Lecture 3 - DNA Synthesis Flashcards
Semiconservative mode of DNA replication and how this makes sense given what you know about the W&C model of DNA structure
- Each parental strand serves as a template for new daughter strands
Direction of DNA synthesis? Evidence?
5’-3’, bidirectional
Denaturation mapping of phage
3 phases of DNA replication
- Initiation
- Elongation
- Termination
DNA polymerases: know what reactions they catalyze
- DNA polymerases are proteins that catalyze DNA synthesis via covalent addition of nucleotides to pre-existing DNA
DNA polymerases: cofactor requirements
- Mg2+ -> as nucleotide comes into catalytic site, Mg 2+ helps position dNTP properly for nucleophilic attack => phosphodiester linkage
- dNTPs -> substrate
- Primer DNA (primer is RNA)
- Template DNA
DNA polymerases: Prokaryotic types
DNA Polymerase I: DNA repair, RNA primer removal
- 5’ to 3’ polymerase activity (catalyzes formation of phosphodiester bond)
- Facilitates nucleophilic attack by 3’ OH of primer strand
- Exergonic elimination of pyrophosphate coupled w/ endergonic formation of phosphodiester bond
- 5’-3’ exonuclease activity: remove RNA primer w/ RNase H, DNA polymerase I fills base gap in bacteria
- 3’-5’ exonuclease activity: removes mismatched bases
DNA Polymerase III: True “replicase” (synthesizes majority of bacterial chromosome)
- Replicates E.coli genome
- Multimeric protein (subunits, beta is important), without holoenzyme, short strands are synthesized (no beta clamp)
- Synthesizes both strands in 5’-3’ direction -> one continuous (leading), one discontinuous (lagging/Okazaki fragment)
DNA Polymerase II, IV, and V: for replication of damaged DNA (idt we need to know)
What is strand directed mismatch repair (SDMMR)?
- Repairs errors missed by proofreading exonuclease of DNA polymerase
- Must recognize/repair error in newly synthesized strand, NOT original base in parent strand
Strand detection:
- Bacteria: more methylated A’s => older => template | less methylated => younger => new strand
-Humans: detects nicks prior to DNA ligase sealing of Okazaki fragments
What are the three proofreading steps that give rise to high-fidelity DNA synthesis?
- 5’ - 3’ exonuclease activity
- 3’ - 5’ exonuclease activity
- SDMMR
What is a holoenzyme?
The complete enzyme complex
Leading vs Lagging Strand? Continuous vs Discontinuous Synthesis? Okazaki fragments?
Both DNA strands are synthesized in the 5’‘3’ direction
One strand is continuously synthesized => leading strand
One strand is discontinuously synthesized in short stretches => lagging strand => Okazaki fragments
Okazaki fragments must be joined together to make discontinuous strand continuous
DNA polymerase: Eukaryotic types
DNA polymerase alpha
- Primer synthesis (complexed to DNA primase)
- Primer extended to ~30 nucleotides (10 RNA + 20 DNA)
DNA polymerase delta
- Lagging strand DNA synthesis
- Nucleotide and base-excision repair
- Processive synthesis of chromosomal DNA (must interact w/ PCNA and Rf-C to be active)
- 3’ to 5’ exonuclease activity
DNA polymerase epsilon
- Leading strand DNA synthesis
- Nucleotide and base-excision repair
What is processivity? What is responsible for processive synthesis of DNA in prokaryotes vs eukaryotes?
Processivity: how well polymerase can stay on track to synthesize longer stretches of DNA
Prokaryotes: Beta subunit “clamp” of DNA pol III
Eukaryotes: PCNA (sliding clamp) and Rf-C (loads PCNA onto DNA) of DNA pol delta
General similarities/differences b/t prokaryotic and eukaryotic DNA replication
All polymerases need primers for free 3’ OH, 1 for leading strand and 1 for every Okazaki fragment
Prokaryotes:
- Faster, but single origin of replication (245 bp, 3x13 bp A/T rich repeat for replication bubble, 4x9 bp repeat for protein binding site)
- No cell cycle => replication is non stop
Eukaryotes:
- Slower, but multiple origins of replication (Autonomously Replicating Sequences ARS 50 bp, core 11-bp AT rich sequence)
- More complex (replisome has more proteins)
- RNA primers and Okazaki fragments shorter
- Occurs only in S phase of cell cycle
- Multiple polymerases at replication fork
- Nucleosomes
- Telomeres
What is DNA primase?
- An RNA polymerase that requires only a DNA template (no primer/free 3’ OH) that creates a primer and RNA/DNA heteroduplex
- The RNA primer provides a free 3’ OH that can nucleophilic attack and form a phosphodiester linkage
- RNA primers are excised and replaced by DNA polymerase I 5’-3’ exonuclease activity in prokaryotes, ribonuclease H1 and ribonuclease FEN-1 in eukaryotes
DNA primase and Okazaki Fragments - elaborate
- 1 primer per Okazaki fragment
- On lagging strand, extension stops when encounters next RNA primer
- Adjacent Okazaki fragment 3’ OH is used as primer for DNA polymerase I
What is DNA ligase?
- An enzyme that catalyzes the covalent closure of nicks in DNA
- Covalently links adjacent Okazaki fragments
- Does not work to fill gaps i.e. where bases are missing => filled by DNA polymerase I in prokaryotes, DNA polymerase delta in eukaryotes, then DNA ligase seals backbone nick
What is DNA helicase?
- 6 subunit enzyme that unwinds DNA using ATP, producing two single strands of DNA
What prevents single stranded DNA from H-bonding to itself and staying extended after unwinding by DNA helicase? Proks vs Euks
In prokaryotes: single-stranded DNA binding protein (SSB)
In eukaryotes: replication protein A (Rp-A)
What is DNA topoisomerase? Type 1 vs Type 2?
- Enzyme that catalyzes transient breaks in DNA to prevent overwinding, positive supercoils
Topoisomerase I: Single stranded break
- Removes supercoils one at a time
- Transient break allows opposite sides of break to spin independently around intact phosphodiester bond
- Reseal break
Topoisomerase II: Introduces negative supercoils
- 2 (-) supercoil added
- Double stranded break
- Ex: DNA gyrase, responsible for negative supercoiling found in E. coli genome
- In eukaryotes -> found where 2 DNA helices cross one another -> breaks both strands of double helix, allows other double helix to pass through gate, reseals double stranded break -> separates two interlocked DNA circles
What is DNA gyrase?
- A topoisomerase II in e. coli
- W/o it, bacterial genome would have a lot of positive supercoil, overwound DNA => block bacterial replication
- Antibiotics work on this to block bacterial genome replication
Components of prokaryotic and eukaryotic DNA replication that serve as accessory proteins to major enzymes discussed
- SSBs in prokaryotes, RP-A in eukaryotes => keep unwound single strand DNA extended
- Sliding clamp: beta subunit of DNA pol III of prokaryotes, PCNA in DNA pol delta of eukaryotes
- Clamp loader: gamma subunit of DNA pol III of prokaryotes, Rf-C in DNA pol delta of eukaryotes
What experiment was used to determine dispersive mechanism of nucleosome replication?
Method: density transfer experiments (use light and heavy isotopes of certain atoms to determine age of histone complex)
Result: Nucleosome on progeny DNA contains old and new histone complexes => nucleosome duplication occurs through dispersive mechanism
Telomeres: General types of sequences associated , where found on the chromosome, issues related to replication of telomere lagging strand, importance of telomerase, relationships b/w telomere length and aging vs. cancers
Sequence: 500-3000 copies of TTAGGG repeat in humans, 12-16 bp single stranded 3’ overhang
Location: Ends of eukaryotic chromosomes
Issues related to replication of telomere lagging strand: Cannot produce terminal Okazaki fragment b/c no primer to provide free 3’ OH (refer to slide 69 lecture 3)
Telomerase:
- Binds to G rich telomere overhang using internal RNA template
- Adds single telomere repeats to parent strand
- After several additions => RNA primer made, DNA polymerase synthesizes new strand
Telomere length and aging vs cancers:
- Older cells have shorter telomere lengths
- Cancer -> tumor cells display increased telomerase activity and longer telomeres vs normal cells
- Progeria -> rare human disease characterized by premature aging i.e. extremely short telomeres
