DNA replication and PCR (10.2) Flashcards
(44 cards)
What can you use to amplify the insulin gene from human DNA?
Can use PCR, then clone it into a bacterial plasma
How can you obtain a fragment of DNA containing the insulin gene for cloning?
–>Cut the DNA to produce “sticky ends”
–> insert insulin gene (PCR product)
–>Glue the sticky ends together (this creates a recombinant plasmid)
–>Put the recombinant plasmid in E. coli
need fairly large quantity of linear DNA containing (only) the human insulin gene
The mode of DNA replication is _______ and ________
semiconservative; bidirectional
3 phases of DNA replication for prokaryotes (goal and end product)
Initiation
Goal: create single stranded template DNA (so that we can begin replication)
End product: replication forks
Elongation
Goal: replicate the template DNA
End product: linear dsDNA
Termination
Goal: stop replicating DNA
End product: circular DNA
What is gyrase?
specific topiosmerase that upwinds supercoiled DNA for DNA replication; requires ATP; it cuts the dsDNA, unwinds one loop and reconnects dsDNA
What do ori sequences contain?
At Rich region, DnaA recognition sequence and GATC sequences (C must be methylated)
Where does DNA replication begin?
At the origin of replication
What happens when DnaA finds DnaA boxes to bind to?
DnaA causes tension to the “B” structure of DNA. H-bonds are broken in the AT-rich sequence of the ori to separate the dsDNA. An initation bubble is created.
What is the helicase? Where is it located?
DnaB which an enzyme that unwinds dsDNA “motor proteins. The replication fork.
What do single-stranded binding proteins do?
bind to single stranded DNA to prevent the H-bonds from reforming/ help keep the initiation bubble from closing
DNA replication initation for prokaryotes
step 1: Gyrase unwinds supercoiled dsDNA to expose ori sequence
step 2: DnaA binds to DnaA boxes within the ori sequences
step 3: Dna proteins bend the dsDNA, creating stress/tension on the structure
step 4: To relieve tension, the hydrogen bonds holding the A:T pairs within the AT-rich region are broken, creating the initiation bubble
step 5:Helicase (DnaB) is loaded on the single stranded DNA by helicase loader (DnaC)
step 6: Helicase moves from 5’ to 3’ to unwind dsDNA
step 7:Initiation is complete once 2 replication forks have been established
DNA polymerase
- Synthesizes new DNA chains. Binds (loosely) to ssDNA. Attach complementary nucleotides to the 3’-OH of a polynucleotide chain. Continue adding complementary nucleotides in a 5’ to 3’ direction
- Helps reshape the dsDNA helix by aiding in H-bond formation between the bases in the old and new DNA strand
- Proof-reads and correct mistakes
- Recognizes mispaired bases, halts polymerase activity, uses exonuclease activity to remove a few bases from the nascent strand
Processivity
the ability of an enzyme to catalyze consecutive reactions without releasing its substrate
Primase
an enzyme that synthesizes short RNA sequences called primers
Primase binds to DnaB helicase, recognizes a specific target sequence that serves as a template for the primer
Helicase+primase
Primosome
How many polymerases does E. coli have? What is the main one?
5 polymerases, Pol3
DNA replication elongation for prokaryotes
step 1: Primase binds to helicase (DnaB), and is carried along as helicase unzips the dsDNA.
step 2: When primase finds a recognition sequence on the ssDNA, primase builds a complementary RNA chain in the 5’ to 3’ direction (using the 3’ to 5’ DNA strand as a template).
step 3: Sliding clamp loader tethers DNA Polymerase to the helicase.
step 4: Sliding clamp loader also loads the sliding clamp onto the DNA. Sliding clamp helps DNA Polymerase remain attached to the template DNA. Each helicase can bind to 2 DNA polymerases.
step 5: The helicase/primase/sliding clamp/sliding clamp loader/DNA polymerase complex is called the replisome.
step 6: DNA polymerase finds the RNA primers, and begins adding free DNA nucleotides to the RNA primer in a 5’ to 3’ direction (using the 3’ to 5 DNA strand as a template).
step 7: One DNA polymerase builds its newly synthesized DNA chain in the same direction that the helicase is moving (“leading strand”).
step 8: One DNA polymerase builds its newly synthesized DNA chain in the opposite direction that the helicase is moving (“lagging strand”).
step 9: The lagging strand synthesis will be blocked whenever the polymerase bumps into a new RNA primer. The discontinuous strand synthesis creates “Okazaki fragments”.
step 10: A different DNA polymerase (Pol1 in E coli) identifies RNA/DNA complexes, removes the RNA component using exonuclease activity (3’ to 5’) , and fills in the gap with DNA (5’ to 3’). This leaves a gap in the phosphate backbone of the newly synthesized DNA.
step 11: DNA ligase seals the gap by connecting the phosphate bridge.
palindromic
sequences that are the same but on opposite strands of DNA
permissive
if Tus and DnaB are on opposite strands, then DnaB dislodges Tus and DNA synthesis continues
non-permissive
if Tus and DnaB are on the same strand, Tus blocks DnaB
DNA replication termination for prokaryotes
step 1: Elongation continues until the replisome meets at the termination sequences.
step 2: Ter1 and Ter2 are ~20 bp sequence motifs that are located on the opposite side of the ori sequence in a bacterial genome. The ter sequences are palindromic.
step 3: Tus proteins bind to each Ter sequence in directional manner. One Tus will bind to the top 5’- 3’ DNA strand. The other Tus will bind to the palindromic sequence on the bottom strand (still binding to a 5’to3’ sequence but in opposite orientation).
step 4: When a replication fork meets a Tus/Ter complex one of 2 things can happen: permissive or non permissive
step 5: When both replication forks are blocked at the Tus/Ter complexes, the helicases are removed, an additional DNA polymerase fills in the gap between the Ter sequences and ligases connect the DNA chains, creating 2 complete circles of dsDNA.
3 phases of DNA replication for eukaryotes goals and end product
Initiation
–>Goal: create single stranded template DNA (so that we can begin replication)
–>End product: replication forks
Elongation
–>Goal: replicate the template DNA
–>End product: linear dsDNA
Termination
–>Goal: stop replicating DNA
–>End product: linear DNAs
What are chromatin remodeling complexes and histone chaperones used for?
temporarily unpackage chromatin and disrupt nucleosomes during eukaryotic DNA replication
What are gyrases used for in eukaryotic DNA replication?
Not used in nuclear DNA replication, but for organellar DNA
