DNA Repair
Judith Westman, MD
Professor, Division of Human Genetics
Department of Internal Medicine
Learning Objectives


Apply knowledge of genetic/genomic variation to explain variation in normal
phenotypic expression, disease phenotypes, and treatment options

Describe the types and extent of variation seen in the human genome,
including both sequence and structural variation in coding and non-coding
sequences (e.g. single nucleotide variants, insertion-deletions, copy number
variants)

Define the terms mutation and polymorphism and describe their role in both
normal human variation and disease

Describe the types of mutations that lead to human disease and their
functional consequences, including but not limited to missense, nonsense,
frameshift, microdeletion, and splice-site mutations
Apply knowledge of the human genome structure and function, including genetic
and epigenetic mechanisms, to explain how changes in gene expression
influence disease onset and severity

Describe the process and regulation of gene expression, including the steps
of transcription and translation

Explain how errors in gene expression can result in disease
Types of Genetic Changes
 Several different types of genetic changes occur within
the genome
 Some produce a greater effect than others
 How are they caused and how can they be repaired?
Polymorphism
 A polymorphism is a change that occurs within the general normal
population. A polymorphism may not necessarily produce a change
in amino acid.
 If no change occurs, it is known as a silent or synonymous change.
We used to believe that silent changes were never harmful. Now we
know that some silent changes may alter miRNA binding sites or
other regulatory regions. While synonymous changes are less likely
to be harmful, it is no longer possible to make definitive predictions.
 If the change causes a change in the amino acid, it is typically
known as a nonsynonymous change or also known as a missense
mutation.
 Either a synonymous or nonsynonymous polymorphism may have
different degrees of effect on the function of the gene product, but
usually within a “normal” range (>50% of product). This normal
range produces a range of normal phenotypes and may have very
difficult to predict functional effects.
Genetic Mutation: Missense
Genetic Mutation: Nonsense
Genetic Mutation: Frameshift
Other Genetic Mutations
Genomic Mutation: Chromosomal
Rearrangement
Genomic Mutation: Nondisjunction
Genomic Mutation: Copy Number
Variants
Nomenclature For Mutations





187delAG = at the 187th nucleotide, deletion of A and G
Insertion (ins)
Single base substitution: GAGGTG
Splice site: 682 +1G>A
Change in polypeptide uses two methods


amino acid single letter abbreviations
 E6V (glutamic acid to valine at amino acid #6)
Amino acid triplet letter abbreviations
 glu6val
 Nucleotide and amino acid nomenclature used
interchangeably
 X used for nonsense mutation
Endogenous Sources of DNA Damage
 The most common causes of DNA damage occur as part
of natural processes within the cell.
Depurination
Deamination of Cytosine
Reactive Oxygen Species
Exogenous Sources of DNA Damage
UV Irradiation
Alkylating and Crosslinking Agents
Replication machinery as source of DNA damage
 Replication slippage in microsatellites
 Replication machinery loses track
 Deletions or insertions of repeat units
Trinucleotide Repeat Mutations
 Trinucleotide repeat microsatellites also happen within
the genome and are prone to the same slippage. In fact,
a special class of disorders exists which involve these
regions. One of the hallmarks of triplet repeat disorders
is that the clinical features appear to worsen in
successive generations - a characteristic called
“anticipation”.
 Key Points:




“Triplet repeat” disorders
Microsatellite slippage
Demonstrate “anticipation”
Successive generations with younger age of onset and
worsening severity.
Anticipation
Fragile X Syndrome
Fragile X Syndrome Mechanism
 In the 5’ untranslated region of the FMR1 gene is a triplet
microsatellite region consisting of a series of CGG units. What
happens with this normal state to cause Fragile X mental retardation
syndrome to occur?
 The microsatellite region, for reasons still not clear, enlarges. But it
enlarges only when passed from a mother to child. It does not
enlarge when passed from a father to his daughter. Once the
expansion is present, it permits the release of miRNAs from the 3’
untranslated region of the gene - all the way on the other side of the
coding sequence. Then these miRNAs come back to the 5’ side and
promote methylation of the 5’ CpG island in the promoter region of
the FMR1 gene. When this region is methylated, transcription of the
FMR1 gene does not occur and no gene product is generated. This
is an example of a gain-of-function mutation (release of miRNAs)
resulting in a loss-of-function of the gene through modification of the
epigenetics.
Fragile X Pedigree
Phenotype in Premutation Carriers
 What about grandpa who had a few too many triplet repeats but not
enough to trigger the release of the miRNAs? We used to think that
he didn’t have any problems. However, now we have recognized
that these individuals develop an adult onset neurodegenerative
disorder with some features of Parkinson disease, known as Fragile
X-associated tremor/ataxia syndrome. In addition, we also have
recognized that women who carry an enlarged premutation FMR1
triplet repeat are at risk for premature ovarian failure with early onset
of menopause in their 20s and 30s.
 Key Points:





Fragile X-associated tremor/ataxia syndrome (FXTAS)
Adult onset neurodegenerative disorder
Affects males >50
Intention tremor, ataxia, Parkinsonism
Premature ovarian failure in female carriers
Fragile X Pedigree: Family History
Tandem Repeats
Unequal Crossing Over
Fusion of Beta-Globin Genes
DNA Repair Mechanisms
Need for DNA Repair
 Without replication repair:
 1 error in every 10,000,000 bases copied
 320 nucleotide errors with every cell division
 16,000 nucleotide errors at cell death
 With repair
 1 error in every 1,000,000,000 bases copied
 3.2 nucleotide errors with every cell division
 160 nucleotide errors at cell death
 ~50 cell divisions before cell death
Base Excision Repair
 The base excision repair system corrects purine loss and
oxygenation of guanine. As we learn more about these repair
mechanisms, we also think of ways we can exploit this knowledge to
help us treat disease more effectively. For instance, cancer cells
are the most rapidly growing cells in a person’s body and are going
through rapid cycles of DNA replication and cell division. If we could
slow down some of these repair mechanisms, it might increase the
likelihood that a cancer cell would develop a genetic mutation that
would result in effective apoptosis of the cell.

Key Points:


Corrects the most common type of DNA damage (purine loss, 8-oxoguanine)
Target for novel cancer therapeutics - slow down BER to permit more
disruption by chemo
Example: MUTYH Associated Polyposis

There are several genes involved in base excision repair. MUTYH is one of them. If a
person has inherited two loss-of-function mutations in the MUTYH gene (biallelic
mutations), they will develop a disorder known as MUTYH associated polyposis
(MAP). In the family pedigree, it will appear as an autosomal recessive type of
pattern. Clinical similarity exists with Familial Adenomatous Polyposis (FAP) and
Lynch syndrome. The base excision repair pathway, and MUTYH specifically,
interacts with the gene products associated with FAP and Lynch syndrome.

Clinical features of MAP include 10-100 adenomatous colon polyps which have an
increased risk to turn into colorectal cancer. The average age at diagnosis of
colorectal cancer in MAP is 47 (range 29-72 years). There may be increased risks of
other cancers (duodenal, breast, leukemia) although these risks are not well
established at present.

Key Points:




MUTYH involved in 8-oxo-guanine repair
Autosomal recessive inheritance
Inherited biallelic loss of function mutations
MUTYH associated polyposis (MAP): 10-100 adenomatous colon polyps with increased risk
for colorectal cancer.
Nucleotide Excision Repair

The nucleotide excision repair pathway targets thymine dimers (those dimers caused
by UV radiation) and large chemical adducts. It is a very complicated pathway with
over 30 proteins involved. If an individual has biallelic loss-of-function mutations of
one of these proteins, they may have a disorder in which they have an increased
sensitivity to the sun and an increased risk for skin cancers. The LOF mutations must
occur within the exact same subunit to cause disease. A person may have one LOF
mutation in an allele of Gene X and a second LOF mutation in an allele of Gene Y
and be completely normal. It is only when both alleles of Gene X are affected does a
problem occur. The inheritance pattern in a pedigree will be autosomal recessive.

Key Points:








Removes bulky DNA distorting lesions --thymine dimers and large chemical adducts
Over 30 proteins involved
Defects in NER cause 4 autosomal recessive disorders
Xeroderma pigmentosum
Cockayne syndrome
Trichothiodystrophy
Cerebro-oculo-facial-skeletal syndrome
All associated with increased sensitivity to sun
Example: Xeroderma Pigmentosum
Complementation
 XP may result when any of 7 different genes involved in the NER
pathway are mutated. Again, BOTH alleles of a single gene must
lose function for disease to occur. Different subunits are responsible
for ethnic differences in the etiology of XP. When single allelic
mutations in different subunits occur and do NOT produce disease,
that is “complementation”.
 Key Points:




Multimeric proteins have different genes encoding each component
Both alleles of a single gene loci must be affected to cause disease
Single allele mutations in two different loci do not produce disease
Ethnic differences in XP


XPA: 90% of Japanese XP patients
XPC: majority of US patients
Mismatch Repair
 Another type of DNA repair mechanism addresses the problems
with base mismatches throughout the genome, including at
microsatellite regions. There are 4 principal genes involved - MLH1,
MSH2, MSH6, and PMS2. The four proteins work in pairs to
recognize mismatches that occur, and then signal additional repair
machinery to complete the repair process.
 Key Points:






Replication errors, mismatched nucleotides, stabilization of microsatellite
regions
4 principal genes
MLH1, MSH2, MSH6, PMS2
Two protein complexes (MLH1/PMS2; MSH2/MSH6)
Heterodimer complexes recognize mismatches
Signals additional repair machinery
Example: Lynch Syndrome
 Lynch syndrome results when a person has a single inherited LOF
mutation in one of the mismatch repair genes. Notice that only one
mutation is inherited here. As a result, the pedigree looks more like
an autosomal dominant disorder. 80% of the inherited mutations are
found in MSH2 and MLH1. If a person has this inherited mutation,
they will tend to accumulate mutations in cells, particularly in the
microsatellite regions. These accumulated acquired mutations can
lead to cancer formation.
 Key Points:




Defective mismatch repair
Mutations primarily in MSH2 and MLH1 (80%)
Lead to accumulation of mutations particularly in microsatellite DNA.
Inherited susceptibility to colorectal cancer, endometrial cancer, and
ovarian cancer
Repair of DNA Breaks
 Here are some disorders with abnormalities in DNA
break repair, followed by the mechanism involved.
Example: Fanconi Anemia
Example: Hereditary Breast/Ovarian
Cancer Syndrome
Example: Ataxia-telangiectasia
Fanconi Anemia DNA Repair Pathway
Recombination Suppression
Example: Bloom Syndrome
ssDNA Break Repair
Thank you for completing this module
 Questions?
 Judith.Westman@osumc.edu
Survey
We would appreciate your feedback on this module. Click on the
button below to complete a brief survey. Your responses and
comments will be shared with the module’s author, the LSI
EdTech team, and LSI curriculum leaders. We will use your
feedback to improve future versions of the module.
The survey is both optional and anonymous and should take
less than 5 minutes to complete.
Survey