Self Study- DNA Replication Flashcards
Requirements for DNA replication (and define each)
- Template- DNA strand that provides info about which nucleotides to add
- Primer- short RNA segments synthesized by a primase and H-bonded to template, provides free 3’-OH to add nucleotides anti-parallel
- Nucleotides- 5’dNTPs
- DNA polymerases- Enzymes capable of synthesizing DNA polynucleotides
Requirements for DNA polymerases
What is released after each nucleotide is added
- Primer and template
- add nucleotides only to 3’ end of replicated strand (5’ to 3’)
- Must be double stranded DNA
- release pyrophosphate (PPi)
DNA Proof-reading ability
How often do errors occur?
- Exists, works as a 3’ to 5’ exonuclease
- Erorrs occur 1:1,000 or 1:10,000 nucleotides, proofreading lowers this to 1:10,000,000
How are primers removed?
DNA polymerase has a 5’ to 3’ exonuclease for this
Semi-conservative replication
Each daughter strand has 1 parental strand and 1 new strand
Replication fork
Helicases
Gyrases
Single-stranded DNA binding proteins (SSB)
- point of separation of parental strands
- Unwind strands so they can separate
- Reduce # of supercoils that would come from unwinding
- Keep 2 strands from annealing during this process
Leading strand vs Lagging strand (and # of primers)
Okazaki fragments
What does DNA ligase do?
- Leading strand has 3’ in direction of replication fork
- Lagging strand has 3’ in direction away from replication fork, synthesized in a series of short pieces in 5’ to 3’ direction called Okazaki fragments
- Leading strand only needs 1 primers while lagging needs multiple (1 for each Okazaki fragment)
- DNA ligase seals the Okazaki fragments together
Prokaryotic DNA polymerases (names, functions, and turnover rate)
Which have 3’ to 5’ exonuclease activity?
Which have 5’ to 3’ exonuclease activity?
What is Klenow fragment?
What is processivity?
Sliding clamp?
Pol I- Lagging strand synthesis (intermediate turnover)
Pol II- DNA repair (low turnover)
Pol III- Leading strand synthesis (high turnover)
- All have 3’ to 5’ exonuclease activity
- Only Pol I has 5’ to 3’ exonuclease to remove primers
- 70 kDa portion of Pol I that has everything except primer-removing exonuclease
- Ability of enzyme to remain on substrate during synthesis (better able –> higher turnover #)
- Protein on Pol III that prevents it from dissociating from template and allowing high processivity
Origin of replication (Ori)
Primosome
Replisome
Where does replication begin in prokaryotes?
Is DNA synthesis unidirectional?
- Points at which DNA replication begins (where DNA polymerase and primase initially bind)
- Contains primases, ligases, helicases, and other proteins that bind to Ori, needed to synthesize primer
- Contains primosome, SSB, Pol III, Pol I, needed for DNA replication
- Begins at single Ori and proceeds in opposite directions until replication forks meet
- No, bidirectional
What is shared between prokaryotic and eukaryotic DNA replication?
- replication fork
- primer synthesis
- Okazaki fragments
- Primer removal
- Gap bridging newly synthesized fragments
- Synthesis is bidirectional
What differences are there between prokaryotic and eukaryotic DNA?
What are replicons and how many per chromosome?
Why are there differences?
- Nucleosomes must disassemble, mking replication form movement slower. To compensate, replication begins at multiple Ori (up to 200)
- DNA segments between Ori, about 100
- Size of DNA (10^9 vs 10^6 kb), packing into chromatin, lower rates of replication fork movement in eukaryote.
- Sidenote: G1 and G2 are very short in prokaryotes
What are eukaryotic DNA polymerases (name and function and processivity)
What does proliferating cell nuclear antigen (PCNA) do?
What is replication protein A (RPA)?
How do both polymerases dimerize and move in the same direction as the replication fork?
When are new histones synthesized?
- Polymerase delta- catalyzes replication of leading strand and serves as proofreader (3’-5’ exonuclease), high processivity
- Polymerase alpha- synthesizes lagging strand, acts as primase, disassembles nucleosome prior to DNA replication (low processivity)
- PCNA is on Polymerase delta, serves as clamp to keep enzyme from dissociating from DNA
- RPA is eukaryotic homologue of SSB, keeps strands from annealing
- Looping of the lagging strands allows this to occur
- During S phase alongside DNA replication, new DNA has nucleosomes with both parental and new histones
DNA replication at end of linear chromosome
What is a reverse transcriptase? What does this have to do with AIDS treatment?
- Gap of 8-12 nucleotides at end where primer removed
- Telomerase, a reverse transcriptase, fills in the gap (even has RNA as part of structure to serve as template)
- Reverse transcriptase is an enzye that uses RNA as a template for DNA synthesis. However, they lack 3’ to 5’ exonuclease so error-prone
- AIDS treatment involves inhibitors of reverse transcriptase. AZT replaces dTTP in viral genome leading to premature viral DNA synthesis, errors persist due to lack of exonuclease
Telomerase activity relationship with cell senescence?
Telomerase activity with tumors?
- Telomerase activity decreases with age
- Telomere length is inversely proportional to age, length can serve as a mitotic clock for somatic cells
- Tumors have high telomerase activity and divide indefinitely (immortal)
DNA recombination (what is it? 3 types and definitions, prokaryotes or eukaryotes? same or different organism?)
- rearranging DNA sequences by breaking and rejoining existing DNA molecules
- Homologous genetic recombination (meiosis)- different alleles can switch between chromatids, leading to genetic diversity
- Conservating site-specific recombination- insertion of bacteriophage DNA into bacterial genome, inserted at specific sites
- Transposition (jumping DNA or transposons)- move across sites within or in another chromasome, can inactivate gene by mutating it, activate gene if in promoter or activator. Is done by bacteria to make them antibiotic resistant
- In both prokaryotes and eukaryotes
- Can be same or different molecules/organisms
