W14L2

Question 1

Non-homologous end joining (NHEJ)

Answer 1

core protein components include:

Ku subunits (Ku70 and Ku80) interact with DNA–PKcs, and potentially form a docking site for other proteins

Artemis is involved in end trimming of the DSB – may lead to microdeletions

LigIV/XRCC4 complex serves to perform the ligation and final step of NHEJ

Question 2

Homologous Recombination vs. Non-homologous end joining

Answer 2

HR accounts for the repair of 10% of DSBs, while NHEJ accounts for the repair of 90% of DSBs

HR is active in S and G2 phases of the cell cycle, NHEJ is active in G1

HR provides a complete repair, NHEJ is error prone as small deletions and insertions occur

HR relies on the pairing of one of the broken strands with a complimentary region on the sister chromatid while NHEJ does not

Question 3

Mutations in key proteins in Mismatched mutant repair

Answer 3

mice lacking Exo1 displayed reduced survival and increased susceptibility to the development of lymphomas

sterile because of a meiotic defect

hereditary nonpolyposis colorectal cancer = HNPCC; is an autosomal dominant disorder

Germline mutations in mismatch repair genes have been found in the majority of families with HNPCC

Germline mutations in EXO1 appear in atypical HNPCC

Question 4

Mutations in key proteins in Base excision repair

Answer 4

XRCC1 = X-ray repair cross complementing 1
- deletion in mice results in lethality before gastrulation
- Biallelic mutations in humans leads to ocular motor apraxia, axonal neuropathy, and progressive cerebellar ataxia
- Point mutations found to increase risk for head and neck cancers

FEN1 = Flap endonuclease 1
- Removes the ”flaps” during DNA repair
- Deletion leads to early embryonic lethality
- Humans mutations results in autoimmunity, chronic inflammation and cancer WHY?
- Leads to frequent spontaneous mutations

Question 5

Mutations in key proteins in Nucleotide excision repair

Answer 5

Human mutations in CSA and CSB genes results in Cockayne syndrome; reccesive genetic disorder
- Cokayne syndrome results in abnormally small head, reduce growth and delayed development

Complete loss of ERCC1 in mice is embryonic lethal
- Mutations in ERCC1 result in cerebrooculofacioskeletal syndrome

Question 6

Mutations in key proteins in NHEJ

Answer 6

Ku70/80 sense double stranded DNA breaks

Mice deficient in Ku70 or Ku80 or both are viable and fertile

Both show growth retardation and severe combined immunodeficiency (Scid)

Both mouse mutants exhibit premature aging

Only Ku70 mouse mutants show susceptibility for cancer

Inhibiting Ku function has been suggested as a therapy for some cancers

Question 7

If altered DNA damage leads to genomic instability and cell death, why the association with cancer?

Answer 7

Compensation by other pathways

May not be as efficient or correct in repair process

Can you target compensatory pathways or factors that affect multiple pathways?
- PARP - Poly(ADP-ribose) polymerase
- Effective in cancers that have mutations in BRCA1/2
- This includes breast and ovarian cancers

If BRCA1/2 is defective, PARP will target repair to a different pathway (MMEJ repair) and away from NEHJ, which is more error prone and likely to lead to further genome instability and cell death

Question 8

Fanconi’s Anemia

Answer 8

Decreased blood cell production

multiple congenital somatic abnormalities, bone marrow failure and cancer susceptibility

Chromosomal spreads are significantly different

This is because the damage is random and repaired differently

Many breaks, improper repair, etc.

Genomic instability

Mutations in BRCA2 and FANC genes
- 29 FANC proteins are part of this pathway

Involved in the resolution of interstrand crosslinks (ICL) between chromosome
- Integral Fanconi complex recognizes these ICLs and recruits enzymes (also FA proteins) to remove them
- Promotes homologous recombination to fix the damaged DNA

Question 9

Translation of Homologous Recombination DNA repair to the lab

Answer 9

Process to create a targeted knockout of a gene in mice requires homologous recombination

Enhancing DSBs in ES cells will increase the potential for homologous recombination to occur

Question 10

CRISPR-Cas9 system for gene editing

Answer 10

CRISPR=Clustered regularly interspaced short palindromic repeats

Much more efficient in targeting (1/3)

Cas9 enzyme is directed by guide sequences to specific areas in the genome

Causes DSBs that can be resolved by NHEJ (common) or HR (rare)

NHEJ leads to INDEL errors (small insertions or deletions that affect the reading frame of your gene)

Essentially knocks out the gene targeted by CRISPR