Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

Genetics > Ch.9 DNA Replication > Flashcards

Ch.9 DNA Replication Flashcards

You may prefer our related Brainscape-certified flashcards:
Biology 101 flashcards
1
Q

S phase of interphase

A

all DNA is copied, and the chromosomes consist now of sister chromatids with identical DNA molecules.
The cell is now ready for division.
Lined up, the DNA from all your cells would reach from Earth to the sun 100 times.
How can a large molecule like DNA make identical copies of itself with relatively few errors?

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

Basic features of DNA replication in vivo (living cells)

A

DNA replication occurs semiconservativly.
Is initiate at unique origins.
Usually proceeds bidirectionally from each origin of replication.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

DNA replication is semiconservative

A

one strand serves as template for the new strand. The new double strand will be composed of old and new strands.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

The Meselson-Stahl Experiment 1958

A
  1. Bacteria cultures in medium with heavy isotope.
  2. Bacteria transferred to medium with lighter isotope.
  3. DNA centrifuged after 1st replication
  4. DNA centrifuged after 2nd replication
    Semiconservative model first replication- heavy and light found in middle
    -2nd replication, light top, heavy light in middle
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

Semiconservative

A

Each single strand serves as a template.
Complementary base pairing determines the sequence of the new strand.
Each stand of the parental helix is conserved, the new molecule is a mixture of old and new.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

Semiconservative replication in eukaryotes (1957)

A

Autoradiography: method for detecting and localizing radioactive isotopes in cytological preparation or macromolecules by exposure to a photographic emulsion sensitive to low-energy radiation.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

Autoradiography

A
  1. duplication with labeled thymidine (incorporated into chromosomes
  2. Autoradiography
  3. Duplication without labeled thymidine.
  4. Autoradiography
    Results in 1 chromatid radioactive and other not.
    (consist of parental strand that was radioactive and new strand that’s not radioactive)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

DNA replication begins at the

A

ORI (origin of replication)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

At site of replication, helix is unwound creating

A

replication fork

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

DNA replication is? (direction)

A

Bidirectional - there are 2 replication forks moving away from each other.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

Origin of replication in bacteria

A

Replication starts at the ori; in E. coli this is called oriC.
Sites of replication are called the replication forks and proceed away from the ori in both directions.
Plasmids will also have ori sites, so that they are replicated by the same mechanism.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

Origin of replication in E. coli

A

Ori are A:T rich regions of DNA.

A:T are only held together by 2 H bonds as opposed to G:C with 3 H bonds. - easy separation of strands.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

Bidirectional replication of the circular E. coli chromosome

A

Replication starts at ori, and the replication forks move away from each other in both direction.
DNA replication is bidirectional.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

DNA replication in Eukaryotes

A

In a linear chromosome, replication bubbles form at many ori sites along the giant DNA molecule.
The bubbles expand as replication moves in both directions.
Eventually the bubbles will fuse and replication of the strands is complete.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

Mechanism of DNA replication in prokaryotes

A

DNA replication is complex process, requiring the concerted action of a large number of enzymes and other proteins.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

Prepriming

A

(1st step in DNA replication)
The replication bubble is a localized region of strand separation.
The bubble is formed through interaction between prepriming proteins with ori.
DnaA protein forms a complex, other proteins join.
DnaB protein (helicase), DnaC protein, DnaJ, DnaK, PriA, PriB, PriC, DNA-binding protein HU, DNA Gyrase, SSB proteins (single strand DNA binding proteins)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

Prepriming steps

A
  1. DnaA protein binds to the four 9 bp repeats in oriC.
  2. Additional molecules of DnaA protien bind cooperatively, forming a complex with oriC wrapped on the surface.
  3. DnaB protein (DNA helicase) and DnaC protein join the initiation complex and produce a replication bubble.
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

Separating the strands

A

Unwinding of DNA starts at the ori
Catalyzed by an enzyme: DNA helicase (DnaB protein).
Problem: what do single stranded molecules of DNA want to do? (close)
This is an endergonic rxn - requires energy in the form of ATP -> ADP (breaking H bonds)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

Preventing the strands from closing again

A

Single Stranded binding protein (SSB) bind the single stranded DNA to keep is separated.
New problem: as the DNA helix unwinds, the DNA in front of the DNA helicase will supercoil.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

The twisting problem

A

Coils in front will become so tight that the 2 strands of DNA will not longer be able to be untangled.
All strands will remain twisted and intertwined, making physical separation impossible.
(untwisting but someone else is holding it so it supercoils).

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

Topoisomerases: solving the twisting problem

A

Enzymes which catalyze transient breaks (temporary) in the DNA molecule.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

DNA topoisomerase 1

A

temporary single strand breaks (nicks)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

DNA topoisomerase 2

A

temporary double strand breaks

Gyrase; member of the topoisomerase 2 family in E. coli

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
24
Q

DNA topoisomerase 1 - single strand break

A

The two ends of the double helix rotate around each other.

Energy of breaking the bond was conserved and is used to seal the nick again.

25
Q

DNA topoisomerase 1 steps

A
  1. One end of the DNA double helix cant rotate relative to the other end.
  2. DNA top 1 covalently attaches to a DNA phosphate thereby breaking a phosphodiester linkage in one DNA strand.
    The original phosphodiester bond energy is stored in a phosphotyrosine linkage making the reaction reversible.
    The two ends of the double helix can now rotate relative to each other.
  3. Re-formation of the phosphodiester bond regenerates both the DNA helix and the DNA top in an unchanged form.
26
Q

DNA topoisomerase 2 (gyrase) produces double stranded breaks in DNA in E. coli

A
  1. DNA molecule with no supercoils
  2. DNA gyrase folds the molecule across itself twice.
  3. Gyrase cleaves both strands, passes the intact helix through the break, and reseals the break.
  4. DNA molecule with two negative supercoils.
27
Q

DNA polymerases and DNA synthesis In Vitro (outside living cells)

A

Much of what we know about DNA synthesis was deduced from in vitro studies.
Molecules which achieve actual synthesis of DNA are DNA polymerases.

28
Q

Chaining nucleotides together 5’ to 3’ direction

A
  1. Free nucleotides (dNTPs - diphosphateenucleotises) attach and form hydrogen bonds with the nucleotides of the parent strand (complementary base pairing; assures accuracy and complementary of the new strand to the parent strand)
  2. Forming a covalent bond between O and P is catalyzed by an enzyme (DNA polymerases). 5’ end attaches to 3’ end
  3. The enzyme can only add nucleotides onto the free 3’ end.
29
Q

Discovery of Polymerase

A

Hypothesized that one could replicate DNA in vitro

30
Q

DNA polymerase requires

A

a free 3’ end

can only replicate if its there

31
Q

DNA polymerase

A

The enzyme which adds nucleotides to the template strand.
Requires a free 3’ end (provided by the RNA primer)
Synthesis of the new DNA strand 5’ -> 3’

32
Q

RNA primers initiate DNA sythesis

A

All known DNA polymerases have an absolute requirement for a free 3’.
Each new DNA strand is inititated by the enyme DNA primase: synthesizes a short RNA primer (a short RNA strand complementary to the DNA template strand).
The RNA primer provides the required 3’ - OH end

33
Q

Continuous DNA synthesis on the Leading strand

A

One end replication can start right away bc free 3’ end.
RNA primer providing DNA polymerase with free 3’.
Continuous elongation in the 5’ to 3’ direction.

34
Q

Discontinuous DNA synthesis on the Lagging strand

A

DNA polymerase can only synthesize in the 5’ to 3’ direction.
But what happens on the other strand? (running the wrong way)
DNA synthesis is discontinuous (in pieces)

35
Q

Semi discontinuous replication

A

Reiji Okasaki discovered that the lagging strand is synthesized in small fragments.
Okasaki fragments (1000-2000 bp)
Each fragment requires a new primer.
This reaction is catalyzed by primase.

36
Q

Discontinuous DNA synthesis on the Lagging strand steps

A
  1. Primase makes RNA primer.
  2. DNA pol 2 makes okazaki fragment 1
  3. DNA pol 3 detaches
  4. DNA pol 3 makes okazaki fragment 2
  5. DNA pol 1 replaces RNA with DNA
  6. DNA ligase forms bonds between DNA fragments.
37
Q

Covalent closure of nicks by DNA ligase

A

Nick: single strand break; no missing bases, but missing phosphodiester linkage.
DNA ligase is the enzyme which catalyzes the closure of the nicks.

38
Q

Different DNA polymerases

A

DNA polymerase are processive enzymes which catalyze the covalent extension at the 3’ end. (synthesize polymers from monomers)
All DNA polymerase require:
-free 3’ end
-template DNA: provides the nucleotide sequence that specifies the complementary sequences of the growing DNA chain.

39
Q

E. coli: at least 5 diff DNA polymerases

A

DNA polymerases 1 and 2: repair function (excision and closure)
DNA polymerase 3 (DNA pol 3): main enzyme of DNA replication
DNA polymerase 4 and 5: repair enzymes

40
Q

DNA polymerase 1: 5’ -3’ Exonuclease Activity

A

Exonuclease: cutting out nucleotides

DNA pol 1: Cuts out the RNA primers.

41
Q

DNA polymerase 1: 3’ -5’ Exonuclease Activity

A

Repair excision for incorrectly paired nucleotides

42
Q

DNA polymerase 1: 5’ -3’ Polymerase Activity

A

Closes the gap after the primer has been excised

43
Q

DNA polymerase 3 is the true DNA replicase of E.coli

A

E. coli DNA polymerase 3 Holoenzyme contains a total of 16 polypeptides

44
Q

The Replication apparatus in E. coli

Primosome

A

Protein complex containing DNA primase and DNA helicase; initiates okazaki fragments on the lagging strand

45
Q

The Replication apparatus in E. coli

Replisome

A

The complete replication apparatus moving along the DNA molecule at the replication fork (outdated)

46
Q

The E.coli replisome updated

A

Replisome is stationary (car wash), DNA is pulled through the replisome (car)

47
Q

Polymerase in Eukaryotes

A

At least 15 diff DNA polymerase have been identified so far.
2 or more for replication.
Replication of DNA in mitochondria: y
The others are repair enzymes.

48
Q

Accuracy of DNA polymerase

A

When processin 1000 bp/sec (DNA pol 1), mistakes are bound to happen. Usually they occur about 1/1000 bases.
Deformation of the double helix, leading potentially to mutations.

49
Q

DNA replication in Eukaryotes

A

shorter RNA primers and okazaki fragments.
DNA replication only during S phase of the cell cycle.
Multiple origins of replication.
Nucleosomes in Eukaryotes.
Telomeres in Eukaryotes

50
Q

The Eukaryotic Replisome

A

similar to bacteria.

Replication happens simultaneously on leading and lagging strand

51
Q

Eukaryotic replication proteins

A

DNA polymerase alpha- DNA primase - initiation; priming of okazaki fragments.
DNA polymerase J - processive DNA synthesis.
DNA polymerase E - DNA replication and repair in vivo.
PCNA (proliferating cell nuclear antigen) - sliding clamp.
Replication factor - C Rf-C - loading of PCNA
Ribonuclease H1 and Ribonuclease FEN-1 - removal of RNA primers.

52
Q

Nucleosomes during chromosome replication

A

Chromatin assembly factor 1 - enzyme that helps to assemble histones and nonhistones into nucleosomes.

53
Q

Telomere Problem

Lagging strand primer problem

A

The missing piece cannot be replaced (bc there is no free 3’ end).
With every replication cycle, the chromosome arms get short and shorter.

54
Q

Telomerase

A

Discovered 1958
An enzyme containing RNA which forestalls the shortening of the telomeres.
In most somatic cells telomerase genes are not expressed (we age bc our chromosomes are shortening, which will cause cell division eventually to stop).
Human cells grown in culture stop dividing after 20-70 generations.
In stem-cell and malignant cancer cells telomerase is active: telomerase inhibitors as possible treatment?

55
Q

DNA cloning: making multiple copies of a gene or other DNA segment

A

Challenge: you want to study a particular gene, for example the gene underlying hGH
DNA molecules are very long.
A single DNA molecule can carry several genes.
Genes occupy only a small portion of the chromosomal DNA (junk DNA not transcribed).
Difference between a gene and the flanking DNA sequences are subtle.
How can you pick out a single gene?

56
Q

Polymerase Chain Reaction

A

A way of replicating targeted sections of DNA.
Amplification of specific genes or other parts of DNA.
1993 Mullis and Smith Nobelprize.
If a certain gene is of interest, it is ideal to isolate it from other DNA in the cell by duplicating so many copies of it that the rest of the DNA becomes neglectable small compared to it.
In vitro DNA replication of a target piece of DNA.

57
Q

PCR components

A

Template DNA
Primer matching to the target piece of DNA (complementary to desired piece).
To synthesize new DNA: Free nucleotides (dNTPs): Excess amounts of Adenine, Guanine, Cytosine, Thymine are added.
Enzyme that catalyzes the rxn: Taq polymerase - works optimally at a much higher temperature than most other polymerases.

58
Q

PCR steps

A
  1. Denaturing
  2. Annealing
  3. Extension (Taq polymerase)
    Usually run between 30 and 40 cycles.
    So many copies of your desired DNA that the rest of the DNA is neglectably small.

Genetics (15 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions