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
