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Biol251 > DNArep > Flashcards

DNArep Flashcards

1
Q

origins of replication

A

Location where replication begins

1 in prokaryotes
many in eukaryotes

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2
Q

oriC

A

prokaryotic origin of replication

consist of repeating AT-rich sequences (9mers and 13mers)

9mers & 13mers less stable, so easy to break apart, enabling unwinding

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3
Q

Bidirectional

A

Replication occurs in both directions away from the origin

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4
Q

ter

A

The termination sequence for prokaryotes undergoing DNA synthesis

forks meet here and replicon gets popped out

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5
Q

Replicon

A
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6
Q

replisome

A

The DNA, the DNApol, and the enzymes involved in preparing DNA

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7
Q

Eukaryotic DNA prep enzymes

A

Helicase
SSBPs
Topoisomerase

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8
Q

Prokaryotic DNA prep enzymes

A

DNA helicase
SSBP
DNA gyrase

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9
Q

Prokaryotic unwinding process

A

protein DnaA binds to a region of 9mers.

The 9mer-DnaA complex associates with a region of 13mers.

This puts strain on the DNA, causing the helix to destabilize.

This exposes an area of ssDNA.

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10
Q

DnaA

A

protein that binds to a region of 9mers to begin unwinding

9mer-DnaA complex associates with 13mers, causing helix to destabilize

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11
Q

9mers and 13mers

A

repeating sequences of base pairs

AT-rich, less stable

9mer binds to DnaA and forms a complex to put strain on DNA and begin unwinding

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12
Q

DNA Helicase

A

made of several DnaB subunits

Recruits the holoenzyme DNApol3 to bind to replication fork

Moves along ssDNA, unzipping helix

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13
Q

DNA gyrase

A

Toposiomerase in prokaryotes

Makes “cuts” along ssDNA and dsDNA to relieve supercoiling

Reseals the cuts in non-coiled conformation

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14
Q

SSBP

A

bind to ssDNA to prevent its reassociation

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15
Q

Primase

A

creates short RNA sequences along the template strand

This allows DNApol to do DNA synthesis, as it gives them a starting point to work from

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16
Q

DNApol synthesis initiation issue

A

DNApol can’t initiate DNA synthesis

It can only bond phosphate groups to hydroxyl groups

So it must have a 3’ end to work off of

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17
Q

RNA primer

A

short RNA sequences along the template strand added by primase

serve as a base for DNApol to begin adding nucleotides to new daughter strand

runs antiparallel to template strand

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18
Q

Ligase

A

After RNA primer has been replaced by DNA by DNApol, ligase rejoins the replacement DNA with the other nucleotides

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19
Q

Basic DNA synthesis process

A

Primase adds primer

DNApol uses primer as a base to begin synthesizing off the primer’s 3’ end

Another DNApol removes the primer and replaces it with DNA nucleotides

ligase rejoins replacements with existing bases

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20
Q

Chain elongation direction

A

5’ to 3’ (concerned with daughter)

DNApol wants to attach phosphate groups to a hydroxyl group

The hydroxyl group of the primer is at its 3’ end

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21
Q

Prokaryote DNApol enzymes

A

DNApol1 (directs & repairs, removes & replaces primer)

DNApol3 (synthesizes new DNA segments)

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22
Q

DNApol1

A

directs DNA repair and synthesis

require the presence of all four dNTPs, and template DNA

Unlike other DNApols, it has a 5’ to 3’ exonuclease activity: removes the RNA primers and fills the gaps after primer removal

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23
Q

DNApol3

A

synthesizes new DNA segments off of the primer

holoenzyme (core enzyme, clamp loader, sliding clamp)

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24
Q

5’ to 3’ exonuclease activity

A

unique to DNApol1

Ability to remove RNA primers while going forward

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25
Q

exonuclease

A

enzymes that work by cleaving nucleotides out of a nucleotide chain

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26
Q

3’ to 5’ exonuclease activity

A

a “backwards” function of all DNApol

the ability to polymerize in one direction, pause, reverse direction, and cut out nucleotides just added

activates when an incorrect nucleotide is inserted, allowing repair

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27
Q

holoenzyme

A

Enzyme has multiple subunits and is a complex of all of the,

28
Q

DNApol3 core enzyme

A

The largest subunit type

can be multiple in a holoenzyme

Necessary for catalytic activity (the reaction the holoenzyme exists to catalyze)

consists of alpha, epsilon, and theta

29
Q

DNApol3 alpha subunit

A

Responsible for DNA synthesis along template strands

part of core enzyme

30
Q

DNApol3 epsilon subunit

A

Possesses 3’ to 5’ exonuclease capability (allows backwards proofreading)

31
Q

DNApol3 sliding clamp loader

A

attaches to the core enzyme
Facilitates the function of the Sliding DNA Clamp

32
Q

DNApol3 sliding DNA clamp

A

Many of its subunits have a donut shape; these can open and shut to encircle the DNA strand

Opening and closing depends on phosphorylation

Leads the way during synthesis; maintains binding of core enzyme to the template

This increases processivity

33
Q

Physical mechanism of synthesis in prokaryotes

A

coordinated synthesis means both strands produced at same time

34
Q

concurrent DNA synthesis

A

both the lagging and leading strand are produced simultaneously by the same holoenzyme

lagging strand template is spooled out to form a loop

allows the physical direction of replication to change

Biochemically the direction of DNA synthesis is the same.

35
Q

5’ end problem

A

IN EUKARYOTES ONLY (LINEAR CHROMOSOMES)

RNA primer on lagging strand will terminate the daughter 5’ end.

The gap left when the primer is removed cannot be filled; the pairs it represents on the template strand are lost.

the daughter strand loses some DNA with every cycle of replication

36
Q

DNA synthesis only happens in 5’ to 3’ direction but is done bidirectionally: How?

A

RNA primer is added in upward increments towards the 5’ end of the daughter strand, and the 3’ template strand. This lets DNA get synthesized “uphill”.

37
Q

Okazaki fragments

A

discontinuous segments of DNA made as part of the lagging strand, using “uphill” RNA primers

38
Q

Semiconservative DNA model of replication

A

One strand is original
New strand made off of it
50% old, 50% new

for each dsDNA, 1 new strand, 1 old strand

39
Q

Conservative DNA model of replication

A

1 old helix, 1 new helix

40
Q

Dispersive DNA model of replication

A

Parental strands are dispersed into two new helices
50% old 50% new, but each strand is a mixture of old and new

41
Q

How do we know about the enzymes implicated in DNA synthesis?

A

Condition mutations (like ts mutations) are used to observe the loss of function associated with mutations to these enzymes

Then their role in DNA synthesis can be analyzed

42
Q

DNApol in eukaryotes

A

alpha, epsilon, delta

all differ in processivities

43
Q

DNApol alpha

A

eukaryotes

synthesizes RNA primers

low processivity; undergoes polymerase switching after laying down primer

44
Q

DNApol epsilon

A

synthesizes DNA on the leading strand

3’ to 5’ exonuclease activity (proofreading)

good processivity

45
Q

DNApol delta

A

synthesizes DNA on the lagging strand

3’ to 5’ exonuclease activity (proofreading)

good processivity

46
Q

Nucleosomes

A

DNA is wrapped around these, and must be stripped off before DNA synthesis can begin

made of eight histone proteins

47
Q

Processivity

A

ability to stay attached to a template strand

48
Q

Histones

A

8 of these make up nucleosomes

new ones are synthesized as DNA is synthesized

49
Q

CAFs

A

chromatin assembly factor

assemble new nucleosomes behind the replication forks

50
Q

Strand invasion

A

The 3’ G-rich telomere strand end will fold and invade into the doublestranded DNA area, forming a T-loop

51
Q

Telomerase composition

A

Made of TERC and TERT

52
Q

Shelterin complex

A

Shelterin proteins form the shelterin complex at looped telomeric ends, helping lock the telomeres into this looped position

53
Q

TERC

A

RNA template contained within the telomerase, used by TERT to create new telomere

54
Q

TERT

A

Reverse transcriptase (can make DNA from RNA) in the telomerase

uses TERC to make new telomere

55
Q

Meselson-Stahl setup

A

Grew E. coli in a medium that had N15 as the only source of nitrogen.

After many generations in the N15 medium, the E. coli were immersed in N14.

The E. coli DNA were put into a centrifuge containing CsCl

CsCl creates a density gradient

different “densities” of DNA (N15 and N14) settled along the gradient where their density was equivalent to the medium

56
Q

Meselson-Stahl results

A

N15 would be closer to the bottom of the tube than N14

Only 1 band of mixed density, disproving conservative model

when strands were denatured and evaluated for density, there were two bands, showing they did not have a dispersive composition

57
Q

Meselson-Stahl conclusion

A

Proved the semi-conservative model in bacteria.

58
Q

Taylor-Wood-Hughes setup

A
59
Q

Taylor-Wood-Hughes results

A
60
Q

Taylor-Wood-Hughes conclusion

A

DNA replication in eukaryotes is semiconservative

61
Q

Polymerase switching

A

Once DNApol alpha has laid down primer, it dissociates from the template and is replaced by delta or epsilon

62
Q

DSBs

A

double-stranded breaks

recognized by the cell and rejoined

63
Q

G-rich and C-rich telomere strands

A

Telomeres in general are G-rich / C-rich (depends on the strand)

G-rich is 3’
C-rich is 5’

G-rich overhangs the C-rich due to 5’ deletions

64
Q

T-Loop

A

A telomeric loop. This hides the ends, preventing ends from getting recognized as DSBs.

65
Q

Telomerase operation

A

extends the G-rich strand (the 3’ overhang)

normal DNApols add RNA primer at the sister strand’s new end and fill the gap normally

primer is removed

the ends have been lengthened

Biol251 (8 decks)

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