Lecture 19: DNA Replication and Repair Flashcards
1
Q
What are the 6 problems that need to be solved during DNA replication?
A
(1) Strand polarity
(2) Unzip DNA
(3) Processivity
(4) Untangle
(5) One copy
(6) Accurate copy
2
Q
Replication
A
- Is semi-conservative
- > Means that each DNA strand is used as a template for the synthesis of a complementary strand
- Damaged DNA, if unrepaired, can persist through cell divisions.
- Errors in copying, if unrepaired, are propagated through cell divisions
- Need to make sure that there’s no errors in any strands so these don’t happen
3
Q
Replication #2
A
- Occurs in the 5’ to 3’ direction
- New nucleotides are added at the 3’ end.
- Chain growth occurs in the 5’ to 3’ direction.
- Addition of deoxynucleotides requires a primer (3’ hydroxyl group).
- This mode of replication results in the antiparallel double helix structure
- Since the DNA is being synthesized in the 5’ to 3’ direction, this means that the DNA is being read in the 3’ to 5’ direction
4
Q
DNA polymerase
A
- What DNA replication is catalyzed by
- An enzyme with fingers (grips the DNA), palm (where catalysis happens) and thumb (holds everything in place) domains
- All nucleic acid polymerases have a similar structure
5’ to 3’ polymerization occurs with an error rate of 1 in 105 nucleotides. Other processes reduce the error rate further
5
Q
Solution to strand polarity
A
- All synthesis is 5’ to 3’
- Because DNA is antiparallel, one strand can be synthesized continuously (leading strand) while the other is synthesized in Okazaki fragments (lagging strand)
- Okazaki fragments are synthesized on the lagging strand.
- DNA Ligase seals gap between successive fragments
6
Q
Solution to unzip DNA
A
- A hexameric (6 identical subunits) complex called DNA helicase unzips the DNA.
- It uses ATP and acts as a rotary engine.
- Unzipped DNA is stabilized by single-stranded DNA binding protein
7
Q
Solution to processivity
A
- A sliding clamp holds the DNA polymerase in place
- The clamp is loaded on DNA by a clamp loader that uses ATP hydrolysis to lock the clamp around DNA
- Helicase is attached to the lagging strand, not the leading strand
- Lagging strand is bent around so both molecules of DNA polymerase are synthesizing in the same direction
8
Q
Rapid rotation of DNA is needed ahead of the replication fork
A
- The rapid rotation of the DNA introduces torsional stress on the DNA molecules
- Built up torsional stress leads to supercoils
- Topoisomerases relieve torsional stress
- Solution to Untangle
9
Q
Nick and swivel mechanism
A
- Topoisomerase I has a tyrosine in its active site which contains a hydroxyl group
- The hydroxyl group performs a nucleophilic attack on a nucleotide and replaces the hydroxyl group on the nucleotide, which breaks the phosphodiester bond between the adjacent nucleotide, creating a nick
- The strand with the nick can now swivel around the opposite strand, which relieves torsional stress
- Once the torsional stress is relieved, the 3’ hydroxyl group next the nick attacks the benzene ring on the tyrosine, which causes Topoisomerase I to detach from the DNA
- The phosphodiester bond then spontaneously reforms and the DNA is restored
- Since bond energies can just be transferred back an forth, Topoisomerase I does not require ATP to relieve strain
10
Q
Gating mechanism
A
- Topoisomerase II (mirror-image enzyme) requires ATP to untangle DNA
- The enzyme attaches covalently to both strands of one DNA helix, creating a gap in the DNA helix.
- The other DNA helix is then passed through the gap and the broken DNA helix is then reattached
- There is a loss of energy in the reversal of the covalent attachment, so ATP is needed in order to restore it
11
Q
Where does DNA replication begin?
A
- Begins at replication origins
- A replication origin is a region of very high AT content (2 hydrogen bonds vs 3 for GC bonds, so easier to break)
- Both directions are synthesized simultaneously, so both directions have a leading strand and a lagging strand
12
Q
Replication origins in prokaryotes
A
- For prokaryotes, there a replication origin and initiator proteins bind to the replication origin first, which allows the loading of the DNA helicase
- DNA helicase will then start unwinding the DNA in one direction and another DNA helicase will be attached to the DNA and start unwinding in the other direction
- It is important to have a single round of replication every time the cell divides in order to prevent the accumulation of DNA
13
Q
Prokaryotic origins have a refractory period
A
- This means that once replication is initiated at the origin, it can’t be initiated again for a little while
- Methylation of adenines on the parent strand at the replication origin regulates DNA replication in prokaryotes
- Once the daughter strands are finished synthesizing, they too are methylated, and once they’re methylated, then the cell can start replication again
- Methylation is mediated by the Dam methylase in prokaryotes
- Solution to One copy, but this only works for prokaryotes
14
Q
When do eukaryotes replicate DNA?
A
- During S-phase of cell cycle
- Eukaryotic DNA replication is more complex than in prokaryotes
- Eukaryotes have large genomes that need to be replicated
- Eukaryotes have many origins of replication
- Replication of DNA needs to occur once & only once per cell cycle
15
Q
Not all eukaryotic origins are used in all S phases
A
- Green replication origins are used most of the time, while red replication origins are used less of the time
- Multiple origins of replication allows the genome to be replicated more rapidly
- ORC-binding site: binds to the origin of replication complex
- Unwinding region: where the DNA helicase binds
- Auxiliary protein binding site: where additional proteins bind
