DNA Repli. Flashcards
What is replication?
-In replication 2 identical doughter DNA molecules are made from a single DNA molecule
What is the purpose of replication?
-Before cells divide they have to double cell structures, organelles and their genetic information
Where is the replication?
-in the nucleus of eucaryotes
Describe the mammalian cell cycle
- starts with G1->rapid growth and preparation for DNA synthesis
- S ph-> DNA synthesis and histone synthesis
- G2 ph-> Growth and preparation for cell devision
- M-> mitosis
- continues from the beginning
- cells from G1 can also go to G0 for no synthesis (quiescent cell) (less cells do so)
- > S phase is as short as 40 min in simple eucaryotes, up to 8 hrs in humans
Name general features of replication
- high fidelity
- semi-conservative
- starts at origin
- bidirectional
- semi-continuous
Which are three mechanism of replication
- initiation
- elongation (verlängerung)
- proofreading and termination
What does high fidelity means in number?
- less than 1 error for every 10^9 nucleotides copied
- > NB: high fidelity: base pairing+proofreading
Which model fits the observations made in DNA replication?
-semiconservative
What did not fit with the conservative and dispersive model for DNA replication?
- conservative: no intermediate (zwischen) DNA ever!
- dispersive: All of the DNA is intermediate
Describe The Meselson and Stahl experiment
-“heavy” DNA (^15N) grow in ^14N medium
->the first generation: one line (btw 15-14) (won through density gradient centrifugation; grow in ^14N medium
->the second generation
had two lines
How is replication origin selected?
- OriC in bacteria: Replication is bidirectional but has one origin
- No OriC in eucaryotes: Replication is bidirectional and has multiple origin
Where is OriC ?
-at A/T rich region
Which bonds occur in OriC ?
-hydrogen, covalent phosphohidester bonds
What is the 2step in replication?
- build daughter DNA stand by adding new complementary base
- >DNA polimerase
Which parts exist in semi-continuous replication?
-replication fork, replication direction, Okazaki fragment, leading strand
What are the Okazaki fragments?
- Many DNA fragments are synthesized sequentially on the DNA template strand having the 5´- end
- > are 1000 – 2000 nt long for prokaryotes and 100-150 nt long for eukaryotes
What is the lagging strand ?
-The daughter strand consisting of Okazaki fragments is called the lagging strand
What is helicase?
-unwinds the parental double helix
Give a summary of DNA replication
- helicases unwind parental double helix
- single strand binding proteins stabile the unwound parental DNA
- the leading strand is synthesized continuously in the 5’ ->3’direction by DNA polymerase
- The lagging strand is synthesized discontinuously; primate synthesizes a short RNA primer, which is extended by DNA polymerase to form Okazaki fragment
- after the RNA primer is replaced by DNA (by another DNA polymerase), DNA ligase joins the Okazaki fragment to the growing strand
Name the Replication Enzymes and proteins (+functions)
- DNA Helicase: unwinds the replication fork
- DNA Gyrase (topoisomerase): prevents supercoiling
- Single Stranded Binding Proteins (SSBP): keep the fork open
- DNA Primase: builds RNA primer
- RNA Primers: starter sequence for DNA Polimerase III
- DNA Polymerases (I, II, III): synthesis and repair
- DNA Ligase: fills the gaps (joins okazaki fragments)
Name the components of the replication apparatus
-dnaA -> binds to origin DNA sequence
-Primasome:
dnaB-> helicase (unwinds DNA at origin)
-dnaC-> binds dnaB
-dnaG->primase (synthesizes RNA primer)
-DNA gyrase -> introduces negative supercoils ahead of the replication fork
-Rep protein -> helicase (unwinds DNA at fork)
-SSB -> binds to single-stranded DNA
-DNA pol III -> primary replicating polymerase
-DNA pol I -> removes primer and fills gap
-DNA ligase -> seals gap by forming 3’, 5’-phosphodiester bond
Name the replication enzymes in eucaryotes
eucaryotes:
- DNA-pol α: initiate replication and synthesize primers, lagging strand synthesis
- DNA-pol β: replication with low fidelity, repair
- DNA-topoisomerase: relases the supercoils
- DNA-pol Y (gamma): polymerization in mitochondria
- DNA-pol ε: polymerization and repair
- DNA-pol δ: repair
- PCNA: (proliferating cell nuclear antigen)
procaryotes:
- DnaG, primase
- DNA-pol I
- gyrase
-DNA-pol I
-DNA-pol III
sliding clamp
How do the functions of Pol δ differ to Pol ε?
-Pol δ would be responsible for the replication of more open chromatin in early S phase whereas Pol ε would replicate more compact chromatin in late S phase
How does the replication machinery work?
- DNA helicase separates the two DNA strands by breaking the hydrogen bonds between them
- This generates positive supercoiling ahead of each replication fork
- DNA gyrase (topoisomerase) travels ahead of the helicase and alleviates (linden) these supercoils - Single-strand binding proteins bind to the separated DNA strands to keep them apart
- Then short (10 to 12 nucleotides) RNA primers are synthesized by DNA primase
- > These short RNA strands start, or prime, DNA synthesis
What does the topoisomerase exactly?
-cuts a phosphodiester bond on DNA, rotates the broken DNA freely around the other strand to relax the constraint (Zwang), and reseals (wiederverschließt) the cut
What is DNA Polimerase III?
- DNA-pol δ
- primary enzyme in elongation (Ausdehnung)
- needs a primer to start, cannot initiate synthesis
- 5´→ 3´ polimerase activity
- 3´→ 5´ exonuclease activity (proofreading)
What is DNA Polimerase I?
- DNA-pol ε
- primary enzyme in correcting mismatched nucleotides
- needs a primer to start, cannot initiate synthesis
- 5´→ 3´ polimerase activity
- 3´→ 5´ exonuclease activity
- 5´→ 3´ exonuclease activity (destroys RNA primer)
- connects the okazaki fragments with ligase
- needs Mg+2 ion
What is semicontinuous replication?
-Continuous synthesis of the leading strand and discontinuous synthesis of the lagging strand represent a unique feature of DNA replication
What are the general features of replication?
- A. Semi-Conservative
- B. Starts at Origin
- C. Bidirectional
- D. Semi-continuous
- E. High fidelity
Define telomere
- the terminal structure of eukaryotic DNA of chromosomes is called telomere
- They are small repeated sequences rich in T and G
- > Telomeres effectively “cap” the end of a chromosome in a manner similar to the way the plastic on the ends of our shoelaces “caps” and protects the shoelaces from unraveling (lösen)
How is the problem: Loss of bases at 5’ ends in every replication solved?
- cell solves this problem by adding DNA sequences to the ends of chromosome: telomeres
- catalyzed by the enzyme telomerase
Where is telomerase used? What is it/ what does it do/ Why?
-The eukaryotic cells use telomerase to maintain the integrity (Einheit) of DNA telomere.
-What is Telomerase?:
It is a ribonucleoprotein: RNA+protein
-What does it do?:
It adds many copies of the telomere sequence (TTTTGGGG) to the 3’end of the template strand after the replication occurs.
-Why:
To prevent shortening of DNA ends and chromosomes
Where else do telomeres participate?
- Telomeres participate in the maintenance of genomic and cellular stability and replication;
- in fact, they protect the genome from degradation, unwanted recombination and chromosomal fusion
What does Telomerase contain?
- Telomerase contains a highly conserved reverse transcriptase [human telomerase reverse transcriptase (hTERT)],
- an associated template RNA [telomerase RNA component (TERC)]
- And a key auxiliary protein known as Dyskerin
