DNA Repair

Question 2

As DNA damage is potentially very deleterious, all organisms have several approaches to dealing with it. The possibilities include:

Answer 2

• a) Remove the lesions and restore genomic integrity (various pathways exist, i.e. repair.
• b) Ignore the DNA modifications to allow survival but with the risk of genomic instability (translesion synthesis bypass).
• c) Halt cell cycle progression to allow more time for DNA repair.
• d) Induce permanent cell cycle arrest (senescence) or execution (apoptosis).

Question 3

What are the main responses to DNA damages?

Answer 3

• DNA repair
• Cell cycle transitions
• Transcription
• Apoptosis

Question 4

DNA Damage Surveillance

Answer 4

• Damage to DNA is continuously monitored by proteins that move along the DNA strands during the processes of replication and transcription:
• DNA polymerase during replication
• RNA polymerase during transcription (leads to transcription coupled repair or TCR)
• Some other proteins not involved in replication and transcription also have a watchdog function (e.g. DNA glycosylases and UV-DDB).

Question 5

Name different types of DNA damage & repair

Answer 5

• Double strand breaks→MRN Complex recruits ATM→DNA damage response (DDR)
• Single strand breaks→ PARP-1→ Base excision repair (BER)
• Base oxidation→ DNA glycosylases e.g. OGG1→ Base excision repair (BER)
• Pyrimide X-links→ RNA Pol & UV-DDB→ Nucleotide excision repair (NER)

Question 6

Why is the double-helical structure of DNA ideally suited for repair?

Answer 6

• The double-helical structure of DNA is ideally suited for repair because it carries two separate copies of all the genetic information—one in each of its two strands.
• Thus, when one strand is damaged, the complementary strand retains an intact copy of the same information, and this copy is generally used to restore the correct nucleotide sequences to the damaged strand.

Question 7

Explain DNA repair

Answer 7

Genetic information can be stored stably in DNA sequences only because a large set of DNA repair enzymes continuously scan the DNA and replace any damaged nucleotides. Most types of DNA repair depend on the presence of a separate copy of the genetic information in each of the two strands of the DNA double helix. An accidental lesion on one strand can therefore be cut out by a repair enzyme and a corrected strand resynthesized by reference to the information in the undamaged strand.

The DDR is an orchestrated signalling cascade that involves recognition > coating of the lesion > error signal amplification > cell cycle checkpoint activation > DNA repair. There is also the mismatch repair system (MMR), which is perhaps more involved in repairing replication errors.

Question 8

Repair of Double strands breaks (DSBs):

What are 2 main pathways for repair of DSBs?

Answer 8

Homologous recombination or end-joining can only occur in the S and G2 phases of mitosis as it requires presence of the 2^nd chromosome in order to copy the correct sequence.

Question 9

Describe recognition of double strands breaks (DSBs)?

Answer 9

• DSBs can cause chromosomal translocations, and are therefore some of the most toxic lesions.
• The key to recognition of DSBs is the MRN protein complex containing 3 proteins: Mre11-Rad50-Nbs1
• MRN recruits other proteins involved in cell cycle control and DNA repair: e.g. ATM and ATR
• They are nuclear serine/threonine protein kinases and part of a family of proteins known as the phosphoinositide-3-kinase-related protein kinases (PIKKs)
• They are rapidly activated and phosphorylate downstream substrates involved in the maintenance of genomic integrity.

Question 10

How is The Mre11-Rad50-Nbs1 (MRN) complex key in the eukaryotic DSB response mechanism?

Answer 10

• The Mre11-Rad50-Nbs1 (MRN) complex is key in the eukaryotic DSB response mechanism, involved in the three facets of DSB repair.
• It acts as a DSB sensor, helps induce cell cycle checkpoint signaling, and helps repair DSBs by both the HR and NHEJ pathways.
• Ataxia-telangeictasia mutated (ATM),
• Ataxia-telangeictasia related (ATR)
• DNA-dependent protein kinase catalytic subunit (DNA-PKcs) proteins are another example of PIKKs. PIKKs are a family of Ser/Thr-protein kinases with sequence similarity to phosphatidylinositol-3 kinases (PI3Ks), but they have roles in responses to stress and DNA damage.

Question 11

Draw a diagram to illustrate that ATM monitors strand breaks and recruits 3 other proteins that lead to cellular effects

Answer 11

3 ptoteins recruited:

• Rad50
• Mre11
• Nbs1

Question 12

Explain ATM and DNA PK control accumulation of DNA repair enzymes

Answer 12

• ATM and ATR accumulate at damage sites within minutes to form foci
• ATM and DNA-PK phosphorylate histone 2AX (gH2AX) at DSB.
• Phosphorylation causes activation
• gH2AX activates DNA repair machinery (RAD50).
• H2AX works with Mediator of DNA damage checkpoint protein 1 (MDC1).

Question 13

Activation of ATM or ATR can also lead to growth arrest

Answer 13

• Damaged DNA also generates signals that block cell-cycle progression in eucaryotes.
• As discussed in detail in Chapter 17, the orderly progression of the cell cycle is maintained through the use of checkpoints that ensure the completion of one step before the next step can begin.
• At several of these cell-cycle checkpoints, the cycle stops if damaged DNA is detected.
• Thus, in yeast, the presence of DNA damage can block entry into the G1 phase; it can slow DNA replication once begun; and it can block the transition from S phase to M phase.
• The DNA damage results in an increased synthesis of some DNA repair enzymes, and the delays further facilitate repair by providing the time needed for repair to reach completion.
• ATM directly interacts with the NBS1 subunit and phosphorylates the histone variant H2AX on Ser139.
• This phosphorylation generates binding sites for adaptor proteins with a BRCT domain.
• These adaptor proteins then recruit different factors including the effector protein kinase CHK2 and the tumour suppressor p53.
• The ATM-mediated DNA damage response consists of a rapid and a delayed response.
• The effector kinase CHK2 is phopsphorylated and thereby activated by ATM. Activated CHK2 phophorylates phosphatase CDC25A which is then degraded and can no longer dephosphororylate CDK2-Cyclin resulting in cell-cycle arrest.
• If the DSB can not be repaired during this rapid response, ATM additionally phophorylates MDM2 and p53 at Ser15.[6] p53 is also phosphorylated by the effector kinase CHK2.
• These phosphorylation events lead to stabilization and activation of p53 and subsequent transcription of numerous p53 target genes including Cdk inhibitor p21 which lead to long-term cell-cycle arrest or even apoptosis.

Question 14

Describe BRCA1 – a repair protein with a link to breast cancer

Answer 14

• BRCA1 is a multifunctional protein that is mutated in breast and ovarian tumours although mutations can be lethal in other tissues.
• BRCA1 is activated by phosphorylation by ATM.
• It is part of a protein complex that repairs DNA by homologous recombination when both strands are broken.
• It directly binds DNA and inhibits nuclease activity (which would cause DNA destruction)
• It associates rapidly with sites of DNA damage (histone H2AX phosphorylation) and increases expression of repair enzymes and survival genes (e.g p21 and GADD45).

Overall BRCA1 promotes transcription-coupled repair (TCR)

Question 15

How does BRCA1 repair double-strand breaks in DNA?

Answer 15

• The strands of the DNA double helix are continuously breaking from damage.
• Sometimes one strand is broken, and sometimes both strands are broken simultaneously.
• BRCA1 is part of a protein complex that repairs DNA when both strands are broken.
• When both strands are broken, it is difficult for the repair mechanism to “know” how to replace the correct DNA sequence, and there are multiple ways to attempt the repair.
• The double-stranded repair mechanism that BRCA1 participates in is homologous recombination, in which the repair proteins utilize homologous intact sequence from a sister chromatid, from a homologous chromosome, or from the same chromosome (depending on cell cycle phase) as a template
• This DNA repair takes place with the DNA in the cell nucleus, wrapped around the histone.
• Several proteins, including BRCA1, arrive at the histone-DNA complex for this repair.
• Regulatory aspect to BRCA1 nuclear ⁄ non-nuclear distribution was first shown by Dr Rao laboratory in 1997
• In the nucleus of many types of normal cells, the BRCA1 protein interacts with RAD51 during repair of DNA double-strand breaks.
• These breaks can be caused by natural radiation or other exposures, but also occur when chromosomes exchange genetic material (homologous recombination, e.g., “crossing over” during meiosis). The BRCA2 protein, which has a function similar to that of BRCA1, also interacts with the RAD51 protein.
• By influencing DNA damage repair, these three proteins play a role in maintaining the stability of the human genome.
• BRCA1 directly binds to DNA, with higher affinity for branched DNA structures. This ability to bind to DNA contributes to its ability to inhibit the nuclease activity of the MRN complex as well as the nuclease activity of Mre11 alone.[15] This may explain a role for BRCA1 to promote higher fidelity DNA repair by non-homologous end joining (NHEJ).
• BRCA1 also colocalizes with γ-H2AX (histone H2AX phosphorylated on serine-139) in DNA double-strand break repair foci, indicating it may play a role in recruiting repair factors.
• BRCA1 is on chromosome 17, while BRCA2 is on chromosome 13, i.e. they are 2 entirely different genes but both increase susceptibility to breast cancer and ovarian cancer.

Question 16

What does excision repair involve?

Answer 16

Excision repair involves identifying an error in the DNA, cutting it out, and replacing with the correct molecular structure (base, nucleotide, etc)

Question 17

General principles of excision repair which apply both to Base and Nucleotide excision repair processes:

Answer 17

• Excision repair enzymes or proteins detect the damaged base.
• An endonuclease nicks the backbone either side of the damaged base leaving a gap between 2 phosphates.
• A DNA polymerase fills the gap.
• A DNA ligase rejoins the nucleotide to the strand to seal the gap.

Question 18

Most of the damage to DNA bases is excised by one of two major DNA repair pathways:

Answer 18

• In base excision repair, the altered base is removed by a DNA glycosylase enzyme, followed by excision of the resulting sugar phosphate.
• In nucleotide excision repair, a small section of the DNA strand surrounding the damage is removed from the DNA double helix as an oligonucleotide.
• In both cases, the gap left in the DNA helix is filled in by the sequential action of DNA polymerase and DNA ligase, using the undamaged DNA strand as the template.
• A deficiency in NER causes a disease called xeroderma pigmentosum.
• Other critical repair systems—based on either nonhomologous or homologous end-joining mechanisms—reseal the accidental double-strand breaks that occur in the DNA helix.
• In most cells, an elevated level of DNA damage causes both an increased synthesis of repair enzymes and a delay in the cell cycle.
• Both factors help to ensure that DNA damage is repaired before a cell divides.

Question 19

Describe the The Base Excision Repair Pathway (BER)

Answer 19

• The BER pathway is one of the most common and universal mechanisms of DNA repair and very important for oxidative damage to bases.
• It is a process of specific recognition of damaged or oxidized bases, initiated by a family of DNA glycosylases.
• These cleave the N-glycosyl bond between the damaged base and 2-deoxyribose to give an abasic site (also called AP).

Question 20

Specific glycosylases include:

Answer 20

• Uracil DNA glycosylase (UDG)
• 8-oxo-guanine DNA glycosylase (OGG1)

But there are about 20 in total

Question 21

Explain the steps in the BER pathway

Answer 21

1. Damaged base
2. Recognized and cut out by a glycosylase to give an abasic site
3. APE1 cleaves the backbone forming a SSB with 3’-OH and 5’dRP ends
4. The 5’dRP is removed and DNA POLb inserts a nucleoside to fill the gap
5. The nick on the 3’ side is sealed by a DNA ligase
6. Complete sequence is now restored.

Question 22

What is involved in the long patch of the mechanism?

Answer 22

The long patch mechanism is more complicated, involving the formation of a 5’ flap and requiring additional enzymes.

APE-1 is Apurinic/apyrimidinic (AP) endonuclease 1.

Question 23

TRUE or FALSE: Single strand break repair (SSBR) is a specialized version of the BER pathway

Answer 23

TRUE

Question 24

SSBR copes with normal and abnormal strand breaks and involves the following steps:

Answer 24

–Strand break detection

–Removal of the 3’ or 5’ terminal blocking group

–Gap-filling repair synthesis

–Nick-sealing by a DNA-ligase

Question 25

–Strand break detection

–Removal of the 3’ or 5’ terminal blocking group

–Gap-filling repair synthesis

–Nick-sealing by a DNA-ligase

Answer 25

–Poly(ADP-ribose) polymerase 1 (PARP1)

–X-ray cross-complementing protein 1 (XRCC1)

Question 26

Describe poly-ADP ribosyl polymerase (PARP) for Single Strand Break Repair (SSBR)

Answer 26

• PARP is a molecular nick-sensor activated by strand breaks
• The DNA binding domain of PARP has strong affinity for single strand breaks (SSBs)
• PARP catalyses transfer of ADP-ribose to proteins associated with DNA structure; 90% on PARP.
• Promotes rejoining of SSBs by BER and NER. NAD is the substrate of PARP enzymes that becomes cleaved forming ADP-ribose and nicotinamide.
• PARP1 also inhibits cell cycle progression and may protect DNA from attack by nucleases

Question 27

PARP-1 is involved in normal BER at which stage?

Answer 27

PARP-1 is therefore also involved in normal BER at the stage when APE-1 has created a nick in the backbone.

Question 28

Diagram illustrating that PARP binding recruits DNA repair enzymes

Answer 28

Question 29

How is PARP involved in cancer therapy?

Answer 29

In cancer therapy, successful inhibition of PARP will drive cells to apoptosis because of an accumulation of strand breaks.

Question 30

What happens once PARP detects a SSB?

Answer 30

Once PARP detects a SSB, it binds to the DNA, and, after a structural change, begins the synthesis of a poly(ADP-ribose) chain (PAR) as a signal for the other DNA-repairing enzymes such as DNA ligase III (LigIII), DNA polymerase beta (polβ), and scaffolding proteins such as X-ray cross-complementing gene 1 (XRCC1). After repairing, the PAR chains are degraded via PAR glycohydrolase (PARG)

Question 31

What is the Nucleotide Excision Repair Pathway (NER)?

Answer 31

NER is a pathway that copes with a very broad range of structurally unrelated DNA lesions

Question 32

•NER is a pathway that copes with a very broad range of structurally unrelated DNA lesions, including:

Answer 32

–Thymine dimers or C-T (cyclobutane pyrimidine dimers)

–Bulky chemical adducts

–Intra-strand crosslinks

–Damage causing helical distortion of the DNA, which includes some oxidative modifications

Question 33

•There are 2 detection mechanisms that trigger NER:

Answer 33

–Transcription-coupled NER, triggered by RNA Pol II stalling when it encounters a lesion.

–Global Genome NER, when lesions in transcriptionally silent DNA regions are detected directly by proteins such as XPC or UV-DDB.

Question 34

Explain the Steps and proteins in NER

Answer 34

1. Damage recognition by XPC (GG-NER) or RNA Pol II (TC-NER)
2. Duplex opening by helicases (XPB / XPD) to unwind the DNA and allow access by repair enzymes.
3. An oligonucleptide containing the damaged section is cut out by endonucleases (e.g. XPA).
4. The gap is filled by DNA Pol and ligase enzymes.

Question 35

Mutations in “genome stability pathways” cause cancer: Xeroderma pigmentosum

Answer 35

A rare disorder transmitted in an autosomal recessive manner. It is characterized by photosensitivity, pigmentary changes, premature skin aging, and malignant tumour development. (NER defect)

Question 36

Mutations in “genome stability pathways” cause cancer: Ataxia telangiectasia

Answer 36

Immune dysfunction and malignancy (mutations in ATR / ATM)

Question 37

Mutations in “genome stability pathways” cause cancer: Fanconi’s anaemia

Answer 37

First malignancy by 14.5 years, 62% of which are haematological. Cancer risk increases with age; by 40, risk is >50% for haematological and solid tumours (defect in the FA pathway, related to NER).

Question 38

Therapeutic targets for anti-cancer agents

Answer 38

• Targets = checkpoints
• Aim: to increase cytostasis or apoptosis
• e.g. DNA damage agent cisplatin causes DNA adduct formation and arrests cells in G1/S and G2/M

Question 39

Describe DNA adducts

Answer 39

• 90% of cisplatin-DNA adducts are 1,2 intrastrand cross-links
• The remaining adducts are comprised of interstrand cross-links and monofunctional cisplatin adducts.
• Used in treatment of metastatic testicular cancers
• Destabilization. Cisplatin binding severely distorts DNA by twisting, unwinding, and shortening the duplex