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Compare prokaryote and eukaryote DNA replication
- Replication fork geometry is practically identical
- Core enzymes at replisome: Basic enzymes are present in both, but eukaryotes have auxiliary enzymes present on top of the basics.
- Genome size; Eukaryotes are much bigger. 10 mb to 3,000 mb vs. 1-10 mb
- Chromosome structure: Eukaryotes have linear chromosomes packaged into chromatin (an obstacle for replication). There are ends of the chromosomes that resemble double stranded breaks.
- Speed of replication forks: 20 times slower in Eukaryotes than prokaryotes. 50 nt/s vs. 1000 nt/s. Slower rate is largely due to new obstacles for replication forks to overcome, but can be compensated for via parallelization of replication.
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Explain bacterial vs. eukaryotic origins of replication
- Bacteria replicate all the time, only limited by nutrient availability. Origins fire as quickly as possible, with little regulation.
- Eukaryotes replicate the genome once per cell cycle, extensive regulation of timing relative to cell division exists.
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Explain eukaryotic origins of replication
- Multiple origins compensate for slower forks and larger genomes.
- Region replicated by one origin is called a replicon. Prokaryotes have one replicon.
- Eukaryotes have thousands of origins (~10k) and therefore thousands of replicons. Not all origins need to fire for replication to complete.
- Multiple origins serve as a regulatory opportunity. We control which origins or how many origins get activated to control replication rate. Cancer cells lack this regulation, favor many origins firing as much as possible.
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Explain Eukaryotic preparation for replication
- The first step is the formation of the pre-replicative complex. (pre-RC)
- The origin recognition complex (ORC) is an initiator protein complex. It binds the origin and recruits remaining replication proteins. It also recruits helicase loading proteins.
- Helicase is recruited (MCM2-7) in the form of a hexameric helicase complex. It is not yet active, just loaded.
- In bacteria, binding of the initiator (DnaA) is directly coupled to DNA unwinding and DNA polymerase recruitment. This is not the case for pre-RC, which must be activated.
- Phosphorylation (post-translational modification) of the Pre-RC is required for the recruitment of auxiliary factors and polymerases, thereby activating RC/Helicase.
- Kinases are required for activation. CDK and DDK are cyclin-dependent kinases.
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Explain eukaryotic initiation of replication
- Stepwise recruitment after CDK/DDK action and the phosphorylation of helicase and other proteins:
- DNA pol epsilon and delta (highly processive DNA polymerases). These cannot yet start, as no primer-template junction is present.
- DNA pol alpha/primase is then recruited, which synthesizes primers and does initial DNA synthesis. However, DNA pol alpha has low processivity.
- Sliding clamp and clamp loader are then recruited to initiate processive DNA synthesis.
- Once DNA synthesis starts, DNA Pol alpha/primase is quickly swapped out for DNA pol delta (lagging) and epsilon (leading). This step is called polymerase switching.
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Why are chromosomes replicated once per cell cycle in eukaryotes?
- Chromosome breakage or loss: If replication is not completed, chromosomes can be torn apart during mitosis.
- Copy number variation: If replication is completed multiple times, some regions may be amplified. There may be different levels of proteins, which may even lead to cancer.
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Explain cell cycle regulation
- Start with one copy of the genome
- S-phase: synthesis is when genome is replicated
- G2 phase.
- M phase: is when chromosome segregation and mitosis occurs.
- G1 phase.
- S-phase
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How do cyclin-dependent kinases regulate DNA replication
- CDKs phosphorylate many proteins that regulate cellular processes, including DNA replication.
- CDKs ensure that replication occurs in the S-phase.
- There is high CDK from S-phase through G2 and M-phase.
- There is low activity in G1 phase.
- In G1, pre-RCs are formed.
- In S, G2, M phases, pre-RCs are activated and the formation of new pre-RCs is inhibited. Origin cannot be re-fired due to this inhibition. Not all pre-RCs are activated, and therefore not all origins are utilized.
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Explain the timing of origin of activation
- Some origins fire every cycle, some fire late.
- This is influenced by chromatin structure established in G1.
- Cells cannot afford to fire all cells at once, which would consume all dNTPs and would also lead to the depletion of other replication factors.
- Taking a snapshot of replicating DNA can indicate early and late origins (large versus small bubbles)
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Explain oncogenes
- Increase proliferative capacity of cancer cells at the expense of proper origin firing control.
- Transcription-replication collision may lead to double-stranded breaks .
- There is an overall increase in genomic instability.
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Explain replication stress
- It is a major challenge for the cell to replicate the genome without damaging it by creating double-stranded breaks at secondary structures and heterochromatin.
- Oncogene-induced replication stress: Oncogenes increase proliferative capacity of cancer cells. However, replication stress is augmented in cancer cells. Overfiring, refiring, asymmetric firing in origins, transcription replication collision are some of the issues that arise from the improper control of origin firing.
- Oncogenes thus compromise the integrity of the genome.
- Genomic instability: Cancer cells thus accumulate a lot of damage in their genome.
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Anticancer drugs target DNA replication
- DNA replication is a common target for chemotherapeutic drugs that aim to prevent tumor growth.
- DNA replication can be targeted at various stages: Biosynthesis of nucleotide precursors, chain elongation, DNA template, DNA polymerase.
- The issues with a brute-force approach like this is that it’s not very specific (normal cells also affected) and cancer cells can develop resistance.
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New generation of anti-cancer therapies targeting DNA replication
- Cancer cells build tolerance to replicative stress. An example would be the overexpression of proteins for repairing.
- Cancer cells then become addicted to the tolerance mechanism.
- If we identify the tolerance mechanism in each cancer cell, we can develop drugs to 1) inhibit tolerance mechanism and 2) induce replication stress.
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Example of combination therapy in clinical trials
- DNA damage sensor kinases are promising targets for anti-cancer therapy.
- ATR is a kinase that is able to detect damage to DNA and increase DNA repair by homologous recombination.
- We can develop ATR inhibitors and replication stress inducers in combination to target cancer cells.
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Explain the “end replication” problem
- In the lagging strand, removal of the last RNA molecule primer will leave a short region of unreplicated ssDNA at the end of the chromosome.
- Lagging-strand synthesis is unable to copy the extreme of linear chromosomes.
- Because eukaryotic chromosomes are linear, the 3’-5’ strand in the lagging strand gets shorter in every cell division.
- Cells avoid this problem by having telomeres, which are “buffer” DNA that contain simple repeat sequences.
- Telomere: tandem repeats (up to 1000 times) of a short TG-rich DNA sequence.
- Different organisms have different telomere signatures.
- Telomeres are made by telomerase, which is ribonuclear (RNA + protein) that adds TG-rich sequences to the 3’ ends of chromosomes.
- Additional 3’ end DNA can act as template for new Okazaki fragment. However, it’s impossible to make a blunt end. There will always be a 3’ overhang.
- Telomerase is an RNA-dependent DNA polymerase (AKA reverse transcriptase). The RNA component of telomerase serves as the template for the telomeric sequence repeat. Once the telomeric sequence repeat is synthesized, telomerase is translocated and continues DNA synthesis
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