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MBG > DNA replication > Flashcards

DNA replication Flashcards

1
Q

three phases of replication

A

initiation, elongation, termination

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2
Q

what is origin of replication

A

where fork is initiated, bound by a trans acting factor, contains signal seqs and stimulates replication

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3
Q

T/F. All organisms have a single replication origin

A

False. Although most bacteria have a single ori, eukaryotes have more and humans have thousands

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4
Q

what are some techniques to look at replication fork structure (3)?

A

2D gels to view topology, EdU labeling/fiber combing, pull out Okazaki fragments with specific antibody

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5
Q

what are some initiation factors (4)

A

helicase, helicase loader, primase, initiators

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6
Q

how are helicase rings activated

A

accessory subunits Cdc45 and GINS activate to begin DNA unwinding

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7
Q

T/F. Helicase is the fastest part of the motor

A

False, synthesis proceeds faster through the combinatorial action of multiple components

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8
Q

How do eukaryotes prevent fork reinitation?

A

DnaA is a product inhibited enzyme - so when it hydrolyzes ATP to ADP, the assembly becomes destabilized and prevents reinitiation

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9
Q

what are two challenges facing the replication fork?

A

topology, chromatin

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10
Q

what are some characteristics of human replication?

A

replication initiates stochasticalyy, initiates within non-transcribed, up to 150kb zones, and terminates dispersively

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11
Q

What does the initiator do, and what is it called in bacteria and eukaryotes?

A

Binds origins/recruits helicase, DnaA in bacteria and ORC in eukaryotes

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12
Q

What does the helicase do, and what is it called in bacteria and eukaryotes?

A

Unwinds DNA, called DnaB in bacteria and CMG (MCM2-7/Cdc45/GINS) in eukaryotes

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13
Q

What does the helicase loader do, and what is it called in bacteria and eukaryotes?

A

Loads Helicase, called DnaC in bacteria and Cdc6/CdtI in euk

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14
Q

What does primase do, and what is it called in bacteria and eukaryotes?

A

Makes RNA primers, DnaG in bacteria and Pol-alpha (Pri1, Pri2, PolAI, PolA2) in euk

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15
Q

T/F. All initiator components of the replication fork use NTPs

A

True

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16
Q

What are the 4 steps of helicase loading in bacteria?

A
  1. DNA initiator binds to DnaA, bends DNA at rep ori
  2. DNA bending allows local unwinding at AT-rich region
  3. DnaC assembles onto DnaB helicase, binds to DnaA
  4. DnaC loads DnaB helicase on, disassembles
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17
Q

What are the 4 steps of helicase loading in eukaryotes?

A
  1. Origin binding (ORC, coinitiates with Cdc6)
  2. Loading (Mcm2-7, Cdt1)
  3. Activation (Cdc45, GINS, DDK kinase)
  4. Unwinding (CMG)
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18
Q

If MCM loading were not limited to G1, what might be a potential problem in S phase?

A

Potentially deleterious re-replication of certain genomic loci

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19
Q

What prevents eukaryotic origins from re-firing?

A

Post translational modifications, proteolysis, nuclear export

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20
Q

Six types of replication elongation proteins

A

helicase, polymerase, clamp, clamploader, primase, SSBs

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21
Q

Helicase in bactera and eukaryotes?

A

DnaB, the “CMG”

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22
Q

Polymerase in bacteria and eukaryotes?

A

Pol III, pol epsilon/pol delta

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23
Q

Clamp in bacteria and eukaryotes?

A

Beta protein, PCNA

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24
Q

Clamp loader in bacteria and eukaryotes?

A

The theta complex, RFC

25
Q

Primase in bacteria and eukaryotes?

A

DnaG, Pol-alpha

26
Q

SSBs in bacteria and eukaryotes?

A

Ssb, RPA

27
Q

T/F. DnaB/CMG encircle and move along both strands

A

False, they move along single DNA strands, in opposite directions

28
Q

What are the members and roles of DNA pol family A?

A

T7 pol, Ecoli pol I, mito pol gamma. Replication, and repair of short segments - the “classic polymerase”

29
Q

What are the members and roles of DNA pol family B?

A

High fidelity replication, 3-5’ exo, members are eukaryotic pols alpha, delta, epsilon, called the “A family catalytic center”

30
Q

What are the members and roles of DNA pol family C?

A

Members E.coli pol III, is a high fidelity, highly processive enzyme class with 3’-5’ exo

31
Q

What are the members and roles of DNA pol family D?

A

PolD, archaeal replication?

32
Q

What are the members and roles of DNA pol family X?

A

Repair pol, low fidelity/processivity, lesion bypass. Members are eukaryotic pols n, i , k and bacterial pols IV, V

33
Q

What are the members and roles of DNA pol family RT?

A

RNA dependent DNA synthesis, viral replication, telomere synthesis - members are the HIV RT and telomerase

34
Q

T/F. DNA pol families A, B, D, Y, and RT are related, but C and X are not.

A

False - A, B, D, Y, and RT are related, and C and X are also related

35
Q

Why do eukaryotes need so many polymerases?

A

Bc there are so many functions they need to perform -priming, making okazaki fragments, repair, mito replication, lagging/leading strand synthesis,

36
Q

What structural feature do pols have to ensure fidelity?

A

“steric gate”, with a snug active site that matches watson/crick pair, disfavors rNTP binding and excludes wobbles/mispairs

37
Q

What two structural features do steric gates of DNA pols contain?

A

A selectivity residue, and a 2’C on ribose

38
Q

T/F. Polymerases read hydrogen bonding to recognize sequence motifs.

A

False, they sense shape complementarity, which is indicative of base-stacking interactions and proper H-bonding, but not H-bonds directly

39
Q

T/F. dNTPs are 50-100X more abundant than rNTPs.

A

False, rNTPs are 50-100X more abundant than dNTPs

40
Q

Why is rNTP misincorporation a problem? (3)

A

U can mispair with G, RNA is more prone to backbone cleavage, and the 2’ OH deforms the DNA helix to alter protein binding

41
Q

Why is rNTP misincorporation a problem? (3)

A

U can mispair with G, RNA is more prone to backbone cleavage, and the 2’ OH deforms the DNA helix to alter protein binding

42
Q

T/F. ATP turnover controls clamp opening and release

A

True, ATP binding leads to conformational change, causing clamp binding/opening, and then ATP hydrolysis causes clamp closure and clamp loader release

43
Q

How do lagging and leading strand polymerases maintain speed with each other in eukaryotes?

A

Random pausing/dissociation events maintain synchrony, it is not directly coupled

44
Q

How are okazaki fragments removed/patched?

A

3 mechanisms:

  1. 3’ - 5’ degradation, nicking and ligase
  2. RNA cleavage with RNaseH, nick and ligase
  3. Flap formation, Dna2/Fen1 cleave, nick and ligase
45
Q

What is the substrate and geometry for RNAse H nuclease action?

A

Substrate - 2 metal ions, strained geometry and incomplete liganding

46
Q

DNA ligase mechanism

A

encircles DNA, ATP hydrolysis to power nick healing

47
Q

how large is the E. coli replisome, and how fast can the fork move?

A

300-400A, and moves at a speed of up to 1kb/s

48
Q

What are three things that can cause forks to terminate prematurely?

A

Protein mediated block, fork collision, and superhelical strain

49
Q

What are catenanes?

A

Interlocking rings of DNA which cannot be separated without breaking covalent bonds, formed during strain of DNA replication and transcription

50
Q

What happens if you don’t resolve catenanes?

A

You get gene misexpression and DNA breakage

51
Q

What is the difference between type I and type II topoisomerases?

A

Type I cuts one strand, and type II cuts both, all form covalent enzyme-DNA intermediates

52
Q

If type IA topos preferentially relax negatively supercoiled DNA, and IIA topos positively supercoiled, where would you expect these two classes to act during transcription

A

The type IA topo would be acting ahead of the RNA polymerase, and IIA would be acting behind.

53
Q

T/F. nucleosomes stabilize positive supercoils

A

False, they stabilize negative supercoils

54
Q

What are 4 types of histone modifications?

A

acetylation, methylation, phosphorylation, and ubiquitylation - make up a “histone code”

55
Q

what are a few things the histone code affects?

A

chromatin accessibility, origin usage, fork speed

56
Q

T/F. The histone code is relatively fixed.

A

False, it is in constant flux

57
Q

How are old and new histones apportioned between daughter nucleosomes

A

Mcm2 has a histon H3-H4 chaperone domain that aids apportionment, preserves epigenetic information between cell divisions, but the segregation mechanism itself is not understood

58
Q

What are 4 characteristics of nucleosomes?

A

Octamer of histones wrapped by DNA, packages DNA in eukaryotes, affects protein recruitment to DNA, and can be post-translationally modified

59
Q

T/F. Initiation, elongation and termination factors are all broadly conserved.

A

False, initiation machineries are broadly conserved, but elongation and termination factors are not

MBG (13 decks)

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