Unit II- DNA Repair and Replication Flashcards
Degree of genome instabilities
1) Base pair changes
2) Small scale insertions, deletions, or inversions
3) Large scale segmental duplications, translocations
4) Large scale repeat expansion (Fragile X syndrome, Huntington’s disease)
5) Whole chromosome loss or gain (Down syndrome, trisomy; Klinefelter’s syndrome, XXY; Patau syndrome, trisomy 13; Triple-X syndrome, XXX)
6) Cancer genome contains all the above changes
Genetic instability and cancer
-genomic instability results from mutations in DNA repair genes and drives cancer development
Duplication and passage of the hereditary material
- each eukaryotic chromosome has multiple origins of replication, one centromere, and two telomeres
- DNA replicates in interphase
- DNA replication is defined as the process by which two complete double helices are produced from an original DNA molecule, and which are identical in nucleotide sequence to each other and the parental double helix
Meselson-Stahl experiment
- labeling 15N-heavy, 14N-light
- when heavy DNA was asked to replicate its DNA in the presence of growth medium containing light isotopes, the replicated DNA is of hybrid density
- showed semiconservative nature of DNA replication
Replication and Origins of Replication
- DNA replication initiates at multiple origins of replication
- 30,000 origins in the human genome
- the activation of these origins follow a temporal program in which some origins activate early whereas others late
- DNA replication is bidirectional in most organism, giving two replication forks per replication origin. Bidirectional synthesis generates a replication bubble
- Y shaped junctions formed by newly synthesized DNA and unsynthesized DNA are called replication forks
- DNA is unwound in front of replication fork
- replication forks move progressively away from the origin in bidirectional manner
Replication fork is asymmetrical
- polymerization in the 5’ to 3’ direction
- DNA synthesis on one of the two strands proceeding in a give direction away from the origin lags behind that of DNA synthesis on the opposite strand called lagging strand and leading strand synthesis, chromosome, the replication fork is thus asymmetrical
- lagging strand is discontinuous and are short, transiently disconnected single stranded DNA fragments called Okazaki fragments (~100-200 bases) are generated by stutter step mechanism
Proteins at replication forks
Origin Recognition Complex (ORC)- protein complex responsible for recruiting other initiation proteins to the origin
Helicase- unwinds double stranded DNA
single-stranded binding proteins (ssDNA) RPA- prevents exposed strands from reannealing, and prevent degradation
Primase- lays down RNA primer, required for leading and lagging strands (multiple)
-DNA polymerase (epsilon for leading strand, delta for lagging) synthesizes DNA
-sliding clamp which is loaded by clamp loader keeps DNA polymerase on template
-RNA primers removed by RNase
-Gap is filled by a repair DNA polymerase
-Gap between adjacent DNA strands is sealed by phosphodiester bonds by ligase
DNA polymerases are self-correcting
-high fidelity enzymes
-exonuclease activity is 3’ to 5’
5’ to 3’ polymerization- 10^-5
3’ to 5’ exonucleolytic proofreading 10^-2
Mismatch repair 10^-2
Total 10^-9
Mismatch repair and mutator phenotype
- Hereditary Non-Polyposis Color Cancer, the most common form of hereditary colon cancer, can be caused by mutations in any one of the seven mismatch repair genes. Mutations in these genes cause the so-called mutator phenotype
- majority in hMSH2 (40%) and hMLH1 (40%)
- mismatch repair deficiency the replication misincorporation errors accumulate
TDP1 (Tyrosyl DNA phosphodiesterase)
-SCAN1 (spinocerebellar ataxia with axonal neuropathy)
APTX (nucleotide hydrolase/ transferase)
AOA1 (Ataxia oculomotor apraxia-1)
DNA ligase I
Immunodeficiency, defects in repair of DNA breaks induced by UV etc
DNA ligase IV
Lig4 syndrome (immunodeficiency, radiosensitivity, pancytopenia, developmental abnormalities, and microcephaly)
FEN1
Cancer susceptibility, autoimmunity
Pre-replication compex (ORC4, ORC4, ORC6, CDT1, and CDC6)
Meier-Gorlin syndrome
Eukaryotic DNA polymerases
B family- chromosomal replication
Pol alpha- primase, RNA primer
Pol delta- 3’- 5’ Exo, lagging
Pol epsilon- 3-5 Exo, leading
Pol gamma (A family)-mitochondrial replication and repair
Replication stress response
- replication forks are vulnerable to DNA damage or nucleotide starvation, etc generally referred to as replication stress
- cells have evolved mechanisms called checkpoints to deal with these stressful events in order to preserve replication fork integrity and genome stability
- abnormal replication intermediate such as extension single-stranded DNA, reversed replication forks, and ultimately double strand breaks
Replication stress checkpoints
DNA damage -> go to stalled fork which causes Fork collapse or the fork is stabilized, both options cause a fork restart.
Or after DNA damage go to checkpoint activation which leads to repair, block ongoing forks and late origin firing, or having dormant origins
Ataxia Telangiectasia
- susceptible to lymphomas
- ataxia (abnormalities of balance), dilation of blood vessels (telangiectases) in skin and eyes, chromosome abberations, immune dysfunction
- sensitive to gamma irradiation
- mutations in the gene ATM (AT mutated), a protein kinase that is important for replication stress response and regulates p53, causing its accumulation
Bloom syndrome
- susceptible to carcinomas, leukemias, and lymphomas
- facial telangiectases, chromosome alterations
- sensitive to mild alkylating agents
- caused by mutations in the BLM gene, a RecQ helicase, that functions in replication stress response
Anticancer or antiviral therapy using replication inhibitors
-nucleotide analog chain terminators AZT, ddC, ddl. cause premature chain termination during DNA replication due to the missing 3’OH group
enzyme inhibitors that target the DNA replication pathway to stop cancer proliferation:
Camptothecin- targets Top I
Hydroxyurea- targets RNR, depleting the cancer cell of deoxyribonucleotides
5-Fluorouracil (5-FU)- targets thymidylate synthetase
DNA damage
-mismatch
-depurination
-pyrimidine dimer
-bulky adduct
-deamination
alkylation
Spontaneous events, exposure to environmental mutagens, DNA replication errors or stress
DNA repair mechanisms
Template-independent damage- Direct reversal:
Pyrimidine dimers: photoreactivation by photolyase
Alkylation: O-methylguanine methyltransferase
Single-strand DNA damage (one intact copy present):
Base excision repair (BER)
Nucleotide excision repair (NER)
Mismatch repair (MMR)
Double-strand breaks:
Homologous recombination
single-strand annealing
Non-homologous end joining (NHEJ)
Single strand DNA damage repair
(BER, NER, and MMR all involve three steps)
1) The altered portion of damaged DNA strand is recognized and removed by enzymes called DNA repair nucleases, which hydrolyze the phosphodiester bonds that join the damaged nucleotides to the rest of the DNA molecule, leaving a small gap in the DNA helix
2) DNA polymerase binds to the 3’ OH end of the cut DNA strand and fills in the gap by making a complementary copy of the information stored in the good strand
3) The break in the damaged strand is sealed by DNA ligase
Only the first step differs
Homologous recombination
double strand break
- dsDNA is exonucleolytically processed to form 3’ ssDNA tails, which invade homologous intact sequences
- DNA strand exchange follows and generates a joint molecule between damaged and undamaged duplex DNAs
- sequence information that is missing at the DSB site is restored by DNA synthesis
- the interlinked molecules are then processed by branch migration, Holliday junction resolution and DNA ligation
Single-strand annealing
- at the DSB site, 3’ ssDNA tails, consisting of direct repeats are generated
- they are aligned and the intervening sequences as well as protruding 3’ ends are removed
Non-homologous end-joining
- following DSB formation, broken DNA ends are processed to yield appropriate substrates for direct ligation
- no homology is necessary for DSB repair by non-homologous end-joining
- breaks can be joined accurately, but more often, small insertions or deletions are created
- most popular mechanism in humans
- recruitment of Ku 70/80 heterodimer to lesion site to bind free DNA ends
- recruitment of catalytic subunit of DNA protein kinase and Artemis, and the phosphorylation of Artemis by DNA-PK
- protein-protein interactions between DNA-PK molecules bridges the DNA ends
- assembly of DNA-PK recruits DNA ligase IV and XRCC4 which polynucleotide kinase (PNK) is localized to break sites
Xeroderma pigmentosum
- severe predisposition to skin cancers
- seven genetic complementation groups, XP-A to XP-G, have been identified associated with defective NER
-loss of function in XPV also results in XP, but is not associated with NER but with defects in lesion bypass of UV-induced
Nijmegan breakage syndrome
- gamma irradiation
- DSB repair, NBS1
- strong predisposition to lymphomas/ Microcephaly, a distinct facial appearance, short stature, immunodeficiency, radiation sensitivity
Cockayne syndrome
- UV light
- Nucleotide excision repair, ERCC6, 8
- Dwarfism, retinal atrophy, photosensitivity, progeria, deafness, trisomy 10
Fanconi anemia
- Cross-linking agents
- FANCD1, FANCD2
- Leukemias/ Hypoplastic pancytopenia, congenital anomalies
Breast and ovarian cancer
- ionizing radiation and other genotoxins
- BRCA1, BRCA2
- hereditary breast and ovarian cancer
