Ch. 22: DNA Replication, Repair, and Mutagenesis Flashcards
What are the 3 general features of DNA replication?
- Semiconservative 2. Bi-directional 3. Semi-discontinuous
What are the 3 possible models of replication?
- Conservative 2. Semi-conservative** (Watson and Crick) 3. Dispersive
What did the Meselson and Stahl Experiment demonstrate? How?
DNA replication is semi-conservative. DNA was labeled with N14 and N15 (heavier-contains 1 more neutron) at pH 7 and 12 (denaturation). After 1st generation, intermediate density at pH=7 (eliminates conservative) and equal amounts of N14 and N15 densities at pH=12 (50%, 50% N14 and N15). After 2nd generation, 2 strands 50% N14/N15 and 2 strands 100% N14. Therefore, at pH=7, 1/2 are at the intermediate density and 1/2 are at the N14 density. At pH=12 (denatured), 2/8 strands N15 and 6/8 strands N14. NOTE: always add the N14 strand as the complementary.
What does bi-directional mean?
DNA has 1+ origins of replication (except bacteria and plasmid DNA, which only have one origin and stop at terminal, but it’s still bi-directional, which allows them to replicate quickly
What does semi-discontinuous mean?
Can only add nucleotide to 3’-OH group (5’->3’ direction). Leading strand (5’->3’) has continuous synthesis. But lagging strand is discontinuous. Make okazaki fragments (200 bp) that are joined by DNA ligase
What are polymerase I’s 3 jobs in bacterial replication?
- DNA repair (5-3 Exonuclease)
- DNA repair (3-5 exonuclease)
- Nick translation (5-3 Polymerase)
- Excise short RNA primer required to initiate DNA synthesis on leading/lagging strand
- Remove mismatched BP during replication
- Fill in gaps in single-stranded DNA that’s already joined in double helix
What is the site where the 3 distinct catalytic abilities of DNA polymerase I reside?
Hans Klenow
What is the smaller fragment’s activity?
5’ exonuclease (DNA repair)
What activities make up the Klenow (larger) fragment of DNA polymerase?
5-3 Polymerase and 3’ exonuclease activity
What activity is unique to DNA polymerase 1?
5’ exonuclease activity
What is the function of a helicase?
Unwind ds DNA
What is the function of single strand DNA binding proteins?
Accessory proteins that bind tightly to ss DNA to stabilize and prevent reannealing
What is the purpose of RNA polymerase (primase)?
Binds to regions where single strand binding proteins are bound and make small RNA primers for DNA polymerases to add nucleotides to (since DNA polymerase can’t initiate DNA synthesis)
What is the function of a ligase?
Seal nick (only if has 3 OH and 5 P terminus, can’t have a missing nucleotide)
What is the function of a topoisomerase?
If moving along circular helix, polynucleotides of unreplicated portion become overwound (positive supercoiling). Topoisomerase introduces negative supercoiling.
What is the function of topoisomerase I?
Introduce negative supercoils by causing single-strand breaks
Then religates strands WITHOUT ATP hydrolysis
What is the function of topoisomerase II?
Introduce negative supercoiling by making double-strand breaks
Then religate strands
NEED ATP HYDROLYSIS (unlike topoisomerase I)
What is the topoisomerase called in E. coli?
DNA gyrase (necessary for in vitro replication of circular DNA)
What are the 3 polypeptides in E. coli DNA polymerase III used for?
alpha subunit: catalytic polymerase function
epsilon: 3-5 exonuclease activity (proofreading)
theta: unknown
Why does the holoenzymes for DNA polymerase in bacteria contain 10 different subunits?
Required for regulation of gene expression
How long does it take for E. coli to replicate? Humans?
E. coli: 40 min. vs Human: 24 hours.
E. coli replicate an entire strand without __________?
Dissociating
Properties of DNA Polymerases of E. coli
Lightest to heaviest?
II, I, III
Properties of DNA Polymerases of E. coli
Slowest to Fastest?
III, II, I
Properties of DNA Polymerases of E. coli
Which have 3c exonuclease activity?
I and II
Properties of DNA Polymerases of E. coli
Which have 5c exonuclease activity?
Just I
Properties of DNA Polymerases of E. coli
What is their biological activity?
I: RNA primer, excision, DNA repair
II: SOS DNA repair?
III: Replicase
Describe alpha DNA polymerase in eukaryotes?
- Location: nucleus
- Only one with associated primase
- No 3’ exonuclease
- Activity: Replication (primase activity, replication initiator)
Describe beta polymerase in eukaryotes?
- Location: nucleus
- No primase, no 3 exonuclease
- Activity: DNA repair (base excision)
Describe gamma polymerase in eukaryotes?
- Location: MITOCHONDRIA
- No primase
- Yes 3 exonuclease
- Activity: mitochondrial DNA replication
Describe delta polymerase in eukaryotes
- Location: nucleus
- No primase
- Yes 3 exonuclease
- Activity: replication (main polymerase at leading/lagging strand
Describe epsilon DNA polymerase in eukaryotes?
- Location: nucleus
- No primase
- Yes 3’ exonuclease
- Activity: replication (leading/lagging strand)
Which DNA polymerase is not in the nucleus?
gamma (in mitochondria)
Which DNA polymerase of eukaryotes has an associated primase?
alpha
Which eukaryotic DNA polymerases have 3’ exonuclease activity?
gamma, delta, epsilon
What are each of the eukaryotic DNA polymerases used for?
- alpha: replication (primase activty, replication initiator)
- beta: DNA repair (base excision repair)
- gamma: Mitochondrial DNA replication
- delta: Replication (Main polymerase at leading/lagging strand)
- epsilong: replication at leading/lagging strand
What is the main polymerase at the leading/lagging strand for eukaryotes?
DNA polymerase delta
What is a Reverse transcriptase?
RNA-dependent DNA polymerase found in HIV and AMV
Catalyzes conversion of RNA templace to DNA double helix
Used to make cDNA and used to quantify level of mRNA synthesis
What are the 3 enzymatic activities of reverse transcriptase?
- RNA-dependent DNA polymerase : makes DNA strand complementary to RNA template
- RNase H: exonuclease that removes and degrades RNA chain in DNA:RNA hybrid from above
- DNA-dependent DNA polymerase: completes ds DNA synthesis (ss-> ds)
What is the problem with reverse transcriptase?
High error rate 1/2,000-4,000 nucleotides (this could be a double edged sword)
Allows for selective advantage of virus that can change (and not be recognized)
Compared to DNA polymerase (1/1,000,000)
Describe the initiation step in E. coli DNA replication?
- Occurs at replication of origin (ONLY ONE) - (rich in AT base pairs so easily separated)
- DnaA binds to origin and causes local denaturation
- DnaB (DNA helicase) and DnaC (required regulator of DnaB) bind to site to take over and unwind
- 2 replication complexes formed at single origin site and proceeds bidirectionally
Describe the elongation step in E. coli DNA replication?
Addition of deoxynucleotides to preceding 3’OH group of RNA primers
Leading strand
- Continuous
- 1st NA primer made by primase at 3’ end. DNA polymerase III can make DNA progressively until end
Lagging strand
- Each Okazaki fragment made and joined to those made previously.
- RNA primers removed by 5-3 exonuclease of DNA polymerase I, which also inputs missing DNA sequence
- Nick between Okazaki fragments (1,000-2,000 bases long) after RNA primers removed are sealed by DNA ligase
- **Okazaki fragments in eukaryotes are 100-200 bp long
Describe the termination step in E. coli DNA replication?
- Occurs at replication termini (Ter) specific site located at opposite end of origin
- Replication terminator protein (tus) - terminator utilization substance - binds to Ter site and induces replication fork arrest
- Inhibit helicase and prevent replisome from passing thru
How does eukaryotic DNA replication termination differ from prokaryotic?
Eukaryotic replication stops when replication forks meet each other or end of chromosome (linear DNA)
Prokaryotic replication stops when Tus protein binds to Ter (circular DNA)
If bacterial DNA undergoes “sexual reproduction” (i.e. factor F is added), how does that affect the origin of replication?
At oriC (original), will be bi-directional
At oriF (add DNA), will be unidirectional.
What is the fidelity of replication error?
1/100,000,000 (10^8)
The fidelity of DNA replication in eukaryotes and prokaryotes is accomplished mainly by ____
Proofreading function of DNA Polymerases
Esp.3-5 exonuclease (eukaryotes)
alpha subunit of E. coli DNA polymerase III Holoenzyme catalyzes nucleotide polymerization. If wrong base added, epsilon subunit of DNA pol III (3-5 exonuclease) removes wrong base and replaces.
What is the incorporation accuracy?
1/1,000,000 (10^6)
What replication system do bacteriophages use?
Rollling circle replication
Describe how rolling circle replication works
- Outer strand nicked
- Free 3’OH end extended by DNA polymerase
- 3’ end lengthened while growing point rolls around template
- 5’ end forms tail
- Use RNA primers to make 5’ end double stranded
- Primer not necessary (??)
- Leading strand covalently linked to template
- Can continue many rounds making concatameric branch (efficient at making copies)
- Template for leading strand never spearates from circular part of molecule
How is the DNA replication of eukaryotes different from that of prokaryotes?
- Highly regulated and activated by extracellular signals
- Require timed activation of cyclin/CDK complexes thru signal transduction
- Multiple initiation sites
- Histone complexes: structural organization of DNA; + charge and made of lyscine and arg, enabling them to bind to phosphate backbone of DNA
- Telomeres: Prevents loss of essential genetic info
- Different DNA sizes
- Linear
Describe the process from G1-> S phase
- External signals delivered to cell during G1 phase (growth factor) and binds to receptor
- Signal transduction cascade
- Transcription factor goes to nucleus to activate synthesis of cyclins
- For cyclin/CDK complex
- Rb phosphorylated and releases E2F (normally inactivates it)
- Stimulate S phase proteins (DNA polymerase, thymidylate synthase)
- Prepare cells for DNA replication
What are the stages of the cell division cycle?
- Go: Resting
- G1: External signal to replicate is transmitted to nucleus; Protein synthesis enables passage to S phase
- S : DNA synthesis
- G2: Cell increases in size
- M: Chromosomes and cells divide (cytokinesis)
G=gap not growth
What are oncoproteins?
Drive the cycle
Tumor suppressor proteins
Inhibit cycle
Process of DNA replication in eukaryotes
- Bind ORC to origins of replication during G1
- ORC serves as platform for other pre-replicative complexes
- ORC converted to initiation complex via CDK and DDK
- Topoisomerase II
- Helicase unwinds ds DNA at origin
- DNA polymerase alpha-primase complex makes 1st primer
- Initiates DNA replication on leading strand and Okazaki fragments on lagging strand
- DNA delta polymerase is the major polymerase on leading strand synthesis
- DNA delta and epsilon polymerase are major on the lagging strand synthesis
- During DNA synthesis, leading strand retains histones. Lagging strad remains free of histone complexes while new histones made and assembled.
What is the first checkpoint in eukaryote DNA synthesis?
G1 to S: to check for DNA damage. If so, arrest in G1 and repair DNA. Activate p53 transcription factor, which makes p21, and stops CDK2-cyclin A formation, so does not proceed to S phase.
What is the 2nd checkpoint of DNA synthesis?
G2 to M: Is DNA replication complete? If not arrest in G2 phase to complete DNA replication. Prevents tyrosine kinase from inactive, so can’t activate CDK2/cyclin B, which leads to mitosis.
What happens to the histones in DNA replication?
Retained on leading strand, but removed on lagging strand while new ones are being made. Histones have greater affinity for ds DNA
What are the functions of telomeres? Sequence?
Help stabilize chromosomes
(TxGy)n
What problem do telomeres solve?
RNA primers at 5’ end of chromosomes are removed by Pol alpha, but it cannot fill the gap because there’s no 3 OH group present (misisng nucleotides). Without telomeres, the DNA gets shorter.
What is so special about germ line cells and their ability to be “immortal”?
They have telomerase (RNA dependent DNA polymerase-so it’s like a reverse transcriptase) that carries a short stretch of RNA with AUCCCAAUC sequence and extends the 5’ end of the lagging strand (parent). DNA alpha polymerase completes task by adding nucleotides to unfinished 3’ side. Normal somatic cells lack this ability so they die eventually.
What induces negative supercoils ahead of the replication fork in prokaryotes? Eukaryotes?
Pro: Gyrase
Eukary: Nucleosome unwinding
What unwinds ds DNA-positive supercoils in prokaryotes? Eukaryotes?
Pro: Helicase
Eukary: Helicase
What adds RNA primers at the starts of Okazaki fragments at 5’ end in prokaryotes? Eukaryotes?
Pro: Primase
Eukary: Primase subunit of DNA pol alpha
What polymerizes the leading strand and lagging strands 5’-3’ in prokaryotes? Eukaryotes?
Pro: DNA pol III
Eukary: DNA pol delta and alpha
What acts as the exonuclease that removes primers and fills DNA gaps in prokaryotes? Eukaryotes?
Pro: DNA pol I
Eukary: DNA pol apha
What links Okazaki fragments in prokaryotes? Eukaryotes?
Both: Ligase
What extends the 3’ ends of DNA strands and fills in the complmentary strand of telemores in prokaryotes? Eukaryotes?
Pro: Circular DNA (not necessary)
Eukary: Telomerase and DNA pol alpha
What are the 3 main classes of DNA replication inhibitors?
- Those that prevent/reduce synthesis of precursors (bases, nucleotides)
- Those that affect template or priming ability of growing strand
- Those that act directly on polymerases or other enzymes needed for replication
What effect do intercalating agents have on DNA synthesis? Examples?
- induce unwinding, lengthening, and stiffening of ds DNA. Inhibit binding of DNA binding proteins. Ca induce mutations during replication, inhibit cell division, and are lethal to actively dividing cells.
- Viscosity increases, Buoyant density decreases, Thermal denaturation temp. increases, Distortion of phophate backbone, Does NOT disrupt Watson-Crick base pairs
- Examples: acridine, eithidium bromine, anthrcyclines, actinomycin D
Bleomycin, Zinostatin
Agents that bind to DNA covalently and cause chain breakage
Alkyl sulphonates, anthramycin, mitomycin, nitrogen mustards
Interstrand cross linkers
Platinum coordination compouds
Binds to guanine and inhibit DNA expression
2’3’-Dideoxyribonucleotides, cordycepin
Agents preventing extension of growing chain
Acyclovir
Inhibit DNA polymerase of HSV
Aphidicolin
inhbit pol alpha and delta (not beta and gamma)
2’ dideoxyazidocytidine
Inhibitor of bacterial primase
Coumermycin, novobiocin, oxolinic acid, nalidixic acid
Effective inhibitors of DNA gyrase in bacteria
Why are topoisomerases important? What do topoisomerase inhibitors do?
Essential for DNA replication and cell growth because relieves torsional stress in DNA by inducing reversible single strand break (no energy req)
- Drug interactions make irreversible double strand breaks-> cell death
Camptothecins
Function and what does it treat
Effective in treating lung, ovarian, and colorectal cancers, myelomonocytic syndrome, chronic myelomonocytic leukemia, acute leukemia, multiple myeloma
- Trap topoisomerase I attached to DNA strand breaks, causing DNA damage in target cell. Cancer cells lack repair system, so permanent chain breakage-> cell death
