Exam 2 DNA repair (15)

DNA Damage by UV Irradiation

-Exposure to UV light can induce pyrimidine dimer formation in DNA. - Two adjacent thymine bases on the same strand become linked to create a cyclobutane ring.

Xermoderma Pigmentosum (XP)

-Hereditary -due to an inability to repair DNA damage that results from UV radiation because of defective NER mechanism -XP patients are at ~1000x greater risk of developing skin cancer and ~100,000x greater risk of developing carcinoma of the tip of the tongue

Range of effects of mutations

-Most will lead to no obvious phenotypes. -Some will have mild effects, such as altered pigmentation -Some can have serious consequences, such as resulting in malignancy.

Repair mechanism for chemical carcinogens

-Nucleotide excision repair. -The lesion is often too bulky for base excision repair to be effective.

Examples of chemical carcinogens

-benzopyrene (found in coffee; coal tar) -acetaldehyde (occurs naturally; also found in tobacco)

What can reactive oxygen species do?

-damage the bases of DNA to cause mispairing -cause the complete loss of a base from a ribose residue; -induce single- and double-stranded breaks

What is a clinical issue associated with BRCA1 and BRCA2 mutations?

-familial breast cancer When mutated/deficient BRCA1 and BRCA2 genes are inherited, affected women are 40-80% more likely to develop breast and ovarian cancer.

Chemical carcinogens: dna adducts

-include natural and synthetic chemicals -many can react with DNA bases, often adding bulky adducts to DNA

How does dna damage occur?

-spontaneously or through chemical & environmental means -Normal metabolic processes in the body can also produce DNA-damaging by-products.

DNA damage by deamination

-the removal of an amine group. -will alter the structure of a base such that it now pairs with different partners during DNA replication.

How do cells deal with a spontaneous deamination of cytosine to uracil?

-they contain DNA glycosylases to remove all unnatural DNA bases created by deamination events. -DNA glycosylases travel along the DNA helix to probe all faces of the nucleotide for damage.

describe how DNA glycosylases are lesion specific? (~8 types in humans)

-uracil glycosylase for removing uracil in DNA (supposed to only be in RNA) -thymine DNA glycosylase for removing pyrimidine mismatches (thymine is a pyrimidine) -8-oxoG DNA glycosylase for removing oxidative base damage

General steps of Nucleotide Excision Repair (NER)

1. A multi-enzyme complex scans the DNA for distortion of the double helix. 2. A nuclease cleaves the phosphodiester backbone on both sides of the distortion. 3. a helicase separates the oligonucleotide containing the damaged DNA from the undamaged, complimentary DNA. 4. The gap is then repaired by DNA polymerase using the undamaged strand as a template and DNA ligase.

endogenous sources of DNA damage

1. DNA replication error 2. spontaneous deamination 3. Base Hydrolysis 4. Oxidation

Common/shared mechanisms of MMR, Base Excision repair, and NER (single strand repair)

1. Enzyme complexes recognize and excise damages (specific enzymes for specific mistakes) 2. DNA polymerase synthesizes proper strand 3. DNA ligase joins new, correct fragment with the existing polymer **net result: repaired DNA

MMR (strand directed mismatch repair) general steps

1. The Msh protein binds to bulge (which indicates a mismatch) in the double stranded DNA 2. The Mlh protein then binds to Msh at the DNA and scans for the closest nick 3. once a nick is found, mlh triggers the degradation of the nicked strand all the way back through the mismatch 4. the gap is then filled in by DNA polymerase and sealed by DNA ligase

exogenous sources of DNA damage

1. UV damage 2. Carcinogens: DNA adducts

How do unrepaired mutations become a permanent part of the genome? (Ex)

1. a guanine residue in a DNA strand is methylated; now, it H-bonds more easily to Thymine then Cytosine 2. When DNA strand is replicated, the mutated guanine pairs with "T;" the other parental strand is unaffected 3. During the subsequent round of DNA replication, half of all new daughter cells will contain the mutation

Base excision repair steps (ex with uracil-containing dna)

1. single "damaged" base is recognized 2. glycosylase cleaves the bond between the damaged base and deoxyribose, leaving behind an AP site (an apurinic or apyrimidinic; "a"=without) 3. Leaving behind an abasic sugar (base is missing; sugar is still present) 4. AP endonuclease then cuts the sugar-phosphate backbone, and exonuclease removes the abasic residue. 5. DNA polymerase adds new nucleotides and DNA ligase seals the nick.

How can the mismatch repair system discriminate between the mother strand and the daughter strand?

in eukaryotes, the presence of single strand breaks in newly replicated DNA appears to specify the strand to be repaired.

What can mutations in germ (sex) cells result in?

inherited disease

What can non-Homologous End Joining (NHEJ) be used for in the occurence of a double strand break?

it can be used to "glue" the DNA strands back together

How often does DNApol introduce an error in DNA reproduction?

it makes 1 for every 10^7 nucleotides added -however, with built in mechanisms to correct mistakes, newly synthesized DNA will have about 1 error in 10^9 or 10^10 nucleotides

If an incorrect nucleotide is added to the growing strand, what will DNA polymerase do?

it will cleave the wrong nucleotide from the strand and replace it with the correct nucleotide before continuing

MLH

looks for nick

What is used to fix a DNA adduct?

nucleotide excision repair

What is used to fix a link between tow bases?

nucleotide excision repair

Repair mechanism for dna damage by UV radiation

nucleotide excision repair -The covalent bond between adjacent bases requires that NER be used rather than base excision repair mechanism.

When do mutations become fixed in the genome?

only once DNA polymerases synthesize new daughter strands

Example of Nucleotide Excision Repair (NER) recognition

recognizes distortion due to pyrimidine dimers where two adjacent thymine (or cytosine) residues are covalently linked (cyclobutane formation/bulky)

Nucleotide Excision Repair (NER)

recognizes structural distortions in the double helix -bulky lesions are removed from the strand with several additional nucleotides ('oligonucleotide')

Depurination

removal of a purine base from the DNA strand -If not repaired, the loss of a base can result in a deletion mutation

What are the normal functions of BRCA1 and BRCA2 proteins?

repairing double-strand DNA breaks

example of a single base change (substitution) causing a disease

sickle cell anemia (glutamic acid (GAG) to valine (GTG))

MSH

sits on mismatch

mutations

when damaged dna isn't repaired and is passed onto subsequent generations, causing permanent changes in dna

how does 3'-->5' exonucleolytic proofreading activity of DNA polymerase work?

Both lagging and leading strand DNA polymerases have an exonuclease activity that can hydrolyze DNA in the 3'-->5' direction (i.e., remove a base that has just been covalently bound to the strand; analogous to a 'delete key') -After the correct base is incorporated, synthesis continues -This proofreading activity reduces the error rate to 1 in 10^9

How does Homologous Recombination (HR) ensure accurate repair?

By using the undamaged sister chromatid or homologous chromosome as a template

How is deamination of cytosine to uracil repaired?

Human cells contain a uracil DNA glycosylase enzyme that scans for and removes uracil bases in DNA (base excision repair)

What can create an apurinic site?

The glycosidic bond between the purine base and sugar in a nucleotide can spontaneously hydrolyze leaving the ribose sugar without a base

Different ways to identify newly synthesized lagging and leading strands

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

What happens when mistakes are missed by DNA polymerase's proofreading activity?

MMR (strand directed mismatch repair) detects distortions in the DNA helix that result from a misfit between non-complimentary base pairs

What do many of the metabolic reactions in the mitochondria generate that can damage DNA?

Reactive Oxygen Species (ROS: O2-, H2O2, OH•)

Hereditary Non-polyposis Colon Cancer (HNPCC)

a condition in which the tendency to develop colerectal cancer is inherited -due to mutations in Msh2 and Mlh1genes (Msh2, 60% of cases and Mlh1, 30% of cases). -Because DNA repair is compromised, 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.

oligonucleotide

a polynucleotide whose molecules contain a relatively small number of nucleotides

How are double stranded damages of DNA repaired?

a. Non-homologous End Joining (NHEJ) b. Homologous Recombination (HR)

How are single stranded damages of DNA repaired?

a. Strand-directed Mismatched Repair (MMR) b. Base Excision Repair (BER) c. Nucleotide Excision Repair (NER)

When does MMR (strand directed mismatch repair) take place?

as dna replication is occurring, before replication is completed

What is the repair mechanism for dna damage by deamination?

bace excision repair

How is deamination of 5-methylcytosine to thymine repaired? (5-methylcytosine is a naturally occurring methylated cytosine involved in gene regulation)

base excision repair

What is the repair mechanism for DNA damage by deamination?

base excision repair

What repairs dna damage by oxidation of bases?

base excision repair

How to repair DNA damage by Base hydrolysis that creates an apurinic site

base excision repair with the action of AP nuclease

Why is NHEJ repair mutagenic and error prone?

because it removes several base pairs at the break site

What does Nucleotide Excision Repair (NER) remove?

bulky adducts (e.g. pyrimidine dimers, carcinogen adducts)

how are the errors introduced during DNA replication resolved?

by 3'-->5' exonucleolytic proofreading activity of DNA polymerase

what can mutations in somatic cells result in?

cancer

example of DNA Damage by Oxidation of Bases

deoxyguanosine oxidation to form 8-oxo-deoxyguanosine

what in DNA occurs at a rate of about 100 bases per cell per day?

A spontaneous deamination of cytosine to uracil

Two pathways can be active with non-Homologous End Joining (NHEJ)

A. may cause small-scale mutation, such as the addition or the deletion of a small number of nucleotides at the break junction, leading to loss of base B. exonucleolytic processing of the double-strand break to reveal stretches of potentially complementary sequence, leading to loss of base - In both cases, DNA ligase seals the gap, and the double-strand break is repaired

What is an absolute requirement for maintaining genomic integrity?

High-fidelity of DNA replication

Which is a better choice for double strand repair: Non-Homologous End Joining (NHEJ) or Homologous Recombination (HR)? Why?

Homologous Recombination (HR) because it accurately repairs a chromosome (using sister chromatid), while Non-Homologous End Joining (NHEJ) is mutagenic and error-prone, because it removes several base pairs at the break site.

How is proofreading by DNA polymerase done?

through 3'-5' exonuclease activity

What is Homologous Recombination (HR) primarily used for?

to repair double- strand breaks during cell division (not replication) when duplicated chromosomes have not yet separated. -HR is beneficial for post-replication repair of DNA too -final product contains a fully restored, undamaged DNA strand; the damaged chromosome is accurately repaired, i.e., error-free.

What does non-Homologous End Joining (NHEJ) require in order to work?

two DNA blunt ends in order to join them back together