2. DNA Replication and Repair Flashcards
Polymerases
Enzymes that synthesize nucleic abides by forming PDE bonds, PPi is released
Nucleases
Hydrolyze PDE bonds:
- Exonucleases: remove nucleotides from ends
- Endonucleases cut within nucleic acid
Required primer DNA polymerase and RNA polymerase
DNA: RNA (or DNA) primer
RNA: None
Proofreading activity polymerases
DNA: Yes (exonuclease 3’->5’), RNA: No
Helicase
Breaks hydrogen bonds
Single stranded DNA binding protein (SSB)
Prevents reassociation of DNA and protects from degradation by nucleases
Primase
Synthesizes (10bp) RNA primer
DNA polymerase III
Begins synthesizing DNA in 5’->3’ direction
Leading strand & Lagging strand
Leading = continuously (move to replication fork) Lagging = Okazaki fragments (move from replication fork)
RNAase H + uncharacterized DNA polymerase (delta)
Removes primers in eukaryotes + fills the gap
DNA Polymerase I
Removes primers in prokaryotes + fills gap
DNA Ligase
Seals the nicks between Okazaki fragments
DNA Gyrase (topoisomerase II)
Provides swivel
Quinolones, function + examples
Block activity of topoisomerase
Levofloxacin, ciprofloxacin, moxifloxacin
Nalidixic acid
Kills bacteria by inhibiting DNA gyrase
Etoposide, teniposide
Inhibitors of eukaryotic topoisomerase II
Eurkaryotic DNA polymerases
Alfa and delta: leading and lagging strand
Gamma: replicates mitochondrial DNA
Beta and epsilon: DNA repair
Epsilon: can substitute for delta
Telomeres
Repetitive sequence at ends of linear DNA
Telomerase (only in eukaryotes)
RNA template, reverse transcriptase activity (hTRT). Only present in embryonic cells, germ cells and stem cells, not in somatic cells. Seen in cancer cells
Inhbition of reverse transcriptase meds
AZT, ddC and ddI
Reverse transcriptase activity in eurkaryotes
- Telomerase (hTRT)
- Retrotransposons (residual viral genomes in human DNA)
AZT (HIV treatment)
- Converted to triphosphate derivative, substrate
- Azide instead of normal hydroxyl group: prevents further replication by causing chain termination. (Reverse transcriptase does not have proofreading activity)
p53
Prevents damaged cells from entering S phase. Associated with Li Fraumeni syndrome and solid tumors
ATM
Kinase essential for p53 activity.
Ataxia telangiectasia
Inactivated ATM, hypersensitivity to X-rays and predisposition to lymphomas
Rb
Retinoblastoma gene, negative regulator cell cycle, binds to E2F and repress transcription of genes
Thymine dimers
Caused by UV light
Remove thymine dimers
By nucleotide excision-repair mechanism (excision endonuclease, aka excinuclease)
Cytosine deamination makes..
Cytosine -> Uracil
Removal of uracil by..
Uracil glycosylase enzyme, then recognized by AP endonuclease that removes damaged sequence (loss of purine or pyrimidine)
Maintain DNA fidelity in G2
Mismatch repair enzymes MSH2, MLH1
Maintain DNA fidelity G1->S
Rb, p53
Maintain DNA fidelity in S phase
DNA polymerase proofreading
Maintain DNA fidelity in G1/G0 phase
Thymine dimer repair (XP, nucleotide excision repair)
Xeroderma pigmentosum (4)
- Autosomal recessive (1/250,000)
- Extreme sensitive to sunlight, skin freckling, ulcerations, skin cancer
- Most common deficiency in excinuclease enzyme
- Measure enzyme level in white blood cells
Heriditary nonpolyposis colorectal cancer
- Aka Lynch syndrome
- Deficiency to repair mismatched base pairs (hMLH1 or hMSH2)
- Can inherit one copy, somatic mutation later
- Intestinal cells high cell division rate
Microsatellite instability
Short tandem repeats, usually in noncoding regions, e.g. TGTGTG. If mismatch repair lacks, number of repeats at locus will vary.
