Lecture 13: DNA Repair Flashcards
Biological scale:
How many feet of DNA in each cell?
Humans receive how many base pairs from each parents?
Human genome is how many chromosomes?
3 feet of DNA in each cell
>3 billion base pairs from each parent
46 chromosomes
What is an absolute requirement of DNA?
High-fidelity of DNA replication is an absolute requirement. Have to replicate accurately and precisely, otherwise genome instability would occur.
DNA Damage
Occurs spontaneously or through chemical & environmental means. Normal metabolic processes in the body can also produced DNA-damaging by products.
If the damage is passed on to subsequent generations, then…
Permanent changes are left in the DNA –> mutations. If not corrected, mutations can be passed on to successive generations of cells and organisms if the mutation occurs
in the germline of multicellular organisms.
Mutations occur if
nucleotides are added incorrectly or if nucleotides base pair
incorrectly due to damage to the template DNA.
Mutations can have many effects, including
- Most will lead to no obvious phenotypes (neutral mutations)
- Some will have mild effects, such as altered pigmentation
- Some will have serious consequences
Mutations in germ (sex) cells can result in
an inherited disease
Mutations in somatic cells can result in
cancer
Mutations occur where a what residue is methylated?
Guanine residue. Methylated guanine is considered damaged.
During replication, the methylated guanine pairs with?
Thymine rather than cytosine; the other parental strand is unaffected.
When DNA polymerases synthesize new daughter strands, these mutations will become?
Fixed in the genome - half of all new daughter cells will now contain the mutation.
Sickle cell anemia mutation
Glutamic acid –> Valine
Single base change substitution causing disease
DNA polymerases actively incorporate bases that are correctly base paired through
conformational changes in the polymerase.
DNA polymerase selectively gives an error rate of about
one incorrect nucleotide in very 104 – 105 nucleotides.
tautomeric shifts
Misincorporation of bases can still occur. There is a spontaneous redistribution of protons and electrons in the bases that lead them to form isomers. These shifts are temporary.
Tautomeric shift example
Keto –> enol (for guanine and thymine)
Amino –> imino (for adenine and cytosine)
Tautomeric shifts causes which mis-matches?
C-A and T-G
In a tautomeric shift, cytosine can become what form? Matches with what?
Can become imino form and base pairs with adenine.
In tautomeric shift, guanine can become what form? Base pair with what?
Guanine can become enol form and base pair with thymine.
In tautomeric shift, adenine can become what form? Base pair with what?
Adenine can become imino form and base pair with cytosine.
In tautomeric shift, thymine can become what form and base pair with what?
Thymine can become enol form and base pair with guanine.
As the DNA polymerase (DNApol) proceeds along the template, it uses
correct base pairing to incorporate the next nucleotide in the chain.
If the template base has shifted to its rare
tautomeric form, an
incorrect base will be added since the hydrogen bonding appears correct.
The rare tautomeric form will shift back to the
common isomer, and the bases will be a mismatch.
DNApol check the? If there is a mismatch, the polymerease?
DNApol checks the last base added. If there is a mismatch, the polymerase will remove the wrong nucleotide using their 3’ to 5’ exonuclease activity.
Polymerase will hydrolyze DNA in the
3’ to 5’ direction (analogous to a backspace key) then, the polymerase will insert the correct nucleotide and synthesis continues. This is referred to as proofreading.
3 to 5 Exonuclease activity increases the accuracy of DNA replication by
100 to 1000 fold
Some lesions in DNA cannot be copied by ___________. An example is:
the replicative DNA polymerase, ex: thymine dimers
The replication fork ______ at these locations, and if DNA synthesis cannot continue the cell will…. (translesion DNA synthesis)
stalls, die
Special DNA polymerases replace the normal polymerases at these lesions. These polymerases have a
larger activity site to accommodate “base pairing” across from lesions.
These special DNA polymerases (ex: eta - poly n) are
error-prone having low fidelity and no 3’ to 5’ exonuclease activity (no proofreading). However, making a few mistakes in the replicated strand is a better alternative for survival.
DNA polymerase eta (Pol η)
specialized polymerase that replicates UV-damaged DNA. When thymine dimers are present, Pol η inserts adenine nucleotides in the newly synthesized DNA, bypassing the lesion and preventing mutation.
Pol η inserts
adenine nucleotides in the newly synthesized DNA,
With selectivity and proofreading, the fidelity of replication is approximately
1 mismatch in every 10^7 to 10^8 nucleotides
. The human genome is made up of how many nucleotides?
3 billion (3x10^9)
To preserve the DNA sequence in subsequent generations, cells have
DNA repair mechanisms to remove any mismatches that are not removed by DNA polymerase proofreading, resulting in less than 1 error in 109 nucleotides.
Error replication image
Okazaki fragments are numbered in the order in which they were
synthesized on each lagging strand (i.e., ‘1’ is
the oldest, ‘5’ is the newest).
The replication bubble is
bidirectional
Mismatches that are missed by the proofreading of the DNA polymerase are repaired by the
DNA mismatch repair (MMR) system.
MMR proteins detect
distortions in the DNA helix that result from a misfit between non-complimentary base pairs. MMR proteins will replace the incorrect base in the newly replicated strand.
The MMR system can discriminate between
the mother strand and the daughter strand
based on the presence of
“nicks” (single strand breaks) in the DNA strand
In eukaryotes, ‘nicks’ in newly replicated DNA appears to
specify the strand to be repaired.
The newly-synthesized lagging strand could be identified by nicks at either end
of Okazaki fragments, whereas the leading strand might be identified by
its growing 3’ end
MSH
MLH
MSH: Sits on mismatch
MLH: Looks for Nick
In humans, the MSH2 proteins finds?
slide along the DNA until they find mismatched bases
MSH2 binds at the site of a mismatch in the double-stranded DNA. MSH recruits the
MLH protein
The MLH protein then scans for the
closest nick in the lagging strand or the 3’ end of the leading strand.
Once a nick is found, MLH introduces
nicks in the daughter strand between the strand break and the mismatch.
MLH recruits
exonuclease 1 (EXO1). EXO1
excises the DNA between the nicks
The gap is then filled in by
DNA polymerase and sealed by DNA ligase.
This repair mechanism takes place as
DNA replication is occurring, before replication is completed
Mutations in MMR genes can result in
hereditary non-polyposis colorectal
(HNPCC) cancer, also known as Lynch syndrome
Lynch syndrome
the most common cause of inherited colorectal cancer.
Non-polyposis means that
colorectal cancer can occur when only a small number of polyps are present (or polyps are not present at all).
~1/300 individuals in the US have a
genetic variant associated with NHPCC
HNPCC is due to mutations in
MSH2 and MLH1 genes (MSH2, 60% of cases and MLH1, 30% cases).
Individuals who inherit on mutant copy of an MMR gene have an increased risk of developing colorectal cancer
as well as other cancers because it is likely they will acquire a mutation in the second copy of the MMR gene during their lifetime.
Cancer is not inherited. What is inherited is a
predisposition (an
increased risk) to develop cancer. Not all individuals with a variant will develop
cancer.
DNA repair becomes
compromised, resulting in the accumulation of mutations in
cells. This will eventually lead to mutations in genes required to maintain control of
cell division
Progression from an adenoma to a carcinoma takes place in only
2-3 years compared to the usual 8-10 years seen in non-HNPCC patients.
Cells have several DNA repair
mechanisms that can function at times other than
during DNA replication. However,
only so much DNA damage can be repaired. If the damage is so severe that it overwhelms the repair pathways, the cell will die.
Repair pathways depends on the type of
damage
Single-strand damages can be repaired by
- Base Excision Repair (BER)
- Nucleotide Excision Repair (NER)
Double stranded breaks are due to
chemical, chemotherapeutics, radiation, etc
Double stranded breaks
- Non-homologous end joining (NHEJ)
- Homologous recombination repair (HR)
BER is used to replace
single bases in DNA that are incorrect, damaged, or missing
A missing base is called
abasic (a=without, without a base)
Abasic sites are called
AP sites, can occur spontaneously
Depurination
the glycosidic bond between the purine base and sugar in a nucleotide can spontaneously hydrolyze, creating an apurinic site (sugar without a base)
Depyrimidination
occurs in cells but less frequently
Under physiological conditions, how many apurinic and apyrimidinic sites are generated in a cell each day?
10,000 apurinic sites
500 apyrimidinic sites
AP sites can be made by
enzymes
Glycosylases
used to remove damaged or incorrect bases generating an AP site (ex: in the case of spontaneous deamination of cytosine in DNA)
Glycosylases are lesion specific: how many types in humans?
11
Uracil-DNA glycosylase removes
uracil in DNA
Thymine DNA glycosylase
removes pyrimidine mismatches
8-oxoG DNA glycosylase
removes oxidative base damage
A spontaneous deamination of
cytosine to uracil in DNA occurs at a rate of about 100 bases per cell per day
Unnatural DNA bases (uracil, 8-oxoG, etc.) incorporated in DNA results in
structural distortion
DNA glycosylases travel along the DNA helix to
probe all faces of the nucleotide for damage and remove all unnatural DNA bases.
BER Steps
- A specific ‘damaged’ base is recognized
- Glycosylase cleaves the bond between the damaged base and the sugar, leaving behind an AP site (an apurinic or apyrimidinic; “a”=without)
- AP endonucleases cut the sugar-phosphate backbone, and exonuclease removes the abasic residue. (‘endo-’ cuts the phosphodiester bonds; ‘exo-’ removes the nucleotides). There is a single-base gap now.
- DNA polymerase adds new nucleotides, and DNA ligase seals the nick.
NER detects
structural distortions in the double helix due to either pyrimidine dimers (where two adjacent thymine or
cytosine residues are covalently linked on the SAME strand) or bulky lesions added to bases due to exposure to carcinogens.
NER system removes a segment of
contiguous nucleotides (“oligonucleotide”) to either side of the damaged base(s).
General Steps of NER
- A multi-enzyme XP complex recognizes the DNA for distortion of the double helix
- A nuclease cleaves the phosphodiester backbone on both sides of the distortion
- A DNA helicase separates the damaged DNA from the undamaged, complimentary DNA. (Depending on the size of the complex and types of damage, the excised base length differs)
- The gap is then repaired by DNA
polymerase using the undamaged strand as a template and DNA ligase
Common/shared mechanisms between BER and NER
- Enzyme complexes recognize distortion in structure and excise damages (specific enzymes for specific mistakes)
- DNA polymerase adds correct nucleotides
- DNA ligase joins new, correct fragment with the existing polymer
Net result: repaired DNA
Double-strand DNA (dsDNA) breaks can occur due to problems with
DNA replication (when polymerase encounters a nick in the template strand), exposure to certain chemicals, ROS, or by ionizing radiation.
NHEJ mechanism, speed, timing, and accuracy
Simple process of ‘gluing” the ends together. Fast process. Does not require a template, so it can happen anytime (mostly in G1 phase of the cell cycle). Error prone.
HRR mechanism, timing, and accuracy
Requires a duplicate chromosome, occurs after DNA replication before separation of chromosomes (S, G2, and M). Accurate.
A classic NHEJ process can be used to glue the
DNA strands back together upon a double-stranded break
When does NHEJ typically happen?
happens in G1 phase of the cell cycle when there is no copy of the chromosome to use as a template.
The Ku70–Ku80 heterodimer binds to the
damage sites and recruits the polymerase, nuclease and ligase complexes
The processing of the ends creates
microhomology between the two DNA ends for the polymerases to work from
This mutagenic and error-prone can result in
Diverse DNA sequences at the repair junction. Repaired DNA generally suffer a deletion of nucleotides
Double-strand break repair - HRR
- Double stranded DNA break
- Resection of the damaged strand
- Undamaged, homologous DNA
- Strand invasion
- Repair synthesis: the homologous DNA provides an undamaged template for DNA synthesis
- Fully restored, undamaged strands (error-free)
Normal functions of BRCA1 and BRCA2 proteins involve repairing
double-strand DNA breaks; mutations in these genes lead to the failed repair of other genes.
Mutations in the BRCA1 and BRCA2 genes
are major contributors to
to inherited (familial) breast cancer. ~1/3 of all breast cancers are due to mutations in one of these genes. The type of cancer is inherited in an autosomal dominant manner.
Cancer is not inherited. What is
inherited is a
a predisposition (an increased
risk) to develop cancer. Not all individuals
with a variant will develop cancer.
Individuals who inherit one mutant copy of a
BRCA gene have an increased risk of
developing cancer, because it is likely they will acquire a mutation in the second copy of
the BRCA gene during their lifetime.
When mutated/deficient BRCA1 and
BRCA2 genes are inherited, affected
women are
40-80% more likely to develop breast and ovarian cancer.
Many sources of DNA damage can alter the
DNA before or after replication,
creating chemically altered bases or segments of DNA that are eventually replicated with the incorrect bases.
Endogenous sources of DNA damage
-DNA replication error
-Spontaneous base deamination
-Base hydrolysis
-Oxidation of bases
Exogenous sources
-Radiation
-Carcinogens
-Chemotherapeutics
Deamination is the removal of an
amine group
Example of deamination
Cytosine to uracil.
Solution: Uracil-DNA glycosylase removes uracil bases in DNA
Demination of 5-methylcytosine (5mC) to
thymine
5mC is a naturally occurring
methylated cytosine. It is involved in gene regulation.
If the mutation is not corrected, the altered base will
pair with different partners during DNA replication
repair mechanism utilized for DNA damage by spontaneous deamination
base excision repair
Base hydrolysis results in
de-purination, removal of a purine base from the DNA strand.
The glycosidic bond between the purine
base and sugar in a nucleotide can
spontaneously
hydrolyze, creating an Depurination apurinic site (AP site).
Replication without a
base
If not repaired, the loss of a base can result in a deletion mutation.
DNA damage by base hydrolysis repair mechanism utilized
base excision repair
DNA damage by oxidation of bases
Many of the metabolic reactions in the mitochondria generate Reactive
Oxygen Species (ROS: O2, H2O2, OH_ that can damage DNA.
ROS can
-damage the bases of DNA to cause mis-pairing
-cause the complete loss of a base from a ribose residue
-induce single and double stranded breaks
example of oxidation of bases
deoxyguanosine oxidized to 8-oxo-deoxyguanosine
guanine oxidized to 8-oxo-guanine
DNA damage by oxidation of bases repair mechanism
base excision repair
DNA damage by UV irradiation
Exposure to UV light can induce pyrimidine dimer formation in
DNA.
Two adjacent pyrimidine bases on the what? Becomes linked to?
On the same strand, become linked to create a cyclobutane ring by exposure to UV light.
DNA damage by UV irradiation repair mechanism
Nucleotide excision repair (NER). The covalent bond between adjacent bases requires that NER be used rather than base excision repair mechanism.
Defective NER mechanism
results in inability to repair DNA damage that results from UV radiation. Accumulation of un-repaired DNA damage leads to the development of cancerous tumors.
Mutations in NER genes can result in
Xeroderma pigmentosum (XP). XP is caused by mutations in genes that are involved in repairing damaged DNA.
XP is a
are genetic disorder that affects ~1/250,000 people. It is inherited in an
autosomal recessive manner.
Since NER will not function, pyrimidine dimers will
form in the presence of UV light and not be repaired. They bulky
dimers are not replicated properly, and translesion synthesis is used to bypass the damage. This results in the accumulation of mutations in the cells, which will eventually lead to mutations in genes required to maintain control of cell division, which can result in cancer.
Carcinogens (cancer-causing agents) include
natural and synthetic chemicals
Many carcinogens can react with
DNA bases, often adding bulky adducts to DNA
benzopyrene
a ubiquitous compound that comes from wood burning; also
found in coal tar, automobile exhaust, in smoke resulting from burning organic
material, and in charbroiled food (found in coal tar)
chemical carcinogen repair mechanism
nucleotide excision repair
topoisomerase inhibitors
intercalate into the DNA (i.e., stack with bases) or bind the topoisomerase-DNA complexes. They inhibit the re-ligation of the DNA by the topoisomerase
Antibacterial chemotherapeutics
Fluoroquinolones
Fluoroquinolones
broad-spectrum antibacterial agents that inhibit bacterial Type II
topoisomerases and not mammalian topoisomerases. Used in human and
veterinary medicine.
Example: ciprofloxacin (“cipro”)
Anticancer drugs
include Type 1 and Type II topoisomerase inhibitors, generating
single- and double-strand breaks, leading to apoptosis (programmed cell death).
Examples of topoisomerase I inhibitors
irinotecan and camptothecin
Examples of topoisomerase II inhibitors
etoposide and doxorubicin
