Replication and Recombination Flashcards
What are the 3 modes of DNA replication? Define it pleaaassee :)
- Semiconservative: Each replicated DNA molecule consists of one “old” and one “new” strand
- Conservative: Two newly synthesized strands come together—original helix is conserved
- Dispersive: Parental strands are dispersed into two new double helices
Explain the Messelson-Stahl Experiment
- 15N-labeled E. coli grown in medium containing 14N
- Each new DNA molecule consists of one old and one newly synthesized strand
- Provided strong evidence that DNA is semiconservative in
prokaryotes - radiolabeling goes from 100% radiolabelled –> 50% radiolabelled –> 25% radiolabelled, etc…..
Understand the band patterns of Meselson-Stahl Experiment
N14 =LIGHT AND UP
N15 = HEAVY AND DOWN
Mix of N14 and N15 = In between
Every generation resulted in what for Messelson-Stahl?
Eventually will deplete the radiolabelled, but it is always halved. First generation is 50%, then 25%, then 12.5%, ETC…
Where does DNA replication begin?
At the ORI (origin of replication)
How many origin and termination points are there in Circular chromosomes
- 1 origin point
- Multiple termination points in a circular chromosome of Prokaryotes
Replication is _____; therefore there are # replication forks
- Replication is BIDIRECTIONAL
- 2 replication forks
Define Replicon
Length of DNA replicated
At site of replication, helix is unwound, creating what?
Replication Fork
DNA synthesis in bacteria involves 5 polymerases. Which one is the most important in E. coli for instance?
- DNA pol 1-5
- DNA pol 3 is the MOST SIGNIFICANT since it synthesizes both leading and lagging strand
Can DNA polymerases generate a strand de novo (from scratch)?
NO! They only work if there is a 3’ hydroxyl sugar to which they can add nucleotides
Can RNA polymerase generate a strand de novo?
YES!!! It just looks at what is given, and provides the complementary
Can polymerases work without Magnesium ion?
NOOOO!!!! Magnesium is an important cofactor for all polymerases
How does DNA polymerase do chain elongation?
- Occurs in 5’ to 3’ direction by adding one nucleotide at a time to 3’ end
- Nucleotide added, two terminal phosphates cleaved off, providing newly exposed 3’-OH
- 3’-OH can participate in addition of another nucleotide as DNA synthesis proceeds
T/F: all synthetic enzymes will have 3’-5’ exonuclease activity
TRUEEEEEEE!!!!!
duh. ‘0’
Which DNA polymerase has exonuclease activity 5’ - 3’?
Only DNA polymerase 1
“Repair polymerase removing RNA primer and fill gap by the respective DNA base in DNA replication”
DNA Pol 2, 4, and 5 are involved in what?
- Involved in various aspects of DNA damage repair
- Repair DNA damaged by external
forces such as UV light
Describe DNA polymerase 3 Holoenzyme
- Note: clamp feature is the Beta clamp
- Subunits each have separate functions
- α 5’-3’ polymerization
- ε– 3’–5’ exonuclease
- θ– Core assembly
What are the 7 key issues that MUST be resolved during DNA replication?
- Unwinding of helix
- Reduce increased coiling generated during unwindng
- Synthesis of primer for initiation
- Discontinuous synthesis of second strand
- Removal of the RNA primers
- Joining of gap-filling DNA to adjacent strand
- Proofreading
Explain DnaA
- Initiator protein encoded by dnaA gene
- Binds to ORI causing conformation change
- Causes helix to destabilize and open up
- Exposes ssDN
Explain DNA Helicase
- Made of DnaB polypeptides
- Hexamer of subunits: Assembles around exposed ssDNA
- Subsequently recruits holoenzyme to bind replication fork and initiate replication
- Helicases require energy supplied by hydrolysis of ATP— denatures hydrogen bonds and stabilizes double helix
Explain Single-stranded binding proteins (SSBPs)
- Stabilize the open conformation of helix
- Bind specifically to single strands of D
Explain DNA gyrase
- Enzyme relieves coiled tension from unwinding of helix (DNA supercoiling)
- Member of larger enzyme group: DNA topoisomerases
- Makes single- or double-stranded cuts
- Driven by energy released during ATP hydrolysis
Explain Primase: RNA polymerase
- Recruited to replication form by helicase
- Synthesizes RNA primer
- Provides free 3-OH required by DNA polymerase III for elongation
Explain DNA polymerase 1
- Removes primer and replaces it with DNA
- Adds DNA bases if needed
- Has both 5’-3’ and 3’-5’ exonuclease!
- Known as a “REPAIR” enzyme
Explain RNA priming
- Universal phenomenon
- Found in bacteria, viruses, and several eukaryotic organisms
DNA pol III ONLY synthesizes in which direction?
5’-3’
Continuous DNA synthesis is known as what? What about Discontinuous?
Continuous = Leading
Discontinuous = Lagging
Explain Okazaki fragments
Lagging strand synthesized as Okazaki fragments, each with RNA primer
Explain polymerase I
Removes primers on lagging strand
Explain DNA Ligase
- Catalyzes formation of phosphodiester bonds
- Seals nicks and joins fragments
- You mutate this enzyme= lethal mutation
Explain Concurrent Synthesis
- Both DNA strands synthesized concurrently
- Concurrent DNA synthesis achieved on
both strands at single replication fork - Lagging strand is looped
- Inverts physical but not biochemical
direction - DNA clamp prevents core enzyme
dissociation from template
Explain Proofreading and Error Correction
- Integral part of DNA replication
- DNA polymerase not always perfect
- Synthesis of noncomplementary base pairs inserted occasionally
- DNA polymerase exonuclease activity of 3’–5’ allows for excise of nucleotides
List the enzymes and Proteins essential to DNA synthesis
- DNA polymerase III core enzymes
- SSBPs: single-stranded binding proteins
- DNA gyrase
- DNA helicase
- RNA primers
Explain Mutations such as Conditional mutations
- Interrupt or impair aspects of replication
- Examples: Lethal mutations
- Ligase-deficient mutations
- Proofreading-deficient mutations
- Conditional mutations: Expressed under
specific conditions
Eukaryotic and Bacterial DNA replication shares many features such as
- Double-stranded DNA unwound at ORI
- Replication forks formed
- Bidirectional synthesis creates leading and lagging strands
- Eukaryotic polymerases require four
deoxyribonucleoside triphosphates, template, and primer
Compare the ORIs of Eukaryotic and Prokaryotic replication
- Eukaryotic replication: Multiple ORIs
- Eukaryotic chromosomes contain multiple ORIs
- Facilitates rapid synthesis of large quantity of DNA
- Larger amount of DNA causes replication to take longer than it would in Bacteria
Eukaryotic DNA replication is MORE complex in what 3 ways?
- More DNA than prokaryotic cells
- Linear chromosomes
- DNA complexed with nucleosomes
Where replication bubble/origin is typically what? A-T or G-C?
It is A-T rich since these only have 2 hydrogen bonds making it easier to break.
Describe Yeast ORI
- Autonomously replicating sequences (ARSs)
- 120 base pairs of consensus sequences
- Consensus sequence: Sequence that is the same in all yeast ARSs
What control timing of DNA replication
Eukaryotic ORIs
Explain the Prereplication complex (pre-RC)
- Assembles at replication ORIs
- Early G1 phase of cell cycle:
- Origin recognition complex (ORC) recognizes ORI and tags ORI as site of initiation
Explain the DNA polymerases involved in nuclear genome DNA replication
- Pol α, δ, ε: Involved in initiation and
elongation - Pol α: Possesses low processivity and RNA primer synthesis during initiation
on leading and lagging strands - Delta is associated with Lagging strand
- Epsilon = Leading strand
Explain Polymerase Switching
- Occurs once the primer is in place
- Pol α and ε replaced by Pol δ for elongation
- Pol δ synthesizes lagging strand
- Pol ε synthesizes leading strand
T/F: 146 base pair nucleosomes wrap around 8 histone proteins?
TRUE!!!
How many histones are there?
- 2 sets of 4 histones
- Known as a Histone Octamer
Define Chromatin
Eukaryotic DNA complexed with binding proteins
For replication through chromatin, what must happen to the nucleosomes?
Nucleosomes must be stripped away before polymerase can begin synthesis
Brief differentiation between Prokaryotes and Eukaryotes which endonucleases
In prokaryotes, there is detachment when replication is occurring. However, in eukaryotes, we have something known as FEN1 (Flap ends endonuclease) which creates this flag which will then be cut off, but detachment NEVER occurs. Then ligase in both would actually fill the gap. Also, what about the ends of our linear chromosomes? That’s when telomerase helps fix this problem.
Define Telomeres
- Inert chromosomal ends that protect intact eukaryotic chromosomes from improper fusion or degradation
- Long stretches of short repeating sequences preserve the integrity/stability of chromosomes
- Telomere t-loops and a complex of six proteins binds and stabilizes chromosome ends
- Forms the shelterin complex
Explain Telomerase
– Eukaryotic enzyme
– Ribonucleoprotein: RNA serves as template for synthesis of DNA complement
1. Telomerase RNA component (TERC)
2. Telomerase reverse transcriptase (TERT)
* Once RNA primer removed on lagging strand, no free 3’-
OH to elongate
* Telomerase adds repeats of six-nucleotide sequence to 3’ end to fill gaps
Stem cells and malignant cells
maintain telomerase activity—
what does this cause?
Telomerase
In Eukaryotic somatic cells, is telomerase active?
NO!
Define Homologous recombination
Genetic exchange at
equivalent positions along two chromosomes with
substantial sequence homology
Genetic recombination involves
- endonuclease nicking
- strand displacement and pairing with complement
- ligation
- branch migration
- duplex separation—generates characteristic Holliday structure
