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S25 Molecular Biology > L6: Replication of DNA > Flashcards

L6: Replication of DNA Flashcards

1
Q

what are the substrates for DNA synthesis?

A

1) primer-template junction
2) dNTPs

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2
Q

substrates for DNA synthesis - primer-template junction

A
  • provides a 3’-OH group to facilitate nucleotide addition
  • needed bc enzymes which catalyze DNA synthesis cannot initiate strand synthesis de novo (on their own)
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3
Q

substrates for DNA synthesis - dNTP

A
  • cleavage of phosphate groups provides energy for DNA catalysis
  • has a sugar, three phosphates (for energy), and a nitrogenous base
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4
Q

what are DNA polymerases?

A
  • class of enzymes which catalyze the synthesis of DNA
  • require a template and primer (3’-OH)
  • synthesis DNA in the 5’-to-3’ direction and in a processive manner
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5
Q

DNA polymerases - active site

A
  • facilitates the addition of a correct nucleotide
  • specific for correctly base-paired dNTP
  • catalysis is far less efficient for incorrectly base-paired dNTPs or rNTPs
  • resembles a hand that grips primer-template junction
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6
Q

DNA Polymerases - Active Site Specificity

A

DNA polymerases ensures active site specificity when incorporating nucleotides during DNA replication

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7
Q

DNA Polymerases: Active Site Specificity - correct dNTP

A
  • it forms the proper base pair with the template strand
  • 3’ OH group of the primer is positioned correctly
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8
Q

DNA Polymerases: Active Site Specificity - incorrect dNTP

A
  • it does not form a proper base pair with the template strand
  • This mismatch prevents proper alignment in the active site.
  • polymerase cannot catalyze the reaction, blocking the addition of the nucleotide
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9
Q

DNA Polymerases: Active Site Specificity - rNTP

A
  • a ribonucleotide present instead of a deoxyribonucleotide
  • 2’-OH group preventing proper positioning with the active site
  • steric gate prevents rNTPs from being incorporated into DNA
  • ensures that only dNTPs (not rNTPs) are used for DNA synthesis.
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10
Q

DNA Polymerases: Active Site - resembles a hand

A
  • fingers enclose DNA when correct base-pair is in place
  • thumb interacts with negative charges and structure of phosphate backbone
  • palm forms hydrogen bonds with minor groove
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11
Q

DNA polymerases - what is processivity

A

number of monomers (nucleotides) added by an enzyme to a growing polymer (nucleic acid) each time it binds

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12
Q

DNA polymerases: processivity - why is it important

A
  • Every time the DNA pol get on DNA, synthesizes millions of nucleotides before falling off
  • Important bc binding is slow and synthesis is fast so DNA pol only wants to do the binding step once
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13
Q

DNA polymerases - high vs low processivity

A
  • high: makes millions
  • low: jump on, make one/two, then fall off
  • increased processivity correlates with faster DNA synthesis
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14
Q

DNA pol - proof-reading capacity

A

many have 3’ exonuclease activity to remove incorrectly incorporated base pairs at end of strand

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15
Q

DNA pol: proof-reading ability - exonuclease vs endonuclease

A
  • endonucleases – cut within DNA strands,
  • exocnulease - cuts nucleotides from the end of a DNA strand
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16
Q

DNA pol: proof-reading capacity - how is it done?

A
  1. error detection
  2. removal of mismatched nucleotide
  3. resumption of DNA synthesis
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17
Q

DNA pol: proof-reading ability - error detection

A
  • DNA pol detects a mismatched base at the 3’ end of the growing strand (near the thumb)
  • Instead of continuing synthesis, the DNA pol shifts the strand to the exonuclease active site (in the palm)
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18
Q

DNA pol: proof-reading ability - removal of mismatched nucleotide

A

3’ to 5’ exonuclease activity removes the incorrect nucleotide from the strand (in the palm)

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19
Q

DNA pol: proof-reading ability - resumption of DNA synthesis

A
  • DNA pol moves the strand back to its polymerization active site
  • DNA synthesis resumes
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20
Q

what is the Replication Fork

A
  • junction between separated DNA template strands
  • forms because both DNA strands are replicated simultaneously
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21
Q

replication fork - what problem does this create?

A
  • DNA is always synthesized 5’-to-3’
  • causes one strand to be synthesized in fragments
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22
Q

replication fork - leading vs lagging strand

A
  • Leading DNA strand is synthesized continuously
  • Lagging DNA strand is synthesized in a discontinuous fashion and leads to the formation of Okazaki fragments
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23
Q

what are the proteins at the replication fork?

A
  • primase
  • helicase
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24
Q

replication fork proteins - primase

A
  • an RNA Pol that can initiate polymerization de novo (on its own)
  • forms short RNA primers (3’ -OH group) to “prime” DNA Pol activity
  • primers must be removed to complete DNA synthesis
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25
replication fork proteins: primase - lagging vs leading strand
- leading DNA strand: requires only one primer - lagging DNA strand: requires multiple primers (one for each Okazaki fragment)
26
replication fork proteins: primase - primer removal
1. Rnas H 2. DNA Pol fills in the spaces 3. DNA ligase repairs final nick to make the strand continuous
27
replication fork proteins: primase - Rnas H
- cleaves bonds between two ribonucleotides - therefore removes all rNTPs except last one bound to DNA, which is removed by a 5' **exonuclease**
28
replication fork proteins - helicase
- its a ring-shaped hex dimer - uses ATP to catalyze the separation of DNA strands
29
replication fork proteins: helicase - what problem does it create?
- single stands can perform intramolecular base pairing - prevents DNA Pol from synthesizing DNA
30
helicase problem - what is the solution?
- single-stranded DNA-binding proteins (SSBs) - cooperate bind and stabilize the separated DNA strand - ssDNA is held in an elongated state suitable for replication
31
What problem does unwinding of the DNA strands create?
- positive supercoiling is created upstream of the replication fork - prevents DNA from unwinding further
32
positive supercoiling during DNA separation - how is this solved?
- topoisomerase - relieves positive supercoiling of dsDNA upstream of the replication fork - it cleaves both DNA strands, moving them around, and reseals them to create negative supercoiling upstream
33
topoisomerase: positive supercoiling -> negative supercoiling - why is it important for this to happen
- positive supercoiling prevents replication - negative supercoiling promotes replication bc it can be used as energy
34
what are the main DNA Pol in *E. coli*?
- 5 DNA Pol: 1. DNA Pol III 2. DNA Pol I 3. 3 other DNA Pol involved in DNA repair
35
what are the main DNA Pol in *E. coli*? - DNA Pol III
- primary enzyme in chromosome replication - processive and has proof-reading capacity
36
what are the main DNA Pol in *E. coli*? - DNA Pol I
- has 5’ exonuclease activity to remove RNA-DNA linkage resistant to RNase H cleavage - synthesizes DNA in its place - not highly processive, but has proof-reading capacity
37
what are the main DNA Pol in eukaryotes?
- 3 essential to replicate the genome: 1. DNA Pol α (alpha)/primase 2. DNA Pol ε (epsilon) 3. DNA Pol δ (delta) 4. other DNA pol: DNA repair
38
DNA Pol in eukaryotes - DNA Pol α (alpha)/primase
- primase synthesizes RNA primer - DNA Pol α initiates DNA synthesis - has low processivity, so is replaced by other DNA Pol
39
DNA Pol in eukaryotes - DNA Pol ε (epsilon)
synthesizes leading strand
40
DNA Pol in eukaryotes - DNA Pol δ (delta)
synthesizes lagging strand
41
what is the sliding clamp?
- doughnut-shaped complex that binds to DNA Pol and increases processivity - it encircles DNA but leaves space for water molecules to facilitate smooth sliding - placed onto DNA via sliding clamp ladder
42
sliding clamp - why is it important?
- w/o clamp: DNA Pol dissociates after 20-100 bp synthesized - w/ clamp: DNA Pol dissociates after thousands-millions bp synthesized
43
what does DNA pol and the sliding clamp do upon reaching dsDNA
- DNA Pol undergoes a confirmational change that reduces affinity for the clamp - the sliding clamp is left behind to recruit proteins (like those for Okazaki fragment repair)
44
DNA synthesis at the replication fork
- holoenzyme - trombone model in *E. coli*
45
DNA synthesis at the replication fork - holoenzyme
a multiprotein complex in which core enzyme activity is associated with additional components that enhance function
46
holoenzyme - DNA Pol III holoenzyme
- a holoenzyme that has these components: 1. three DNA Pol III enzymes 2. one sliding clamp loader with three τ (tau) proteins
47
DNA synthesis at replication fork - trombone model in *E. coli*
- **helicase** is linked to the lagging strand and separates DNA - one ***DNA Pol III*** works continuously on leading strand - lagging strand is "spooled out" before **Primase** synthesizes the primer - **sliding clamp** is loaded onto lagging strand and DNA Pol III unit begins - **DNA Pol III** reaches preceding Okazaki fragment, dissociates (leaving sliding clamp) and is recruited to next lagging strand primer
48
initiation of DNA replication
- origin of replication - replicator - initiator
49
initiation of DNA replication - origin of replication
physical site at which DNA unwinding and replication is initiated
50
initiation of DNA replication - replicator
- *cis-*acting DNA sequences that directs replication initiation - usually AT-rich and therefore unwinds readily
51
initiation of DNA replication - initiator
- *trans-*acting protein that binds to replicator and initiates replication - humans: ORC, *E. coli*: DnaA
52
initiation of DNA replication - difference between *cis-*acting and *trans-*acting
- *cis-*acting: bound to the DNA - *trans-*acting: can move around and binds to the *cis-*acting protein
53
initiation of DNA replication in *E. coli*
1. DnaA binds 2. DnaC places DnaB on ssDNA 3. primase is recruited 4. replication bubble formed (only one)
54
initiation of DNA replication in *E. coli* - DnaA binds
- DnaA initiator protein directly binds 9-mer replicator sequences - when associated with ATP, DnaA induces strand separation at 13-mers
55
initiation of DNA replication in *E. coli* - DnaC places DnaB on ssDNA
- DnaC: DNA helicase loader - DnaB: helicase
56
initiation of DNA replication in *E. coli* - primase is recruited
primase is recruited followed by the assembly of DNA Pol III holoenzyme
57
initiation of DNA replication in *E. coli* - forms replication bubble
origin of replication is in the middle of the bubble
58
initiation of DNA replication in *E. coli* - in rapidly growing cells
- origin of replication reinitiate DNA replication before cell division - results in some DNA being replicated twice - allows fast division but means multiple rounds of replication are occurring simultaneously - typically good for prokaryotes
59
initiation of eukaryotic DNA replication
- eukaryotic chromosomes replicate only once per cell cycle - it is unable to reinitiate DNA replication before the cell divides
60
initiation of eukaryotic DNA replication - explain how DNA is unable to reinitiate DNA
helicase activity is tightly controlled: - helicase loading occurs at G1 phase of cell cycle - loaded helicase are only activated by two protein kinases - these kinases are active at S phase of cell cycle
61
initiation of eukaryotic DNA replication - timing of helicase loading and activation
- allows only one round of DNA replication per cell division - helicase loading is regulated by CDK (protein kinase) levels
62
initiation of eukaryotic DNA replication: timing of helicase loading and activation - CDK (protein kinase levels)
- decrease during G1, helicase is loaded but not activated - increase during S-G2-M, inhibits helicase loaded and activates previously loaded helicase
63
end replication problem - telomeres
- region at the end of a eukaryotic chromosome - does not contain genes - instead contains short DNA repeats
64
end replication problem - replication of telomeres
- leading strand synthesis: continuous and complete - lagging strand: discontinuous and shortened
65
end replication problem: replication of telomeres - why is this a problem?
- results in telomere shortening - DNA gets shorter with every cell cycle - it is fine if its just the telomeres but can effect DNA and genes
66
end replication problem - telomere shortening
- the replication fork will reach the end of the linear chromosome - but the last RNA primer on the lagging strand has been removed - the primase needs something to sit on but the DNA is too small so it cannot be primed or replicated - ssDNA will be degraded or lost
67
solution to end replication problem of lagging strand
telomerase
68
solution to end replication problem of lagging strand - telomerase
- a novel DNA Pol that does not require an exogenous template - it is a **ribonucleoprotein** with an RNA component (telomerase RNA or TER) that can act as a template - it will then exploit head-to-tail repeats of TG-rich sequences at telomeric ends of linear DNA - has a reverse transcriptase subunit (TERT) that synthesizes DNA
69
telomerase - how does it work?
1. unreplicated strand has 3' overhang 2. telomerase binds to overhand and synthesizes DNA 3. telomerase catalyzes repeated additions of the same shirt sequence to terminus 4. DNA Pol can then replicate DNA and complete the lagging strand
70
telomerase - how does it relate to aging and gametes
- not all cells have telomerase and DNA gets shorter as we age - gametes have high concentrations of telomerase (don't want to pass shortened DNA to progeny)

S25 Molecular Biology (23 decks)

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