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2. wk > DNA Repli. > Flashcards

DNA Repli. Flashcards

1
Q

What is replication?

A

-In replication 2 identical doughter DNA molecules are made from a single DNA molecule

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2
Q

What is the purpose of replication?

A

-Before cells divide they have to double cell structures, organelles and their genetic information

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3
Q

Where is the replication?

A

-in the nucleus of eucaryotes

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4
Q

Describe the mammalian cell cycle

A
  • 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
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5
Q

Name general features of replication

A
  • high fidelity
  • semi-conservative
  • starts at origin
  • bidirectional
  • semi-continuous
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6
Q

Which are three mechanism of replication

A
  • initiation
  • elongation (verlängerung)
  • proofreading and termination
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7
Q

What does high fidelity means in number?

A
  • less than 1 error for every 10^9 nucleotides copied

- > NB: high fidelity: base pairing+proofreading

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8
Q

Which model fits the observations made in DNA replication?

A

-semiconservative

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9
Q

What did not fit with the conservative and dispersive model for DNA replication?

A
  • conservative: no intermediate (zwischen) DNA ever!

- dispersive: All of the DNA is intermediate

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10
Q

Describe The Meselson and Stahl experiment

A

-“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

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11
Q

How is replication origin selected?

A
  • OriC in bacteria: Replication is bidirectional but has one origin
  • No OriC in eucaryotes: Replication is bidirectional and has multiple origin
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12
Q

Where is OriC ?

A

-at A/T rich region

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13
Q

Which bonds occur in OriC ?

A

-hydrogen, covalent phosphohidester bonds

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14
Q

What is the 2step in replication?

A
  • build daughter DNA stand by adding new complementary base

- >DNA polimerase

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15
Q

Which parts exist in semi-continuous replication?

A

-replication fork, replication direction, Okazaki fragment, leading strand

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16
Q

What are the Okazaki fragments?

A
  • 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
17
Q

What is the lagging strand ?

A

-The daughter strand consisting of Okazaki fragments is called the lagging strand

18
Q

What is helicase?

A

-unwinds the parental double helix

19
Q

Give a summary of DNA replication

A
  • 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
20
Q

Name the Replication Enzymes and proteins (+functions)

A
  • 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)
21
Q

Name the components of the replication apparatus

A

-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

22
Q

Name the replication enzymes in eucaryotes

A

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

23
Q

How do the functions of Pol δ differ to Pol ε?

A

-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

24
Q

How does the replication machinery work?

A
  • 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
25
Q

What does the topoisomerase exactly?

A

-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

26
Q

What is DNA Polimerase III?

A
  • 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)
27
Q

What is DNA Polimerase I?

A
  • 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
28
Q

What is semicontinuous replication?

A

-Continuous synthesis of the leading strand and discontinuous synthesis of the lagging strand represent a unique feature of DNA replication

29
Q

What are the general features of replication?

A
  • A. Semi-Conservative
  • B. Starts at Origin
  • C. Bidirectional
  • D. Semi-continuous
  • E. High fidelity
30
Q

Define telomere

A
  • 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)
31
Q

How is the problem: Loss of bases at 5’ ends in every replication solved?

A
  • cell solves this problem by adding DNA sequences to the ends of chromosome: telomeres
  • catalyzed by the enzyme telomerase
32
Q

Where is telomerase used? What is it/ what does it do/ Why?

A

-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

33
Q

Where else do telomeres participate?

A
  • 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
34
Q

What does Telomerase contain?

A
  • 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

2. wk (9 decks)

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