and other genetic disease syndromes.

The BRCA genes, for instance, associated with breast cancer risk, are critical DNA repair factors - and their mutation is the basis for familial genetic predisposition to not only breast cancer but also prostate cancer in men!
but most cancers, about 85% of them, are what the
doctors call 'sporadic' which means they are not
due to familial factors, inherited gene mutations!
rather, these 'sporadic' cancers are due to 'new'
mutations in a large set of potential cancer risk factors, including protein kinase and other gene families.
again pointing
out the importance
of ...
While DNA repair is crucial, this process is also a way for new mutations to be fixed in the genome!
This is because DNA repair is usually quite error-prone!
Homologous recombination can allow 'perfect' repair, but
this is not the general rule for all cells subject to DNA damage.
DNA damage, artist's rendition
so many things can damage DNA
http://dx.doi.org/10.1590/S1984-82502012000100012
some selected references:

Chlamydia paper:
http://www.cell.com/cell-host-microbe/abstract/S1931-3128(13)00193-5

comet assay
http://www.ncbi.nlm.nih.gov/pubmed/17406208

RNA dependence of DNA repair (ncRNAs from site of damage
http://www.ncbi.nlm.nih.gov/pubmed/24156824

direct sequencing of DNA damage/modifications
http://www.genomeintegrity.com/content/2/1/10
http://pacb.com/applications/base_modification/

53bp1
http://www.ncbi.nlm.nih.gov/pubmed/24326623

Stress and DNA damage:
http://www.ncbi.nlm.nih.gov/pubmed/21857681

DNA damage and its repair
is particularly affected by DNA damage,
which can occur in many ways, even
through intrinsic cellular processes.
BRCA and ATM are examples of just two from the long list of these genes also linked to diseases, the first particularly associated with breast cancer, but also, in the 'loss of function' case, predisposing people to other malignancies, and the second causative for a severely disabling neurodegenerative disease,
ataxia telangiectasia.

Repair is clearly essential for resilience and health.
chlamydia can both
increase DNA damage and
decrease repair
(promoting, ultimately, carcinogenesis)!
In his 2015 Nobel lecture, Lindahl highlighted specific atoms prone to damage in nucleotide strands.
Chronic Stress results in accumulation of DNA damage, due to p53 export from the nucleus and its degradation, dependent on Beta-arrestin. These molecular pathways
were defined already in 2011 (Hara et al, Nature).
- the active maintenance of all* the genetic elements in the cells of an organism for proper dynamic function (aka health) -
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