DNA replication ppts Flashcards
what are examples of nucleic acids?
DNA/RNA
what are nucleic acids made up of?
polymers consisting of monomers (nucleotides)
what are nucleotides made of?
1) a pentose sugar
2) a phosphate group
3) a nitrogenous base
DNA replication is always _____ to _____
5’ to 3’
DNA is always read ____ to _____
3’ to 5’
Energy for formation of phosphodiester (sugar) bond is released by the breaking of the ______ bond.
triphosphate
The ____’ (prime) end of the free nucleotide is added to the ____’ (prime) end of the nucleotide chain that is already formed.
5, 3
We talk about DNA being double stranded and aligned in an anti-parralel orientation.
Does this mean that both strands of DNA are identical to each other?…
no, they are complimentary
how many bonds between A and T?
2
How many bonds between G and C?
3
Explain basic steps associated with DNA replication
Precursors, enzymes, direction
Specifically the need of a RNA primer
- DNA replications is initiated by RNA primer
- DNA is copied by DNA polymerase
- Leading strand is synthesized continuously
- lagging stand synthesized in Okazaki fragments
- RNA primers are removed and Okazaki fragments are joined by DNA ligase
Explain when/where does DNA replication occur in prokaryotes? In eukaryotes?
multiple origin of replication in eukaryotes
only one OR in prokaryotes
Which chromosomal features are essential to both bacterial and eukaryotic chromosomes?
origin of replication
The origin of replication in eukaryotes is at ____ locations next to _____ genes
defined, important
What is meant by bidirectional replication?
in prokaryotes, the origin of replication is bidirectional, meaning it has two replication forks
in what direction is the leading strand going?
3’ to 5’ (toward the fork)
in what direction is the lagging strand going?
5’ to 3’ (away from fork)
what is the purpose of helicase?
binds to double stranded DNA and breaks the hydrogen bonds between the nucleotides, separating the strands
what does unwinding cause?
unwinding induces supercoiling ahead of the replication fork
what is the purpose of DNA topoisomerase?
to break DNA and allow it to uncoil, then reseal the break
what first imitates replication?
RNA primer - polymerase will not synthesize without it
How long is an RNA primer? How is it made?
5-10 bases long, primes
why use RNA?
If there is a mistake, when DNA is added back into it to make a new strand, the body will notice an error and fix it
how often is there an origin of replication in linear DNA?
every 3000 bp
What is the problem with the replication of linear DNA.
Removal of the RNA primer at the end will leave a 3’ overhang
- this leaves a gap from RNA removal at tellers because no new DNA synthesis could fill them in
Telomerase
ribonucleoprotein (made of RNA and Proteins) “TTAGGG”
Which of the following is analogous to telomeres?
the two ends of a shoelace
In what type of cell would you expect to normally see telomerase activity
Embryonic Cells
What components are needed to replicate DNA?
A. Free Nucleotides (ATP, GTP, CTP, TTP) B. Primers to direct DNA polymerase C. DNA polymerase D. Template DNA E. All of the above are needed
E
What are the necessary components of PCR reactions?
The various components required for PCR include a DNA sample, DNA primers (oligonucleotides), free nucleotides called ddNTPs, and DNA polymerase.
Why must the DNA polymerase be highly thermally stable?
because during PCR, temperature rise and fall for denaturing, annealing, and synthesis. That is why helicase isn’t used in PCR
What are some advantages and limitations of PCR?
amplifies a lot of DNA
oligonucleotides
short pieces of synthetic DNA can be manufactured that contain any sequence,
…template specific!
How is DNA strands separated in PCR
heat
why can’t you use helicase in vitro?
…DNA denaturing conditions such as high heat or low salt concentrations irreversibly denature or inactivate most polymerases,
…dNTPs are not affected by denaturation,
…primers are not affected by denaturation.
What are the temperatures of the thermal cycle, and what steps occur at each of these temperatures?
94 degrees - Denature
60 degrees - Anneal
72 degrees - Synthesis
Exponential synthesis requires:
as few as 1 DNA templates required,
excess dNTPS,
excess primers,
multiple cycles.
DNA helixes run ____
anti-parallel
What is the correct model for DNA replication?
semi-conservative model
how many origin of replications do bacteria have?
1, but they have two replication forks - the forks run in opposite directions (bidirectional)
what initiates replication in bacteria?
binding of DnaA proteins
where is bacterial DNA first separated?
AT rich region, separated by helicase
what all is needed for linear DNA replication?
RNA primer, helicase, topoisomerase, single stranded binding proteins, DNA polymerase III and I, DNA ligase
DNA helicase
breaks hydrogen bonds
Topoisomerase II
alleviates positive supercoiling
Single stranded binding proteins
keep the parental strands apart
Primase
synthesis RNA primer
RNA primer
initiates replication - 10-12 bp in length
DNA polymerase III
synthesizes new strand of DNA
DNA polymerase I
knocks off RNA primers and fills in their gaps with DNA
DNA ligase
covalently links the Okazaki fragments together
what are dNTPs?
deoxiribonucleotiside triphosphate (nucleotides)
what removes mismatched bases?
an exonuclease on the 3’ end of the DNA polymerase
in eukaryotes, what gets rid of RNA primers on lagging strand?
Flap endonuclease, removes flaps created by DNA squiggle
- cannot remove a flap if its too long
the ends of eukaryotic chromosomes are replicated by
telomerase
how does telomerase work?
additional DNA sequences are attached to the ends of telomeres, where telomerase recognizes the sequences and synthesizes additional repeats of telomeric sequences
what is the problem with telomerase?
activity of telomerase decreases with age
what are the steps of telomerase?
1) binding to the 3’ overhang
2) polymerization - telomerase synthesizes a new 6 nucleotide repeat
3) moves 6 nucleotides to the right to make another sequence (translocation)
4) complimentary strand is made by primate, polymerase, and ligase
what part of telomerase contains the sequence complimentary to the telomeric repeat sequence?
TERC - telomerase RNA component
telomerase reverse transcriptase (TERT)
uses RNA template to synthesize DNA
