DNA Damage & Repair

Phenotype

Observable characteristics of a person, an organ, or a cell

Genotype

combination of alleles that a person possesses at a single locus (or at a number of loci)

Disease phenotype

specific manifestations that arise in response to the differential expression of just one or a small number of genes that may be harmful

Character or trait

Observable manifestations that are not disease-associated

• Multiple characters/traits make up a phenotype

• Ex. blue eyes; blood group O

Genetic variation

Changes in the base sequence of our DNA → change in phenotype

Environmental factors and epigenetic effects contribute to the ___

Development of disease or change in phenotype

Epigenetics 

Addition of chemical moieties to DNA (ex. methylation, acetylation), leading to a particular gene to be silenced or activated

• Change in phenotype can happen even w/o change of DNA sequence through epigenetic modification

Mutation

Process that produces altered DNA sequences AND the outcome of that change

• Both a verb and a noun: process of making the change and the outcome of the change

Mutation → DNA variants (alternative forms of DNA) → >1% polymorphisms, <1% rare variants

• Depending on how prevalent the specific mutation is worldwide, it’s called single nucleotide variant (less common; <1%) or single nucleotide polymorphism (more common; >1%)

4 consequences of Mutations

• Normal phenotype (ex. height)

• Disease phenotype

• No obvious effect on the phenotype

• Very rarely, some beneficial effect

Mutations originate as a result of changes in our DNA that are __

Not corrected by cellular DNA repair systems

DNA changes are occasionally induced by radiation and chemicals in our environment, but the great majority arise from ___

endogenous sources

Types of Genetic Variation

No change in DNA content

Change in DNA content → Net loss/gain of DNA sequence

Genetic Variation: No change in DNA content

• Ex. SNPs: one nucleotide is replaced w/ another nucleotide

• Most common DNA changes are on a small scale and involve only a single nucleotide or a very small number of nucleotides

  • Small scale change (point mutations) often have no obvious effect on the phenotype (silent/neutral mutations)

Single Nucleotide Polymorphisms (SNPs)

• Change in a single nucleotide

• Most common type of genetic variation in the human genome

  • SNP variation accounts for a lot of physical variations like blood type

  • Account for ~75% of DNA changes

• Estimated that there is 1 SNP/1000 base pairs in the human genome

  • ~3 million SNPs/person

Genetic Variation: Change in DNA content; Net loss/gain of DNA sequence

• Ex. Trisomy, deletion

• Change in the copy number of whole nuclear DNA molecules are almost always harmful

  • Most are embryologically lethal except: Down syndrome, 21 trisomy; Edward’s syndrome, 18 trisomy; Turner Syndrome, X; Kleinfelter’s syndrome, X

  • Many result in spontaneous abortion and some give rise to developmental disorders

ABO Blood Group

Gene inactivation in normal individuals

• Most genetic variation has a neutral effect on the phenotype, but a small fraction is harmful

Immune system genes

• Polymorphic: undergo somatic rearrangements to produce different variants

• Genes involved in identifying microbial pathogens → Constant positive selection to maximize diversity in the proteins involved in antigen recognition

Variation typically happen d/t 3 major events

1. Recombination

2. Independent Assortment

3. Various mutational events

Variation: Recombination

Prophase I of meiosis I: homologous chromosomes exchange of chromosomal segments

• More common in the subtelomeric regions

• Important for producing the variation that happens at the sperm or egg stage → No two eggs or sperm are the same

Variation: Independent Assortment (of paternal and maternal homologs)

Metaphase I: random lining up and separating, movement of 1 chromosome is not dependent on another one (completely random)

Variation: Various mutational events

• Endogenous chemical damage to DNA

• Chemical damage to DNA caused by external mutagens

• DNA replication errors

• Chromosome segregation and recombination errors

3 Types of Endogenous Chemical Damage to DNA

• Hydrolytic damage

• Oxidative damage from normal cellular metabolism

• Aberrant DNA methylation

Hydrolytic damage

Disrupt covalent bonds that hold bases to sugars, cleaving the base from the sugar to produce an abasic site → loss of purine bases (depurination)

• Abasic site: location in a DNA or RNA molecule where a nucleotide has lost its nitrogenous base

• Hydrolysis: use of water to break a bond

  • Sometimes break bonds that are not supposed to be broken leading to hydrolytic damage

Oxidative Damage

• Most significant are superoxide anions (O2–), hydrogen peroxide (H2O2), and hydroxyl radicals (OH)

• Too much ROS → DNA strand break

Aberrant DNA methylation

• Many cytosines in our DNA are methylated by methyltransferases

• Cells also use S-adenosyl methionine (SAM) as a methyl donor in a non-enzymatic reaction to methylate different types of molecules

  • Sometimes SAM can inappropriately methylate DNA to produce harmful bases → unwanted gene silencing

3 Types of External Mutagens

1. UV radiation (sunlight): covalent bonding b/w pyrimidines

• Bind to the same strand instead of the other strand → unwanted linkage

2. High energy irradiation (X-Rays): Generate ROS → DNA strand break

3. Mutagenic chemicals (ex. cigarette smoke, automobile fumes) → bulky DNA adducts inserting itself b/w DNA strands → obstruction and distortion of the double helix

Types of DNA Repair

Single stranded break repair:

• Base excision repair (BER)

• Nucleotides excision repair (NER)

• Mismatch repair (MMR)

Double stranded break repair:

• Non-homologous end joining (NHEJ)

• Homologous recombination (HR)

Base excision repair (BER)

• Repairs lesions where a single base has either been modified or excised by hydrolysis to leave an abasic site

  • Only can fix small lesions (1 base), it is fast and precise, and basically like spell check coming in to replace 1 mistyped letter

• Available throughout the cell cycle

Base excision repair (BER) Process

(1) To replace a modified base by the correct one: specific DNA glycosylase cleaves the sugar–base bond to delete the base, producing an abasic site

(2) Endonuclease and phosphodiesterase remove residual sugar-phosphate from the abasic site

• Endonuclease clears the area

• Phosphodiesterase cleaves the phosphodiester bond

(3) DNA polymerase fills gap

(4) DNA ligase seals

DNA mismatch repair (MMR)

• Repairs erroneous insertion, deletion, mis-incorporation of bases during DNA replication and recombination

  • Like you wrote a sentence poorly and deleted it to rewrite it → fixing typos

• More important in S phase but not restricted to only S phase

DNA mismatch repair (MMR) Process

(1) Mismatch recognized on the daughter strand

(2) Identifies which one was the good strand and which was the wrong one → acts during/after replication

(3) Mismatch and surrounding nucleotides fully excised, creating a large gap

• Fold an area with error and then clears it

(4) DNA polymerase fills gap

(5) DNA ligase seals

Nucleotide excision repair (NER)

• Repair bulky, helix-distorting DNA lesions (UV induced T-dimers)

  • Like a sentence riddled with so many spelling mistakes due to someone smashing your keyboard that you just delete the chunk and rewrite it

• More important in G1 phase but not restricted to only G1 phase

Nucleotide excision repair (NER) Process

(1) Lesion detected and damage site is opened

(2) DNA cleaved some distance away on either side of the lesion, generating an oligonucleotide of about 30 nucleotides containing the damaged site

(3) Damaged oligonucleotide discarded

(4) DNA polymerase fills the gap, using the template strand as a guide

(5) DNA ligase seals

Homologous recombination (HR)-mediated DNA repair

• Highly accurate repair mechanism → requires a homologous intact DNA strand to be available to act as a template strand

• Operates in S and G2 phase (before mitosis), using a DNA strand from the undamaged sister chromatid as a template to guide repair

Homologous recombination (HR)-mediated DNA repair Process

(1) Double stranded DNA break

(2) Proteins come together and cleave the area

(3) Overhang falls close to the homologous strand (template) and use that to fill the sequenced area (creates a loop-like structure and then extends the strand using the homologous chromosome as a template)

(4) Goes back to original strand

(5) Polymerase fills the gap

(6) Ligase seals it

Non homologous end joining (NHEJ)

• No template strand needed → broken ends are fused together

• Always available to cells

  • Does not have the requirement of a template strand which only is available after DNA replication (S and G2 phase)

• Most important for the repair in G1 phase, before the DNA replication

Xeroderma Pigmentosum

Inability to repair damage caused by UV light (NER)

Xeroderma Pigmentosum Mechanism

Defective NER → Unrepaired UV-induced mutations → Xeroderma Pigmentosum → predisposition to skin cancer

• Malfunction of nucleotide excision repair (NER) → thymine dimers remain → block replication → unrepaired UV-induced mutations → Xeroderma Pigmentosum → predisposition to skin cancer

Inheritance of Xeroderma Pigmentosum

autosomal recessive

Clinical Features of Xeroderma Pigmentosum

• Dry skin, pigment accumulation (ex. freckles), clouding of cornea, keratitis (inflammation of cornea), cancer of the eyelets or conjunctiva, etc.

  • Cannot repair the damage caused by sunlight → sensitive to sunlight → produce lots of melanin and predisposed to cancer

• Before children turn 10 years old, typically have a diagnosis of cancer

  • Typically skin cancer (ex. basal cell carcinoma, squamous cell carcinoma, malignant melanoma) d/t sensitivity of UV rays

• 30% of kids w/ the disease can also have severe progressive neurological dysfunction (ex. seizures, cognitive disability, difficulty speaking, difficulty hearing, movement disorder, etc.)

Onset of Xeroderma Pigmentosum

Typically diagnosed ~2years old

• Signs appear in infancy or early childhood

Xeroderma pigmentosum with vs. without neurological dysfunction 

With: live to ~20 years old (longevity shortens)

Without: live to ~30 years old

Severe Combined Immunodeficiency (SCID)

Deficiency in both B and T lymphocyte functions (both antibody and cell mediated immunity are gone)

Inheritance of SCID

X-linked recessive or autosomal recessive

Lab Findings in SCID

low IgG, IgA, and IgE levels

Most common form of SCID

Mutations in the gene encoding the common gamma chain (γc), a protein that is shared by receptors for interleukins (ILR2 receptor)

• ILR2 receptor detects signals from the outside and sends messages to the inside

  • Not functioning → can’t activate cytotoxic T cells or B cells → immune cells malfunction → no cell-mediated or antibody-mediated immunity → cannot fight infections

Most severe form of SCID

Defects in non-homologous DNA end joining (NHEJ) mechanism (double-strand break repair)

Treatment of SCID

W/o bone marrow transplant, they can’t survive the first 2 years of life

• If the bone marrow transplant happens before 3 months of age, longevity increases significantly

Clinical manifestations of SCID

“BE SURE”

• Bone abnormalities

• Ear infections (8+ years)

• Sinus infections (2+ years)

• Unexplained failure to thrive

• Recurrent pneumonia

• Excessive time on antibiotics

Hereditary Non-polyposis Colorectal Cancer (HNPCC) aka Lynch Syndrome

Most common form of hereditary colorectal cancer

Cause of HNPCC aka Lynch Syndrome

mutations in genes involved in DNA mismatch repair (MMR proteins)

Inheritance of HNPCC aka Lynch Syndrome

Autosomal dominant

Clinical Features of HNPCC aka Lynch Syndrome

Propensity to develop right-sided, flat adenomas at a young age

• Develop adenomas (glandular benign tumors) at the same rate as individuals in the general population; however they are more likely to progress to adenocarcinoma (cancer)

  • 50-70% risk of developing colorectal cancer and other cancers (ex. stomach, hepatobiliary, small intestine, urinary tract, ovarian, endometrial)

Treatment of HNPCC aka Lynch Syndrome

High dose aspirin (300mg) reduces the progression from adenoma to adenocarcinoma but it is not clinically practiced d/t risk of bleeding

Prevalence of HNPCC aka Lynch Syndrome

1/300 Americans

Bloom Syndrome

Mutation in BLM gene → defective ReQ helicase → defective unwinding of DNA → defective homology mediated DNA repair → Bloom syndrome

• BLM gene codes for ReQ helicase

• ReQ helicase participates in the unwinding of DNA in the DNA replication process and is also required for the homology mediated DNA repair mechanism

Inheritance of Bloom Syndrome

Autosomal recessive

Clinical features of Bloom Syndrome

Short stature (<5ft)

Butterfly-shaped rash

High pitched voice

Long, narrow face

Small lower jaw

Prominent nose and ears

People w/ Bloom Syndrome live until their ___

• 20-30s

  • Rare and lethal disorder d/t high risk for cancer (ex. Skin, colorectal, leukemia, lymphomas)