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BIOL 308 Molecular Biology > DNA Replication (contd.) > Flashcards

DNA Replication (contd.) Flashcards

1
Q

RNase H function

A
  • removes most of RNA primers’ nucleotides (except last one)
  • auxiliary role for longer primers
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2
Q

DNA pol I function, directions(!)

A
  • removes the last ribonucleotide in primers (that RNase H misses)
  • fills gap with DNA (5’-3’)
  • slow (10-20nt/sec)
  • both exonuclease activities:
    – 5’-3’ (removes ribonucleotides)
    – 3’-5’ (proofreading)
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3
Q

what enzyme seals the gap between DNA fragments

A

DNA ligase

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4
Q

is DNA pol I:
RNA-dependent DNA pol (RNAP) or DNA-dependent DNA pol (DNAP)

A

DNAP! makes DNA from DNA template

basically replaces ribonucleotides with deoxyribonucleotides

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5
Q

endonuclease vs exonuclease

A

endonuclease cuts from the middle and cuts bonds between 2 bases (like inside the nucleotides)

exonuclease cuts at free ends only

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6
Q

DNA ligase function

A
  • enzyme that links DNA fragments on lagging strand following primer removal and Pol I synthesis
  • creates phosphodiester bond (b/w 3’OH + 5’PO4 of adjacent nucleotides)
  • NEEDS ATP
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7
Q

topoisomerase role in DNA rep

A
  • recognize + regulate supercoiling
  • 2 types (I/II); type II (gyrase) NEEDS ATP
  • introduces negative supercoils around oriC (necessary for initiation by DnaA)
  • after helicase unwinds, positive supercoils form ahead of growing fork; TOPO II converts them into negative supercoils!
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8
Q

loop is growing; lagging core displaces ____ and finishes ____ ____

A

SSB
okazaki fragment

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9
Q

primase is positioned by ____ to make a RNA primer for lagging strand

A

helicase

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10
Q

replication fork for lagging strand (what happens after lagging core finishes an okazaki fragment)?

  • primase, beta clamp, lagging core, gamma complex, RNase H, DNA pol I, ligase
A
  • new primer made, primase released
  • beta clamp released finished okazaki fragment from lagging core
  • lagging core stays connected to leading core through tau subunit (dimer)
  • gamma complex loads beta clamp to new primer
  • lagging core binds to beta clamp, starts DNA synthesis
  • RNase H and DNA pol I clean up ribonucleotides, DNA pol I fills gaps
  • ligase connects fragments
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11
Q

what protein initiates by binding to oriC?

A

DnaA

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12
Q

what enzyme separates strands? what is its escort

A

helicase, DnaB (DnaC as escort)

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13
Q

what proteins keep strands apart during DNA rep?

A

single-strand binding proteins (SSB)

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14
Q

what enzyme is necessary to convert +ve supercoils to -ve?

A

topoisomerase II (gyrase, needs ATP)

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15
Q

what enzyme makes primers for both leading and lagging?

A

primase (DnaG, RNA polymerase)

lots of primers for lagging, one primer for leading

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16
Q

how does primase know where to make a primer?

A

primase positioned at right place through helicase - PRIMOSOME

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17
Q

what is a DNAPIII holoenzyme

A

the 2 cores; one for leading and one for lagging

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18
Q

why is DNAPIII processive?

A

beta clamp

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19
Q

what are the factors that load/unload beta clamp onto DNAPIII?

A

gamma complex

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20
Q

why is there synchronized replication of both strands?

A

2 core DNA pol are kept together by tau subunits

21
Q

what enzymes work together to clean up RNA primers?

A

RNase H (except last one) + DNAPI (removes last one and fills in w/DNA)

22
Q

what enzyme functions so newly made strands don’t have nicks?

A

DNA ligase

23
Q

why is replicating bacterial genome called a theta structure?

A

the replication bubble looks like the theta symbol (bidirectionality), and end as two catenated loops
- forks meet at farthest point from oriC, following two termination forks (1 each)

24
Q

how replication of circular DNA completes

A

since they are stuck together (2 loops)
- decatenation
- denaturation of unreplicated terminus, followed by supercoiling
- replication can complete before or after decatenation
- topo IV decatenates in vivo
- topo II can decatenate in vitro

25
Q

euk vs prok DNA replication speed

A

euk: 50 bp/sec (more complex chromatin structure)
prok: 1000 bp/sec

26
Q

how is euk diff from prok in DNA rep?

A
  • speed (euk slower)
  • multiple replicons
  • euchromatin first
  • “domino model” of origin activation during S phase
27
Q

what is origin of replication in yeast?

A

autonomous replication sequence (ARS)

28
Q

what is conserved in ARS?

A

A-domain of ARS (11bp consensus seq)
- mutation abolishes func

29
Q

what recognizes euk origin (name)

A

eukaryotic origin recognition complex (ORC)

30
Q

what is ORC made of?

A

6 proteins
- 5 subunits have winged-helix domain (WHD) and AAA+ domain (contains RecA fold and lid domains)

31
Q

what are cyclins? what do they bind to? conc?

A

main proteins in cell cycle regulation
- bind to cyclin-dependent kinases (sets off cell cycle phase)
- diff ones are more active during diff cell stages
- gets degraded after triggering Cdk

32
Q

licensing factor

A
  • factor located in nucleus, necessary for replication
  • Cdt1 - geminin, regulates lvls
  • new rep needs new licensing factor to enter in nucleus (as old one is degraded)
  • re-entry occurs in subsequence mitosis when nuclear membrane breaks
33
Q

what are the polymerases for euk? their roles?

A

pol alpha - prime DNA synthesis (rep and repair)
pol delta - DNA rep of lagging strand (rep and repair)
pol epsilon - DNA rep of leading strand (rep and repair)

34
Q

alpha pol = ?

A

primase

35
Q

T/F: Pol I and III are in eukaryotes

A

FALSE, only prokaryotes

36
Q

pol alpha’s interactions with pol epsilon and pol delta

A

pol alpha makes RNA primers,
- pol epsilon takes over for pol alpha on LEADING strand
- pol delta takes over for pol alpha on LAGGING strand

37
Q

what is MCM?

A

helicase

38
Q

what is cdc6?

A

helicase loader

39
Q

what is FEN-1? coordinated by?

A

endo/exonuclease - removes primer
(RNase H)
coordinated by PCNA (sliding clamp)

40
Q

what is PCNA?

A

sliding clamp

41
Q

what is RFC?

A

clamp loader

42
Q

euk DNA rep completion steps, coordinated by?

A
  • FEN-1 removes primers from both strands (coordinated by PCNA)
  • pol delta completes gaps on lagging strand (coordinated by PCNA and RFC)
  • DNA ligase I seals gaps together on lagging strand
  • nucleosomes are reassembled
43
Q

why do we need telomeres and telomerases? what are they/their functions?

A

we lose bp with every replication
- telomeres contains repeating seq (TTAGGG) w/ protruded ss 3’end that can fold into loop, acts as a safety buffer
- telomerases - repairs and replicates telomeres

44
Q

is telomerase RNA-dependent DNA pol or DNA-dependent DNA pol

A

RNA-dependent!

45
Q

telomerase details

A
  • ribonucleoprotein (protein-RNA complex)
  • carries its own template RNA
  • has reverse transcriptase activity
  • tandem repeat (TTAGGG), results in 3’ overhang of lagging strand template
46
Q

what is replicative cell senescence?

A

shortening chromosome ends, losing genetic info
- after many generations, descendent cells inherit defective chromosomes
- eventually stop dividing, stop cell cycle

47
Q

telomeres - shelterin

A

TTAGGG are bound by shelterin-telosome protein complex (protection + regulation of telomeres);
subtelomeric regions are adjacent to telomeres, also repetitive DNA

48
Q

telomeres - methylation

A

subtelomeric DNA is VERY methylated
- these modifications at telomeres and subtelomeres negatively regulate telomere length (prevent telomerase activity)

BIOL 308 Molecular Biology (14 decks)

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