MB - DNA Replication (Eukaryotes) Flashcards
What are the overall similarities between eukaryotic and prokaryotic DNA replication? (4)
- Use helicases to unwind the duplex and create replication forks
- Use SSBs to hold the ssDNA apart
- Use RNA primers for the polymerase/clamp
- Use various DNA polymerases for the synthesis of the new DNA on leading and lagging strands
What are some broad differences between eukaryotic and prokaryotic DNA replication? (5)
- Occurs in the nucleus, not the cytoplasm
- More genetic material to replicate (can be four orders of magnitude greater)
- More than one chromosome (46 in humans)
- Linear chromosomes (except mitochondrial)
- Additional packaging (e.g. nucleosomes)
What are some specific differences in eukaryotic DNA replication? (5)
- Multiple origins per chromosome (10s-1000s)
- DNA polymerases are much slower
- No DNA polymerases with 5’-3’ exonuclease
- Polymerases synthesising leading and lagging strands are not physically linked together
- Okazaki fragments are much shorter
How often does initiation occur per cell cycle?
Must only happen once per cell cycle
- Ensured by a 2-step process
Where do Origins of replication arise from?
Sections of DNA called Autonomously Replicating Sequences (ARS)
What happens during initiation/licensing in the G1 phase of the cell cycle? (3)
1) An Origin Recognition Complex of proteins (ORC) binds to the A region
2) Accessory proteins (licensing factors) accumulate in the nucleus during G1
- Cdc6 and Cdt1 bind to ORC
3) Two helicases are loaded by Cdt1
- Cdc6/Cdt1 leave
What happens during initiation/activation in the S phase of the cell cycle?
The pre-replication complex must be activated
- Activated by additional proteins + DNA polymerases
What polymerases are involved in DNA elongation in eukaryotic replication? (5)
Polymerase α
- Has own primase activity
- Does not associate with PCNA (so not progressive)
- No proofreading
Polymerase β
- Involved in repair
Polymerase γ
- Replicates mtDNA
- Has proofreading
Polymerase δ (lagging strand)
- Associates with PCNA (sliding clamp protein)
- Has proofreading
Polymerase ε (leading strand)
- Associates with PCNA
- Has proofreading
What are 2 methods of editing in elongation?
RNA “flap” produced by Pol δ or ε can be mostly digested by RNAse H1
- 1 RNA nt left
(like E.coli version)
Flap 1 endonuclease (FEN1) binds PCNA and can remove any incorrect nt
- Cuts off a section of ssDNA/RNA
What is the end replication problem in DNA replication?
Affects the lagging strand
- Last RNA primer may not be at the extreme 3’ end of the DNA→missed section
How is the end replication problem addressed in eukaryotic cells?
Telomeres
- Multiple repeats of a simple sequence (TTAGGG) located at the 3’ ends of chromosomes
How do embryonal cells, cancer cells, stem cells and other types divide more times than the Hayflick limit?
→ Synthesis of new telomeric DNA
What happens when the telomeric DNA is gone? (2)
→Gene loss
→Cells stop dividing, a point called the Hayflick limit
How is DNA replication handled in mitochondrial DNA? (4)
- 2-10 copies of circular mtDNA per mitochondrion
- Unidirectional replication (1 fork)
- Pol γ synthesizes leading strand
- Lagging strand is RNA Okazaki fragments
What are the replication mechanisms used by bacteriophages, RNA genomes, and retroviruses?
Bacteriophages
- Small circular genome
- Some use a “rolling circle” mechanism to continuously synthesize new DNA
- Sigma (σ) replication
RNA genomes
- Have an RNA-dependent RNA polymerase called RNA replicase
- +Strand RNA is copied directly to make - strand (which is used as a template for more + strand)
- Can be self-priming – no primer is required
- No proof-reading → highly error-prone
Retroviruses
- Virally encoded reverse transcriptase creates a DNA strand using RNA as a template and tRNALys as a primer
