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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
exonuclease
enzymes that work by cleaving nucleotides out of a nucleotide chain
26
3' to 5' exonuclease activity
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
27
holoenzyme
Enzyme has multiple subunits and is a complex of all of the,
28
DNApol3 core enzyme
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
DNApol3 alpha subunit
Responsible for DNA synthesis along template strands part of core enzyme
30
DNApol3 epsilon subunit
Possesses 3' to 5' exonuclease capability (allows backwards proofreading)
31
DNApol3 sliding clamp loader
attaches to the core enzyme Facilitates the function of the Sliding DNA Clamp
32
DNApol3 sliding DNA clamp
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
Physical mechanism of synthesis in prokaryotes
coordinated synthesis means both strands produced at same time
34
concurrent DNA synthesis
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
5' end problem
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
DNA synthesis only happens in 5' to 3' direction but is done bidirectionally: How?
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
Okazaki fragments
discontinuous segments of DNA made as part of the lagging strand, using "uphill" RNA primers
38
Semiconservative DNA model of replication
One strand is original New strand made off of it 50% old, 50% new for each dsDNA, 1 new strand, 1 old strand
39
Conservative DNA model of replication
1 old helix, 1 new helix
40
Dispersive DNA model of replication
Parental strands are dispersed into two new helices 50% old 50% new, but each strand is a mixture of old and new
41
How do we know about the enzymes implicated in DNA synthesis?
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
DNApol in eukaryotes
alpha, epsilon, delta all differ in processivities
43
DNApol alpha
eukaryotes synthesizes RNA primers low processivity; undergoes polymerase switching after laying down primer
44
DNApol epsilon
synthesizes DNA on the leading strand 3' to 5' exonuclease activity (proofreading) good processivity
45
DNApol delta
synthesizes DNA on the lagging strand 3' to 5' exonuclease activity (proofreading) good processivity
46
Nucleosomes
DNA is wrapped around these, and must be stripped off before DNA synthesis can begin made of eight histone proteins
47
Processivity
ability to stay attached to a template strand
48
Histones
8 of these make up nucleosomes new ones are synthesized as DNA is synthesized
49
CAFs
chromatin assembly factor assemble new nucleosomes behind the replication forks
50
Strand invasion
The 3' G-rich telomere strand end will fold and invade into the doublestranded DNA area, forming a T-loop
51
Telomerase composition
Made of TERC and TERT
52
Shelterin complex
Shelterin proteins form the shelterin complex at looped telomeric ends, helping lock the telomeres into this looped position
53
TERC
RNA template contained within the telomerase, used by TERT to create new telomere
54
TERT
Reverse transcriptase (can make DNA from RNA) in the telomerase uses TERC to make new telomere
55
Meselson-Stahl setup
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
Meselson-Stahl results
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
Meselson-Stahl conclusion
Proved the semi-conservative model in bacteria.
58
Taylor-Wood-Hughes setup
59
Taylor-Wood-Hughes results
60
Taylor-Wood-Hughes conclusion
DNA replication in eukaryotes is semiconservative
61
Polymerase switching
Once DNApol alpha has laid down primer, it dissociates from the template and is replaced by delta or epsilon
62
DSBs
double-stranded breaks recognized by the cell and rejoined
63
G-rich and C-rich telomere strands
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
T-Loop
A telomeric loop. This hides the ends, preventing ends from getting recognized as DSBs.
65
Telomerase operation
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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