Lecture 2 Flashcards
(16 cards)
Evidence for semiconservative mechanism of DNA replication
Heavy nitrogen (15N) used to make parent DNA, which was then allowed to replicate amongst normal nitrogen (14N). All double-stranded molecules in first generation were 15N-14N. Some molecules in successive generations were 15N-14N, others were 15N-15N, and most were 14N-14N.
DNA replication, step 1 of 7
Helicases unwind DNA to single-strand templates
DNA replication, step 2 of 7
Replication protein A stabilizes single strand
DNA replication, step 3 of 7
DNA polymerase ε adds nucleotides 5’ –> 3’
DNA replication, step 4 of 7
RNA primase lays down primers in lagging strand for DNA polymerase α
DNA replication, step 5 of 7
PCNA and DNA polymerase δ attach to primers to synthesize lagging strand
DNA replication, step 6 of 7
RNAse H digests RNA primer, and Pol α continues
DNA replication, step 7 of 7
Ligase forms phosphodiester bond to seal together DNA fragments
Proofreading DNA polymerases
Pol δ and Pol ε
Pol α does not have proofreader => 100,000-fold increase in errors
DNA defects to repair (4)
Spontaneous mutation, mismatch, thymine-thymine dimer, double-strand lesion
Spontaneous mutation (2 types)
Depurination - Hydrolysis of A or G base from sugar
Deamination
cytosine + H2O —> uracil + NH3
5-methylcytosine + H2O —> thymine + NH3
Repair of spontaneous mutation (name of mechanism and 4 steps)
Base-excision repair:
1) DNA glycosylase removes T/C base from sugar
2) APE1 makes 1 cut at sugar (endonuclease)
3) AP lyase removes sugar, creating gap
4) DNA polymerase β and DNA ligase add 1 T/C and seal gap
Repair of mismatch (name of mechanism and 3 steps)
Mismatch excision repair:
1) MSH2 and MSH6 attach around mismatch, recruiting MLH1 endonuclease and PMS2
2) DNA helicase and DNA exonuclease remove segment of daughter strand
3) Gap repair by DNA polymerase and ligase
Repair of thymine-thymine dimer (name of mechanism and 4 steps)
Nucleotide excision repair:
1) Initial damage recognition
2) Opening of double helix
3) XP-F and XP-G endonucleases remove segment surrounding dimer
4) Gap repair by DNA polymerase and ligase
Lesser repair of double-strand lesion (name of mechanism and 3 steps; 1 extra fun fact)
Nonhomologous end joining (NHEJ):
1) DNA-PK with KU80/KU70 heterodimer covers broken single-stranded ends
2) Other proteins remove protruding nucleotides, making blunt ends
3) Ligase joins ends together
Potential for frame shifts and incorrect end joining (translocation)
Better repair of double-strand lesion (name of mechanism and collection of 4 facts)
Homologous recombination:
Borrows from intact non-template strand
Single-strand break uses a Holliday structure
Double-strand break uses 2 Holliday structures
Result = crossing over (recombinant type or parent type)
