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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
- realized that the two strands of a double helix will only be stable if they run in opposite directions (complementary and antiparallel strands)
- base pairing can explain DNA replication

  • 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 and Chargaffs Rule

structure, chargaffs, each full turn

A
  • (2 polynucleotide molecules held w/H bonds)
  • polynucleotide chain: deoxyribose sugars are linked w/ po4 groups=sugar-phosphate backbone
    full linkage w/ riding phosphate=phosphodiester bond
  • TWO strands are held together
    Chargaff’s Rule:
    A-T
    C-G
  • he observed that the number of purines=number of pyrimidines, and the number of A=T, C=G, hence the pairing
  • Each full turn of a double helix is 10 nucleotide base pairs (20 total)
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12
Q

Nucleotide subunits of DNA
what is on 3’/5’ end

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
  • base pairs lie in flat planes perpendicular to the long axis of DNA helix=repeating pattern in X-ray diffraction pattern
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14
Q

How does DNA replicate

A

Semiconservative replication
(technically proposed by Watson+Crick) but confirmed by Mehelson-Stahl

  • 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

(before the transfer to the medium w/ light nitrogen and after each round of replication in 14N following the transfer, a sample of cells were taken and their DNA was extracted)

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
after rep 1: 15-14N hybrid
after rep 2:
- light nitrogen DNA
- hybrid 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

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18
Q

Enzymes of DNA replication

A

Helicase- unwinds DNA

Primase- synthesizes RNA PRIMER (starting point for nucleotide assembly by DNA polymerase)
because DNA polymerase can only add nucleotides to the 3’ end of an existing strand, so w/o an existing strand It cant begin a new strand

DNA polymerase- assemble nucleotides into a chain (at 3’ end), remove primers, and fill resulting gaps (complementary pattern)
- can add a nucleotide only to 3’ end, therefore when assembling a new DNA strand, 3’OH is always exposed at newest end, and the oldest end has an exposed 5-P
- DNA polymerase assemble nucleotides in 5’-3’ direction

DNA ligase- closes remain single-chain nicks, therefore makes covalent bond

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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
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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

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21
Q

sliding DNA clamp

A
  • helps enzyme tethered onto DNA strand
22
Q

Enzyme activities
What is. Origin of replication
What is replication fork
What is dna helicase

A

Origin of replication
- Ori determines the start point of replication
-determined by specific DNA sequences recognized by initiator proteins
- RNA primase then synthesizes the RNA primer at the ori to start DNA 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 and topoisomerase

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:
  • cuts the DNA ahead of the replication fork by turning the DNA on one side of the break in the opposite direction of the twisting force and rejoins the 2 strands
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 + telomerase (+why we need them)

A
  • ends of eukaryotic chromosomes
  • short sequences of DNA (5’TTAGGG3’) 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)

WHY WE NEED IT!
primarily for lagging strand
- when RNA primer is removed, a gap is left at 5’ end of new strand (there is no 3’) therefore, DNA polymerase has no existing nucleotide chain to elongate at the end of a nucleotide chain
- because of this inability to replicate the ends, chromosomes would shorten during each replication cycle

  • would occur on both 5’ ends of the chromosome (opposite strands of helix), new shortened DNA strands are a template for the next round and the resulting chromosomes will be shorter still
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 it provides the 3’ end for DNA poly 3 to attach its nucleotides so it can tether and synthesize DNA

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

TELOMERASE DOES NOT PREVENT MECHANISM THAT CAUSES SHORTENING OF CHROMOSOMES, IT ACTS AGAINST IT BY LENGTHENING CHROMOSOMES

SUMMARY:
Telomerase binds to the 3’ end of the chromosome and extends the telomere by adding repetitive DNA sequences using its RNA template. After telomerase finishes, primase adds a primer to the new telomere, and replication of the chromosome end is completed. A short single-stranded region remains after primer removal, which is typically repaired to prevent loss of genetic material.

35
Q

Telomere shortening and telomerase in somatic/germ cells

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

telomerase activity in cells
- in somatic cells they’re inactive so somatic cells can only divide so many times before dying
- only active in rapidly dividing cells of early embryo, stem cells, during WBC differentiation, and in male germ line to ensure the chromosomes of gametes have telomeres restored before next generation
- in cancers, they are reactivated to allow for indefinite divisions

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 built in 3’-5’ exonuclease to remove mistake)
  • the enzyme will resume DNA synthesis in the forward direction to insert the correct nucleotide
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 (DNA strand is cut on either side of mismatch)

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

43
Q

Process of DNA replication

A
  1. DNA helicase unwinds DNA, primases synthesize short RNA primers in 5’-3’
    - topoisomerase prevents twisting ahead of fork
  2. DNA polymerase 3 adds DNA nucleotides to RNA primer, continuing the 5’-3’ direction of synthesis
  3. DNA helicase continues to unwind DNA as polymerase synthesizes the leading strand. primase synthesizes a new RNA primer on lagging strand near point of unwinding
    - Primer is extended by the addition of DNA nucleotides by DNA polymerase 3
    - New DNA synthesis stops when the polymerase reaches 5’ end of Okazaki
  4. DNA Poly 1 removes RNA primer of previously synthesized Okazaki using 5’-3’ exonuclease, and replacing RNA nucleotides w/ DNA using 5’-3’ polymerizing activity
    - enzyme leaves the template
    - there’s a break in the backbone btwn newly synthesized fragments
  5. Ligase seals break btwn 2 laggings strands

6.DNA helicase continues to unwind g DNA and synthesis proceeds

44
Q

What would happen between the two strands during replication

A

breakage of H bonds, allowing them to unwind and separate where each strand would then act as a template for the synthesis of the other one

45
Q

antiparallel nature of DNA results in

A

template strand read in 3’-5’
DNA polymerase assemble nucleotides in 5’-3’
= overall direction of new synthesis is in 5’-3’

46
Q

DNA Polymerase 1 vs 3

A

1: removes RNA primer at 5’ end of previous, newly synthesized Okazaki fragments to replace RNA nucleotides
- fixes nucleotide errors and removes RNA primers

3: Extends primer by adding DNA nucleotides, synthesizes the complementary strand
- proofreading during replication

47
Q

what is an exonuclease

exonuclease activity relative to DNA polymerase 1 and when it stops

A

activity of enzymes where it removes nucleotides from the end of a molecule; the primer is digested from 5’ end towards 3’ end

  • when its used by DNA polymerase 1, its stopped when it meets the first DNA nucleotide that was synthesized in the Okazaki fragment
  • the DNA base replacing the last RNA base of the primer ends up beside the first DNA base of the Okazaki fragment
48
Q

Replisome

A

enzyme for replication into a DNA replication complex

49
Q

Ori’s for prokaryotes vs eukaryotes

A

prokaryotes: 1 ori because they have circular DNA

eukaryotes: several linear chromosomes with hundreds of Oris
=many

50
Q

Repair of DNA damage

A

DNA damage- because its for 1 strand
mutation-2 strands

3 TYPES OF REPAIR

  1. Proofreading- correcting errors made by DNA polymerase w/exonuclease
  2. Mismatch repair- correcting errors made during replication that escape proofreading w/nuclease
  3. Excision repair- correcting DNA damage like the ones caused by chemicals and radiation

Usually fixed by:
1. Recognition of DNA error and removal
2. Replacing the removed DNA by new DNA synthesis using a repair DNA polymerase
3. Sealing new DNA to old w/ligase

51
Q

Excision repair to correct DNA damage

A
  • non bulky damage=no significant distortion of helix
  • identify and removed damaged base w/nuclease
  • replace with normal base complementary to pairing partner on other side of DNA strand
    (similar to mismatch repair)

After proofreading, the base-excision repair is the most important mechanism to fix incorrect or damaged bases

Nucleotide-Excision repair
- portions recognize bulky distortion
- remove segment of DNA strand w/ bulky damage
- repair DNA polymerase + ligase replace removed DNA w/ new DNA and seal it

52
Q

ATP coupling in DNA synthesis

A
  • the energy for DNA replication comes from the hydrolysis of nucleoside triphosphates (dNTPs), where the removal of two phosphate groups (pyrophosphate) releases energy to drive polymerization
  • polymerizationL process of linking monomer
  • Helicase uses this energy for its unwinding job

=== Energy coupling links an exergonic reaction (e.g., ATP hydrolysis) to drive an endergonic process (e.g., DNA synthesis).

BIOL 1101 (18 decks)

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