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BIOL 1101 > Lecture 11: DNA structure, replication, and organization > Flashcards

Lecture 11: DNA structure, replication, and organization Flashcards

1
Q

What is genetic material

A

DNA

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2
Q

Griffiths Experiments

A

Observation: a substance from a killed infective pneumonia could transformed noninfective living pneumonia to be infective

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3
Q

what is meant in Griffiths experiment by transformation

A

process of genetic change

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4
Q

Mice injected with pneumonia (Griffiths experiment)

A

1) Mouse injected with living, infective S cells= dead mouse, S cells are in their blood and virulent (yes pneumonia)

2) Mouse injected with noninfective R cells=mouse live, no R cells in blood, nonvirulent (no pneumonia)

3) Mouse injected with heat killed S cells (S bacteria is dead)=mouse live, S cells in their blood but not virulent (no pneumonia)

4) Mouse injected with heat-killed S cells AND live R cells= mouse die, living S cells in blood, showing living R cells can be converted to virulent S cells w/ factor from the dead S cells

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5
Q

what does the S cells being in the blood but not being virulent mean?

A

S cells must be living to be virulent (unless other R cells are present where they can convert)

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6
Q

What is the transforming molecule
(we see in the mice experiment)

A
  • either DNA or protein
  • most scientists at the time believed it was protein

= eventually figured out DNA was the transforming molecule

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7
Q

Avery’s Experiments

A
  • wanted to determine if DNA or protein was the transforming molecule

In Experiment 1 he used proteases to break proteins (won’t affect DNA)
and he used bacteria to test it (not mice this time)
HE SAW
- DESTROYED PROTEIN: STILL HAD TRANSFORMATION
- DESTROYED DNA: NO TRANSFORMATION

  • but his experiment was still questioned and rejected

  • added proteases to the heat-killed virulent bacteria to destroy proteins, but if the exchange btwn R and S still happened, DNA must be the transforming molecule
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8
Q

Point of Hershey and Chase’s Experiment
- and what did they use?

A
  • verification of whether the transforming molecule was DNA or proteins
  • used bacteriophage T2
  • used 35S and 32P (radioisotopes)
  • showed that bacteriophage DNA (not protein) enters the bacterial cells to direct the life cycle of the virus
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9
Q

Process of Hersheys and Chase’s Experiment

A

1) they infected e.coli growing with radioactive Sulfur isotopes with bacteriophage T2 (NO SULFUR IN DNA ONLY IN PROTEINS, SO THE PROTEIN IS RADIOACTIVE NOT THE DNA)
2) Fresh E.Coli cells were infected with radioactive phages
3) Cells were mixed and components were analyzed
= NO radioactivity!
- therefore, protein cant be the transformative molecule

THEN…
1) same process but with phosphorus radioactive isotopes, and instead w/ radioactive DNA and non-radioactive proteins
= Radioactivity!
- DNA is the transformative molecule and the hereditary material that’s passed

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10
Q

DNA strucure
- and who discovered it

A

Watson and Crick (technically Rosalind Franklin discovered it w/ her X-ray)
- discovered that DNA has 2 polynucleotide chains twisted around each other into a right handed double helix

  • Each chain has
    1) deoxyribose
    2) PO4 group
    3) a base (A,C,T,G)
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11
Q

The Double Helix Model

structure, chargaffs, each full turn

A
  • deoxyribose sugars are linked w/ po4 groups=sugar-phosphate backbone
  • TWO strands are held together
    Chargaff’s Rule:
    A-T
    C-G
  • Each full turn of a double helix is 10 nucleotide base pairs (20 total)
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12
Q

Nucleotide subunits of DNA

A
  • DNA= sugar phosphate backbone
    w/ phosphodiester bonds

PHOSPHATE at 5’ carbon of sugar
HYDROXYL at 3’ carbon of sugar

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13
Q

How did Rosalind Franklin find the structure of DNA

A
  • X-ray Diffraction analysis of DNA
  • saw a helical structure
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14
Q

How does DNA replicate

A

Semiconservative replication

  • two strands of parental DNA molecule will unwind
  • each is a template for the synthesis of a complementary copy
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15
Q

All of the theories of DNA replication?

A

a) semiconservative replication
- one old strand + one new strand

b) conservative replication
- old go together + new go together

c) dispersive replication
- mix of old and new

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16
Q

Meselson-Stahl Experiment

A
  • attempting to determine if DNA replicates semiconservatively

1) bacteria grown in radioactive heavy nitrogen is incorporated into DNA bases

2) Bacteria transferred to light nitrogen medium is allowed to grow and divide, and all the DNA is light

(sample from heavy nitrogen is transferred to light sample for the first replication and then transferred to sample B for second replication)

3) DNA extracted from bacteria cultured in heavy and light nitrogen

4) DNA mixed with CsCl and centrifuged

5) We saw a mix of the
- light nitrogen DNA
- hybrid DNA
- heavy nitrogen DNA
= can only support semiconservative of dispersive theory

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17
Q

How does the Meselson-Stahl Experiment bring it down to only support semiconservative theory

A
  • from sample with the heavy DNA they saw
  • after 1 replication: 15N-14N hybrid DNA (new)
  • after 2 replications: 14-14N layer and 15-14 hybrid layer

meaning it supports semiconservative replication only

18
Q

Enzymes of DNA replication

A

Helicase- unwinds DNA

Primase- synthesizes RNA PRIMER (starting point for nucleotide assembly by DNA polymerase)

DNA polymerase- assemble nucleotides into a chain (at 3’ end), remove primers, and fill resulting gaps (complementary pattern)

DNA ligase- closes remain single-chain nicks

19
Q

w/ 3 PO4 we can

A
  • build phosphodiester bonds, because they’re all negative they want to separate (exergonic, and spontaneous)
  • remove 2 po4s
  • energy released
  • harness that energy to fuel reaction =dATP
20
Q

OH on the 3’ end will

A

participate in the reaction, its where we add a nucleotide to another to form complementary strand

21
Q

sliding DNA clamp

A
  • helps enzyme tethered onto DNA strand
22
Q

Enzyme activities

A

Origin of replication
- Ori determines the start point of replication

Replication Fork
- where replication occurs

DNA helicase
- recruited by proteins bound to the origin, uses energy of ATP hydrolysis to wind DNA to produce replication fork

23
Q

Importance of SSBs

A
  • single stranded binding proteins that coat and stabilize single-stranded DNA preventing the two strands from reforming the double stranded DNA
  • topoisomerase keeps DNA relaxed
24
Q

RNA primer

  • and the process of primase and polymerase working together
A

primase synthesizes a short RNA primer to a initiate a new DNA strand (new DNA = antiparallel strands)

  • short sequence of RNA that has a 3’ end w/ OH- (bc DNA polymerase can only add nucleotide to an existing 3’ end)
  • primase leaves; DNA polymerase takes over
  • new DNA extended from primer from DNA polymerase
25
Q

Assembling Antiparallel Strands

A

Leading=continuous
- allows DNA strand to be made continuously in the direction of unwinding

Lagging=discontinuous
- DNA is built in fragments=Okazaki fragments
- copied in short lengths that run in the direction opposite to unwinding
- discontinuous replication produces short lengths that are linked together

26
Q

Process of DNA replication

A

1) DNA helicase unwinds DNA, primates synthesize short RNA primers in 5-3 direction
- direction of leading
- opposite of lagging
Topoisomerase prevents twisting ahead of the replication fork of the DNA unwinds

2) DNA polymerase 3 adds DNA nucleotides to the RNA primer, continuing to the 5-3 direction of synthesis

3) DNA helicase continuously unwinds DNA, DNA polymerase 3 continues the leading strand.
Primase synthesizes a new RNA primer on the lagging strand template near point of unwinding, the primer is extended by addition of DNA nucleotides by DNA polymerase 3
- New DNA synthesis stopes when the polymerase reaches the 5’ end of the previously synthesized Okazaki fragments

4) DNA polymerase 1 removes RNA primer of previously synthesized Okazaki fragments 1 nucleotide at a time and replaces the RNA nucleotides with DNA nucleotides
- the enzyme leave the template when these tasks are done
- a break between the backbone remains between the newly synthesized fragments

5) DNA ligase seals the nick between the 2 lagging strands

6) DNA helicase continuous to unwind the DNA and the synthesis proceeds as before with more synthesis of leading strand and synthesis of a new fragment to be added to lagging strand

27
Q

the naming system is specific to?

A

prokaryotes

28
Q

which enzyme connects Okazaki fragments

A

DNA ligase

29
Q

Primer is extended by which enzyme

A

DNA polymerase

30
Q

REVIEW MAJOR ENZYMES OF DNA REPLICATION SLIDE!

A

ok

31
Q

DNA synthesis

A
  • begins at sites that act as replication origins
  • only activates once during S phase
  • proceeds form the origins as 2 replications fork move in opposite directions
32
Q

telomeres

A
  • ends of eukaryotic chromosomes
  • short sequences repeated hundreds to thousands of times
  • repeats protects against genes on chromosomes shortening during replication
  • telomerase: enzyme that prevents chromosome shortening by adding telomere repeats (only in eukaryotic DNA bc of linear DNA)
33
Q

Why do we need RNA primer

A

Recall:
- DNA polymerase only adds to 3’ end of new strand, which is why we need RNA primer bc w/o primer DNA polymerase can’t work

34
Q

Telomere importance

A

1) chromosome end after primer removal (we need to replicate otherwise offspring will have missing genes)

2) telomerase binds to single stranded 3’ end of the chromosome by complementary base pairing btwn RNA of telomerase and telomere repeat w/ telomerase it extends the chromosome

3) Telomerase synthesizes new telomere DNA using telomerase RNA as template

4) Telomerase moves to 3’ end of newly synthesized telomere DNA

5) Telomerase synthesizes more new telomere DNA using telomerase RNA as the template

6) Telomerase leaves the extended template strand and a primer is added to the telomere by primase

7) New end of the chromosome after replication

8) Short, single-stranded region remains after primer removal

35
Q

Telomere shortening

A
  • not bad because it has 0 genes
  • telomerase enzymes work far slower (cell aging) leading to issues
36
Q

Mechanisms of correct replication errors

A

a) proofreading: depends on the ability of DNA polymerases to reverse and remove mismatched bases

b) DNA repair corrects errors that escape proofreading

37
Q

Mutations are

A
  • good
  • bad
  • or neutral
38
Q

When happens if there is a replication error

A
  • a base can be mispaired for ex.
  • DNA polymerase reverses and removes the most recently added base (uses exonuclease)
  • the enzyme will resume DNA synthesis in the forward direction
39
Q

process of proofreading via DNA polymerase

A

1) polymerization activity of DNA polymerase adds DNA nucleotides to the chain in 5’ to 3’ direction using complementary base pairs

2) rarely, DNA polymerase adds a misplaced nucleotide

3) DNA polymerase recognizes the mismatched base pair, the enzyme reverses using its 3-5 to remove mispiared nucleotides

4) DNA polymerase resumes its polymerization activity in the forward direction extended in the new chain in 5-3 direction

40
Q

DNA Repair Mechanisms

A
  • set of DNA polymerase enzymes:

1) recognize distorted regions caused by mispaired base pairs

2) Remove mispaired base region from the newly synthesized nucleotide chain

3) Resynthesize correctly using original template chain as a guide

41
Q

Mismatch Repair

A

1) repair enzymes recognize mispaired base and break DNA chain

2) Enzyme remove several bases included the mispaired one leaving a gap in DNA chain

3) Gap is filled w/ DNA polymerase using template strand as guide

4) Nick left after gap filling is sealed with DNA ligase to complete the repair

42
Q

What are nucleoases

A

enzyme capable of cleaving the phosphodiester bonds that link nucleotides together to form nucleic acids

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