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Biochem > DNA Replication > Flashcards

DNA Replication Flashcards

1
Q

G1 phase

A
  • period of cell growth
  • many structural components and metabolic enzymes are synthesized here
  • towards the end DNA polymerases and other replication enzymes are synthesized
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2
Q

G2 phase

A
  • checkpoint that checks to make sure cells are ready to divide
  • prevents cells from entering mitosis with DNA damages since last division, providing opportunity for DNA repair and stopping proliferation of damaged cells
  • -some synthesis of cell materials occurs here, (tubulin monomers for microtubule assembly)
  • synthesis of wall & membrane components
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3
Q

general characteristics of DNA replication

A
  • chemically unidirectional (5’ -> 3’)
  • spatially bidirectional (2 forks at once in opposite directions)
  • semi-conservative
  • semi discontinuous
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4
Q

semi-discontinuous

A

DNA polymerase can’t stay on DNA strand the whole time without falling off and new polymerase coming on

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5
Q

origin of replication

A

the region of DNA that first separates and replication begins

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6
Q

bi-directional replication

A

DNA replication proceeds in both directions away from origin of replication

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7
Q

replication fork

A

point of separation of double-stranded DNA at which incorporation of nucleotides occurs during DNA replication

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8
Q

semi-conservative replication

A

where each separated polynucleotide strand serves as a template for the synthesis of a single new complementary strand

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9
Q

okazaki fragments

A
  • the name given to discontinuous fragments of DNA synthesized in lagging strand
  • 100-200 nucleotides in eukaryotes
  • 1000-2000 nucleotides in prokaryotes
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10
Q

overview of DNA replication in E. coli

A
  1. initial unwinding, separation, and stabilization of duplex DNA (several proteins)
  2. primer synthesis (primase- RNA template)
  3. DNA synthesis (DNA polymerase)
  4. Replace RNA primers with DNA
  5. seal gaps between okazaki fragments on lagging strand (ligase)
  6. termination of synthesis (“ter” protein, telomerase)
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11
Q

initiation

A

-unwind and stabilize duplex DNA to form replication fork

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12
Q

initiation factors

A

DnaA proteins bind to origin of replication

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13
Q

helicase

A

DnaB protein catalyzes ATP-deendent unwinding of duplex DNA

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14
Q

topoisomerases

A
  • prevent supercoiling & tangling of DNA during unwinding
  • bind ahead of replication fork, nick supercoiling DNA, releases stress by allowing uncoiling
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15
Q

formation of replication fork

A
  • 20-40 DnaA proteins bind to oriC sequence forming nucleosome-like structure
  • localized melting causes a small segment of DNA to open up
  • DnaB (helicase) enters oriC region and unwinds DNA
  • single stranded binding proteins attach to keep strands apart and protect against nucleases
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16
Q

oriC

A

origin sequence, where initiation of replication begins

-contains four 9 base pair repeats

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17
Q

primase

A
  • an RNA polymerase synthesizes a short RNA primer
  • DNA polymerase CANT initiate DNA synthesis-can only add nucleotides to the end of a chain that is base-paired with template strand
  • RNA polymerase can initiate synthesis without a primer
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18
Q

DNA polymerase I function

A
  • fills in gaps
  • repairs miss-matched pairs
  • replaces primer RNA during replication with DNA
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19
Q

DNA polymerase II function

A
  • thought to also be involved in some repair processes

- prevalent during stationary phase

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20
Q

DNA polymerase III function

A
  • the main polymerase of E. coli

- extends RNA-primed chain

21
Q

DNA ligase

A

joins DNA (okazaki) fragments

22
Q

E exonuclease repair function

A
  • enzyme recognizes mispaired bubbles in DNA
  • backs up, excises, then polymerase resumes replication
  • 3’ to 5’ exonuclease
23
Q

theta subunit function

A

unknown

24
Q

core polymerase composition

A

alpha, epsilon, and theta subunits

25
Q

beta subunit

A
  • sliding clamp that helps keep DNA polymerase bound to template during many rounds of nucleotide addition
  • converts polymerase from distributive enzyme to progressive enzyme
26
Q

T proteins

A

dimerize 2 DNA polymerase III cores

27
Q

clamp loading complex

A

responsible for placing doughnut shaped B subunit around DNA template

28
Q

processivity

A

frequency with which an enzyme dissociates from template during DNA replication

29
Q

alpha subunit function

A

5’ to 3’ polymerase

30
Q

E. coli replisome components

A
  • DNA polymerase III (dimer)
  • primosome (helicase + primase)
  • ssbp
  • dimer + looped lagging strand allows synthesis in same direction
31
Q

Eukaryotic replication

A

-3 polymerases total (some have dual function)

32
Q

polymerase delta

A

-lagging strand

33
Q

polymerase epsilon

A

leading strand

34
Q

polymerase alpha

A

makes primase

35
Q

P domain

A

-acts as sliding clamp in eukaryotic replication

36
Q

eukaryotic replication of linear chromosome

A
  • bidirectional and proceeds from several fixed origins
  • forks advance until they meet another fork traveling in the opposite direction
  • origins are programmed to initiate replication at fixed times in S phase
  • requires that replication origins become active at different times
  • chromatin must be dismantled prior to replication fork
37
Q

telomere

A
  • series of repeated TTAGGG DNA sequences located at the ends of linear eukaryotic chromosomes
  • each time a cell divides, some of the telomere is lost due to exonuclease activity
  • eventually little/no telomere remains but degradation continues
  • cell dies
  • typically one telomerase is G rich and the other is C rich.
38
Q

telomeres and cloning

A

premature aging and death of animal clots thought to be related to cloning from cells/nuclei with partly reduced telomeres

39
Q

telomerase

A
  • enzyme that restores telomere sequence
  • thought to restore chromosome/cell longevity
  • some cancers may be due to over-active telomerase
40
Q

hayflick’s limit

A

50 +or- 10

-number of cell divisions on average

41
Q

problems with synthesis completion on the 5’ end of linear genomes

A
  • excision of RNA primer would leave gap that can’t be filled bc no 3’ primer terminus to extend
  • viruses have evolved 3 strategies to overcome
  • eukaryotes solve problem using telomeres
42
Q

termination of DNA replication in prokaryotes

A
  • ter binding proteins bind to ter sites on opposite side of DNA loop in the bacterial chromosome
  • inhibits helicase and prevents further progression of replication forks
43
Q

termination of DNA replication in eukaryotes

A
  • DNA polymerase runs off the ends of DNA
  • replication bubbles fuse as polymerases collide
  • involves telomeres (sequences at ends of chromosomes that buffer against the loss of critical coding sequences following a round of DNA replication)
44
Q

retroviruses

A
  • use reverse transcriptase to produce DNA from RNA template
  • RNA viruses steal tRNA from host cell to use as primer
  • starts to extend primer towards opposite end of viral genome
  • Rnase H cuts off portion of viral genome to allow RNA to circularize
  • RNA synthase keeps synthesizing circular DNA
  • eventually enters host cell’s genome
  • cuts somewhere using integrase and incorporates itself
45
Q

template dna in vitro pcr

A

provides desired sequence of gene to be expressed

46
Q

buffer with Mg2+ included in vitro pcr

A

provides environmental conditions necessary to carry out rxn

47
Q

dna polymerase (Taq) in vitro pcr

A

catalyzes dna elongation

48
Q

dNTPs in vitro pcr

A

allows DNA polymerase to make copies of template DNA

49
Q

primers in vitro pcr

A

hybridize with specific portion of template DNA to initiate synthesis of new strands

Biochem (12 decks)

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