Lecture 19: Genetic Disease and DNA Repair

Example of Chromsome Translocation

- The Philadelphia Chromosome (chromosome 9 and 22) - 95% of people with myelogenous leukemia (CML) have this abnormality occur between 8 and 14

What is unique about benzopyrene and how is does it cause mutations in DNA?

- benzopyrene must first be oxidized within cells before it can be an alkylating agent - once oxidized it binds with GC base pair

What do reactive oxygen species do to DNA?

- damage bases - single stranded breaks - double stranded breaks

How does nucleotide excision repair work?

- exonuclease removes large section of DNA that is flanking the lesion (mutation) - DNA polymerase and DNA ligase then fills the gap

Chromosome Rearrangements

- homologous recombinations - translocation - transposable elements

Other affects of point mutations..

- interference with splicing - can be in promoter and result in decreasing or increasing expression

What does nitrogen mustard do?

- non-specific DNA alkylating agent - CAN CROSS LINK DNA STRANDS - similar to mustard gas - Chlorine groups attack adjacent bases

How does repair system know which strand is correct and which is incorrect?

- parental strand is METHYLATED - when repair system encounters mismatched base pair, it scans DNA for methylated base. Then replaced entire stretch of nucleotides in nascent (new) strand from point of recognition to and including mismatched base.

How does the mismatch repair system know which is correct?

- parental strand is methylated, new strand not methylated yet

What are the disadvantages of NHEJ?

- some DNA can be lost in process - error prone, mutagenic - can lead to cancer and immunodeficincies

Depurination

-example of spontaneous DNA damage - glycosidic bond in a purine nucleotide spontaneously hydrolyzes - DNA in a human cell can lose as many as 5,000 purines (A and G) per day from their deoxyribose due depurination - sugar phosphate backbone lacking a base at this site is referred to as "apurinic" or "apyridinic site"

List come common forms of DNA damage...

-missing, altered or incorrect base - bulges due to insertions or deletions - UV induced pyrimidine dimers - Strand breaks at phosphodiester bonds or deoxyribose rings - covalent cross-linking of strands (intra and inter)

Alkylating Agents

-react with oxygen or nitrogen of various bases in DNA - some react directly, others need to be metabolized before they are reactive (i.e. benzopyrene) - bifunctional alkylating agents can cross-link the DNA strands (i.e. nitrogen mustard) - alkylating agents are used in cancer chemotherapy

What does unrepaired DNA damage result in?

1. Aging 2. Mutations which can cause cancers

What are two types of damages formed from UV light?

1. Cyclobutane pyrimidine dimers 2. 6-4 Photoproduct Two common UV products are cyclobutane pyrimidine dimers (CPDs, including thymine dimers) and 6,4 photoproducts. These premutagenic lesions alter the structure of DNA and consequently inhibit polymerases and arrest replication. Dimers may be repaired by photoreactivation or nucleotide excision repair, but unrepaired dimers are mutagenic. Pyrimidine dimers are the primary cause of melanomas in human beings.

Which proteins are important for homologous recominbation?

BRCA1 and BRCA2 - help recruit other proteins/enzymes

Why does homologous recombination occurs in S,G2, and M phase but not G1?

Because G1 generally does not have a duplicated set of chromsomes so there is not a sister chromatid (homologous chromsome) to use as a template.

Homozygosity

Both copies of the gene in question are mutated

Repair of Double Stranded Breaks

Cannot be repaired by previously mentioned methods Repaired by one of two systems (Non-Homologous End Joining Repair or Homologoous Recombination Repair)

Mutation

Change in DNA sequence

Point Mutation

Change in a single base pair

What does AP nuclease do in base excision repair?

Cleaves backbone - exonuclease removes the deoxyribose-phosphate and several additional residues, and the gap is then repaired by DNA polymerase and DNA ligase

Deamination

Cytosine in DNA can be deaminated to Uracil. Uracil will then pair with adenine base in DNA.

Deamination results in which base turning into which?

Cytosine is deaminated to become Uracil. (amino group is replaced with carbonyl group (ketone)) (uracil then pairs with adenine)

What is Spontaneous damage (endogenous)?

DNA damages occur spontaneously or in presence of reactive metabolites.

DNA methylation review...

DNA methylation is a biochemical process that is important for normal development in higher organisms. It involves the addition of a methyl group to the 5 position of the cytosine pyrimidine ring or the number 6 nitrogen of the adenine purine ring (cytosine and adenine are two of the four bases of DNA). This modification can be inherited through cell division.

Deletion or Insertion Mutations

Deletion/insertion of 1 or 2 baes can result in a frameshift mutation causign the protein being translated in the wrong reading frame. - some can be huge and delete entire genes.

Ionizing Radiation

Developes double strand breaks.

Is UV damage exogenous or endogenous?

Exogenous

When is NHEJ generally used?

G1

Transcription Coupled Repair Mechanism

Genes are copied from DNA to make messenger RNA to instruct protein synthesis by RNA polymerase II. When RNA polymerase finds a lesion in the DNA, like those caused by UV light it stops and the gene is not transcribed. TCR is associated with RNA polymerase II, and the stalled RNAP is the trigger for TCR both in prokaryotes and eukaryotes.

Dominant

If heterozygous affect the patient clincally. The mutation is dominant.

Xeroderma Pigmentosum

Inherited recessive disease characterized by inability to repair DNA damage

Somatic Mutation

Leads to abnormal somatic cells. - series of such mutations can lead to loss of normal controls on cell division →cancer.

What has a greater error rate: NHEJ or Homologous Recombination repair?

NHEJ Homologous Recombination is preferred.

Germ Line Mutation

Occur in egg or sperm and are inheritied.

Heterozygosity

Presence of one copy of mutated gene and one copy of wild type gene.

What does DNA glycosylase do in base excision repair?

Removed damaged base by glycosidic bond. - this creates an AP site (apurinic site) where the sugar phosphate backbone is intact but the base is missing

General summary of NHEJ

Requires proteins for the direct joining of broken ends of DNA. Ku proteins are heterodimers that recognize and bind the broken ends. Additional proteins are then required for the processing and ligation of the broken ends.

What is SAM and what does it do?

S-adenosyl methionine - non-enzymatically methylates bases

Methylation

S-adenosyl methionine (SAM) non-enzymatically methylates bases.

How does the double stranded nature of DNA protect the cell from permanent damages?

The complementary nature of DNA ensures that information lost through damage to one strand can be recovered from the other.

Recessive

The disease appears only if individual is homozygous

What bond is broken in Depurination?

The glycosidic bond. - spontaneously hydrolyzed

What does Homologous recombination use for a template for the broken double stranded DNA?

The sister chromatid. (homologous chromosome)

6-4 Photoproduct

These occur less frequently than cyclobutane pyrimidine dimers but are much more mutagenic. (caused by UV)

Why are Ku proteins important in Non-Homologous End Joining repair?

They recognize and bind broken ends of DNA. Potentially recruit other proteins for processing and ligating. - Ku proteins are heterodimers

Transcription Coupled Repair

Transcription-coupled repair is a DNA repair mechanism which operates in tandem with transcription. The activity of TCR has been known for 20 years, but its mechanism of action is an area of current research. Failure of the transcription-coupled repair is the cause of Cockayne syndrome,[1] an extreme form of accelerated aging that is fatal early in life.

What is an "apurinic site"

Where the DNA was depurinated.

DNA Damage slide

While DNA is relatively stable, it is susceptible to spontaneous damages even under normal cell conditions which would lead to a mutation if left unrepaired.

What happens if mutation in BRCA1 and BRCA2?

Women are predisposed to breast cancer and ovarian cancer.

What are the two methods for repair of double stranded DNA breaks?

1. Non-Homologous End Joining Repair 2. Homologous Recombination Repair

How many purines can be lost per day due to depurination?

5,000 (A and G)

What is mismatch repair?

A mechanism that excise and replace mismatched regions. - scans duplex DNA for mis-paired bases, excise and replaces entire stretch of nucleotides in the new strand

Nonsense Mutation

A point mutation in which a pre-mature terminating codon is present and can result in a truncated protein.

Missense mutation

A point mutation in which it occurs in the coding region and can lead to an AMINO ACID SUBSTITUTION. i.e. Glutamate switched to Valine in sickle cell

Silent Mutation

A point mutation in which there is no effect due to the "redundant nature of the genetic code"

Mutagens

Agesnts (chemical or radiation) that damage DNA and increase the rate of mutations.

Base Excision Repair

In biochemistry and genetics, base excision repair (BER) is a cellular mechanism that repairs damaged DNA throughout the cell cycle. It is responsible primarily for removing small, non-helix-distorting base lesions from the genome. The related nucleotide excision repair pathway repairs bulky helix-distorting lesions. BER is important for removing damaged bases that could otherwise cause mutations by mispairing or lead to breaks in DNA during replication. BER is initiated by DNA glycosylases, which recognize and remove specific damaged or inappropriate bases, forming AP sites. These are then cleaved by an AP endonuclease. The resulting single-strand break can then be processed by either short-patch (where a single nucleotide is replaced) or long-patch BER (where 2-10 new nucleotides are synthesized).[1]

Does the mismatch repair system remove just the mismatched nucleotide?

No, it replaces entire stretch from point of recognition up until and including the mismatched base.

What is Non-Homologous End Joining Repair?

Non-homologous end joining (NHEJ) is a pathway that repairs double-strand breaks in DNA. NHEJ is referred to as "non-homologous" because the break ends are directly ligated without the need for a homologous template, in contrast to homologous recombination, which requires a homologous sequence to guide repair. The term "non-homologous end joining" was coined in 1996 by Moore and Haber.[1] NHEJ typically utilizes short homologous DNA sequences called microhomologies to guide repair. These microhomologies are often present in single-stranded overhangs on the ends of double-strand breaks. When the overhangs are perfectly compatible, NHEJ usually repairs the break accurately.[1][2][3][4] Imprecise repair leading to loss of nucleotides can also occur, but is much more common when the overhangs are not compatible. Inappropriate NHEJ can lead to translocations and telomere fusion, hallmarks of tumor cells.[5] NHEJ is evolutionarily conserved throughout all kingdoms of life and is the predominant double-strand break repair pathway in mammalian cells.[6] In budding yeast (Saccharomyces cerevisiae), however, homologous recombination dominates when the organism is grown under common laboratory conditions.

Nucleotide Excision Repair

Nucleotide excision repair is a DNA repair mechanism.[1] DNA constantly requires repair due to damage that can occur to bases from a vast variety of sources including chemicals, radiation and other mutagens. Nucleotide excision repair (NER) is a particularly important mechanism by which the cell can prevent unwanted mutations by removing the vast majority of UV-induced DNA damage (mostly in the form of thymine dimers and 6-4-photoproducts). The importance of this repair mechanism is evidenced by the severe human diseases that result from in-born genetic mutations of NER proteins including Xeroderma pigmentosum and Cockayne's syndrome. While the base excision repair machinery can recognize specific lesions in the DNA it can correct only damaged bases that can be removed by a specific glycosylase, the nucleotide excision repair enzymes recognize bulky distortions in the shape of the DNA double helix. Recognition of these distortions leads to the removal of a short single-stranded DNA segment that includes the lesion, creating a single-strand gap in the DNA, which is subsequently filled in by DNA polymerase, which uses the undamaged strand as a template. NER can be divided into two subpathways (Global genomic NER and Transcription coupled NER) that differ only in their recognition of helix-distorting DNA damage.

What enzymes are used in homologous recombination?

Same enzymes usually used to perform recombination between homologous chromosomes during meiosis.

What does base excision repair often fix?

Spontaneous deamination (cytosine bases converted to uracil which then pair with adenine rather than guanine)

What is nucleotide excision repair used for commonly?

UV light damage (Thymine dimers and 6-4 photoproducts)

Cyclobutane pyrimidine dimers

UV light induces linkages with reactions localized on the C=C double bonds.

What disease can alkylating agents be used to treat?

Various cancers. - purposely mutate bases in cancer cells

When does Homologous recombination usually occurs?

When a double strand break occurs during S, G2, or M phase.