DNA_repair

Describe the relationship between mutations, DNA repair and cancer. Give examples of heritable human diseases that are caused by defective DNA repair pathways.

- If DNA damage is not properly repaired, the result is mutations. If mutations occur in genes encoding components of the DNA repair machineries or DNA damage sensing and signaling pathways the result is more mutations and genomic instability - accumulation of changes to the DNA. -If there are mutations in these pathways such that the cell cannot kill itself or stop dividing however, the result is increased risk of developing cancer. Clearly DNA damage and repair is essential for keeping cancer in check. For example: mutations in genes that mediate NER lead to human genetic diseases including Cockayne Syndrome (CS), Xeroderma pigmentosum (XP), and Trichothiodystrophy (TTD).

What are the types of DNA damage?

1. Change of the base: • Spontaneous base loss (depurination, depyrimidination) • Spontaneous deamination (A, C, G)--can cause mutation • Sunlight (UV)-induced thymidine dimers • Base alkylation (e.g. O6-meG)--can cause mutation • Base oxidation by reactive oxygen species (e.g. 8-oxo-dG) 2. Change in DNA structure • Bulges in DNA double helix caused by: --Insertion/deletion of nucleotides --Bulky chemical adducts --Replication errors (mismatch) --Intra/inter-strand crosslinks • DNA strand breaks • Stalled DNA replication forks Note: one DNA damage can be repaired by various repair pathway

How do eukaryotic cells distinguish b/n old and new strand of DNA in mismatch repair?

1. DNA nicks (okazaki fragments) that are more abundant on the newly replicated strand of the lagging strand; there are nicks in the leading strand as well? 2. the new leading strand will have ribonucleotides monophosphate, which is processed into nicks by RNase H2.

what type of machinery is used to repair the damage

1. Direct reversal of the damaged bases: ex: MGMT (removal of methyl group by methyltransferease) 2. Excision of damaged, mispaired, or incorrect bases: a.NER b.BER c.MMR 3. Tolerance/bypass of base damage (trans-lesion synthesis) 4. Strand break repair of damage to the DNA backbone a. single-strand break repair (SSBR) b. double-strand break repair (DSBR) ----->Non-homologous end joining (NHEJ) ----->Homologous recombination (HR)

What are the two ways NER recognizes DNA damage

1. Global Genome NER: --Recognizes damage anywhere in the genome. --Defects cause cancer. e.g.Xeroderma pigmentosum(XP) - skin cancer 2. Transcription-Coupled NER --Recognizes damage within region of active transcription. --defect causes CNS disorder e.g. Cockayne syndrome(CS) CS causes sun hypersenitivity, premature aging, impaired dev't, neurological degeneration Note: the first step of NER is different in the above two ways but steps 2-5 are the same. so a defect in shared step causes skin cancer and CNS disorder

Describe the double strand break repair mechanism

1. Homologous recombination (HR) a. Extended sequence homology needed b. Accurate 2. Nonhomologous end-joining (NHEJ) a. No homology needed b. Often inaccurate, resulting in deletion/insertion

Explain the basic steps of nucleotide excision repair

1. Recognition and binding of the damaged site by a multi-protein complex (two different ways depending on local transcription activity). 2. Local unwinding of the DNA duplex by helicases (parts of the TFIIH protein complex) to form a bubble of ~25 bases. 3. Double incision of the damaged strand by two endonucleases and removal of a ~12-30 base oligonucleotide containing the lesion. 4. Filling in the gap by a DNA polymerase. 5. Rejoining the two ends by a DNA ligase.

What are the steps common to all three excision repair mechanisms

1. Recognition of the damaged/mismatched nucleotide. 2. Endonuclease-mediated cutting of the phosphodiester backbone flanking the damaged/mismatched nucleotide. 3. Nuclease-mediated removal of the DNA fragment containing the damaged/mismatched nucleotide. 4. DNA polymerase-mediated synthesis of the missing nucleotides by copying nucleotide sequence from the intact DNA strand. 5. DNA ligase-mediated sealing of the remaining nick in the phosphodiester backbone.

Explain the basic steps of mismatch repair

1. The mismatched base pair is recognized shortly after DNA synthesis by MSH (MutS Homolog) and MLH (MutL Homolog) proteins. **MutS surveys and recognizes mismatch and then recruits MutL, helicase for unwinding; MutL chews away the mistmatch 2. A helicase assists with the unwinding of the double helix. 3. An endonuclease cleaves the phosphodiester backbone of the new strand of DNA. 4. An exonuclease chews away the new DNA strand including the mismatch nucleotide 5. DNA polymerase repairs the resulting single strand gap by incorporating complimentary base pairs, and 6. DNA ligase seals the phosphodiester ***mutations in MMR genes causae HNPCC (hereditary non-polyposis colorectal cancer: lynch syndrome)

What are the molecular consequences of failure in DNA repair?

1. Thymine dimers 2. Uracil mis-incorporation 3. Bulky chemical adducts 4. Double strand breaks

what are the sources and nature of damage to DNA

1. cancer, radiation therapy, chemotherapy 2. uncorrected errors made during DNA replication 3. damage that occurring to replicating or nonreplicating DNA: --oxidative damage* caused by products of normal metabolic activity of the cells *are caused by reactive oxygen species (ROS) generated during metabolism --cleavage of a DNA strand caused by radiation and chemicals --chemical alteration to the base (e.g alkylation) -- G to 06-methylguanine * point mutation--> G:C --> O5meG:T --> A:T --loss of a base (depurination/depyrimidation) via hydrolysis --loss of an amine group of the base (deamination) via hydrolysis -- C to U and G to A *leads to point mutation --sunlight induced thymine-dimers aka pyrimidine dimers

What are the two DNA checkpoint kinases that sense DNA damage, DNA double-strand break, and/or stalled replication fork

ATR and ATM ATR and ATM then recruit? ChK1 and Chk2 for downstream repair action

Why is DNA repair important?

DNA damage can be caused by both exogenous and endogenous sources, and if unrepaired, can lead to mutations that cause many human diseases, including cancer. Note: significant fraction of our genome encodes proteins involved in DNA repair. 3% of our human gene is dedicated to DNA repair *DNA damage is also used to treat cancer through radiation therapy and chemotherapy

Describe the mechanism that enables replication to continue in the face of DNA lesions that other repair pathways fail to remove, and know the unfortunate consequence of this process for the cell.

If a cell encounters so much DNA damage of the type that normally blocks DNA replication (such as UV-induced thymidine dimers) that the excision repair systems cannot fix it all, cells resort to a pathway called lesion bypass or translesion synthesis. Lesion bypass allows cells to continue replicating and dividing in the face of immense damage. However, it is highly mutagenic because alternate DNA polymerases that lack 3' to 5' proofreading exonuclease activity are used to replicate past the DNA lesion. The result is an error rate 100-10,000 higher than normal DNA replication.

Explain the concept of DNA damage checkpoint and its role in maintaining genome stability.

It is a cellular surveillance mechanism that halts cell cycle progression when DNA is compromised to allow time for DNA repair. It is a signaling pathway composed of damage sensors, signal transducers (kinases) and effectors. Central to lesion detection is a pair of homologous protein kinases called ATM and ATR, which is recruited to site of DNA damage and initiate the sequential recruitment and activation of downstream proteins. ATM and ATR become activated in human cells undergoing earlier stages of tumorigenesis to delay or prevent cancer. Mutations disrupting this checkpoint result in genomic instability and malignant conversion.

How is a single strand break repaired

PARP: Poly(ADP-ribose) polymerase is activated by a single-strand break and adds poly(ADP-ribose) chains to proteins. PARP: - Amplification of damage signal. - Focal enrichment of repair proteins. - Change in local chromatin structure. **works in BRCA 1 and 2 ?? breast cancer

Explain the basic steps of base excision repair

Step 1. Modified base is recognized by a specific DNA glycosylase, which hydrolyzes the N-glycosidic bond, yielding an AP site. Step 2. An AP site-specific endonuclease (APE1) cleaves the sugar-phosphate backbone 5' to the AP site. Step 3. Another endonuclease cuts 3' to the AP site, removing the deoxyribose phosphate. Step 4. The resulting gap is filled by DNA polymerase, and the nick sealed by DNA ligase. Note: each glycosylase recognize only a particular base damage, a particular inappropriate base, or a particular mispairing.

Describe the three excision repair mechanisms to repair DNA damage

all three repair machineries take advantage of the double-stranded nature of the DNA molecule to copy the correct information from the intact strand of DNA to the damaged strand. 1) Base excision repair (BER) **repairs base damages that do not distort the DNA. **uses base-specific glycosylases to remove the damaged base. 2) Nucleotide excision repair (NER) **repairs base damages that distort the DNA. **removes an oligonucleotide that contains the damaged base. 3) Mismatch repair (MMR) **removes misincorporated nucleotides during DNA replication made by DNA polymerase **distinguishes between the template strand and the new strand.