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BIOL 308 Molecular Biology > DNA Replication > Flashcards

DNA Replication Flashcards

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1
Q

origins of mutations: human males vs females

A

human males: much more divisions in germ cells
human females: fewer divisions in germ cells

frequency of mutations: (male sperm 10-50X female egg)

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2
Q

DNA replication
- enzymatic/structural/regulatory

A

enzymatic: nucleic acids as substrates
- DNA: restriction enzymes, topoisomerases, telomerases, methylases, polymerases, etc
- RNA: tRNA synthetases, polymerases, polyadenylases

structural: change in DNA/RNA structure
- helicases, histones, TBP (tata-binding proteins)

regulatory: binding to nucleic acids
- in both transcription (DNA binding) and translation (RNA binding)

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3
Q

Type of protein-nucleic acid interactions (4)

A
  • ionic
  • H bonds
  • van der waals
  • hydrophobic interactions

*also stabilize tertiary and quaternary protein structures

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4
Q

replication is _____ (style of conservation)

A

semiconservative

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5
Q

replication origin (prok vs euk)

A

prok: bacterial genomes and plasmids often have only 1

euk: multiple origins

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6
Q

replicon meaning

A

entire region of DNA replicated from one origin
(a piece of DNA which replicates as a single unit)

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7
Q

experiment that confirmed bidirectionality in DNA replication

A

pulse-chase experiment
- pulse of ^3H-T (tritium isotope), followed by “chase” of unlabeled T
- light up areas showed bidirectional growth (going in two directions)

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8
Q

discovery of enzymes in DNA rep

A
  • studied prok w genetic and biochemical approaches
  • make a mutant pop, screen for DNA rep mutants
  • purify enzymes needed for rep
  • assay: add labelled dNTP to necessary DNA rep enzymes, template DNA, and measure how much label is in new DNA
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9
Q

what does DnaA do in DNA rep?

A

initiation

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10
Q

what do single-strand binding proteins do in DNA rep?

A

they sheathe strands (so don’t reanneal)

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11
Q

what is helicase AKA?

A

DnaB

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12
Q

what is primase AKA?

A

DnaG, RNA polymerase

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13
Q

what are clamp loader and sliding clamp AKA?

A

clamp loader - DnaC
sliding clamp - beta clamp

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14
Q

replisome meaning

A

all the proteins that function at the replication fork, as part of DNA synthesis

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15
Q

3 problems in copying DNA by DNA pol:

A
  1. DNA pol can’t break inter-chain H-bonds at point of origin (need other molecules)
  2. DNA pol can’t start chains, only elongate (needs primer @3’OH)
  3. DNA pol can only add from 3’OH end (chain always grows in 5’-3’ direction)
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16
Q

3 ways to generate 3’ ends (for DNA rep), used by diff organisms

A

specific RNA pol
- synthesize small segment of RNA
- in DNA rep for prok and euk

nicked DNA
- happens in rolling circle replication; some phages do this

primed nucleotide
- some viruses can do this; also at end of euk rep (telomerase)

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17
Q

leading strand vs lagging strand

A

leading strand - continuously made from a single primer, 5’-3’

lagging strand - DIScontinuous, from multiple primers, also 5’-3’

18
Q

what are okazaki fragments, what are they joined by?

A

lagging strand segments

joined by DNA pol I and ligase

19
Q

how many replication forks in replication bubble?

A

2 replication forks in 1 replication bubble

20
Q

processivity meaning

A

how fast and continuous the polymerase copies (high processive pol are more efficient)

21
Q

initiation of replication in E. coli (origin of rep name, bp#, characteristic)

A

OriC, ~245bp

A-T rich 13 bp (3)
repetitive 9 bp (DnaA boxes, 4)

22
Q

initiation depends on ______ of oriC

A

methylation

23
Q

initiation depends on methylation of oriC:

A
  • N6 of adenine is methylated in sequence GATC (copies exist in oriC)
  • note: methylations can happen on C and A residues in DNA (prok)
24
Q

methylation is epigenetic/trans-generational?

A

epigenetic
- template keeps methyl but new strands don’t, so not passed to future strands (hemi-methylated DNA)

25
Q

what methylates origins for initiation

A

Dam methylase

26
Q

DnaA function, full desc in E. coli DNA rep

A
  • initiates replication at oriC
  • recognize 4 DnaA boxes (9bp) in oriC
  • 10-20 DnaA proteins and 4 DnaA boxes form an initial complex
  • starts replication only if DNA is NEGatively supercoiled (easy to unwind)
    – opens at the 13bp, NEEDS ATP
27
Q

DnaB function, full desc in E. coli DNA rep

what protein escorts it?

A

AKA helicases!
- separate strands, NEED ATP
- hexamer, can clamp around either ss of DNA
- NEEDS DnaC to escort DnaB to DnaA (form pre-priming complex or pre-primosomal complex)
- moves toward ds region of rep fork
- processive (doesn’t fall off until end of strand or unloaded)

28
Q

what does SSB stand for?

A

single-stranded DNA-binding protein

29
Q

what does SSB (single-stranded DNA binding protein) do?

A
  • small protein that binds to ssDNA
  • cooperative binding: binding of one molecule promotes binding of next one
  • prevents strands reannealing and internal pairing (no hairpins)
30
Q

DnaG AKA, function, full desc in E. coli DNA rep

A

AKA DNA primase
- makes RNA primers for DNA pol to build off
- lagging strand primase (DnaG) works with DnaB (helicase) to form next priming site (PRIMOSOME)

31
Q

what happens at the end of initiation?

A

binding of DNAPIII, polymerization starts

32
Q

are DNA pol I major enzymes for DNA rep in vivo? why or why not?

A

no, some mutants result in viable and normal rate DNA rep

33
Q

what is the most important polymerase in DNA rep?

A

DNA pol III - key enzyme, synthesis of DNA from RNA primers

34
Q

what does DNA pol II do?

A

repair

35
Q

how are nucleotides added? (movement of a molecule)

A

addition of nucleotide triphosphate;
alpha phosphate connects backbone while base pairs match. other 2 phosphates disconnect (catalysis)

36
Q

how many daltons in pol I and pol III?

A

pol I: 1 polypeptide, 109kDa
pol III: 10 diff polypeptides, >600kDa

37
Q

what is DNAPIII’s core made of? (3 subunits & their functions)

A

alpha - active site for base addition (5’-3’)
epsilon - 3’-5’ exonuclease (activity that proofreads)
theta - function unclear

38
Q

what is function of beta subunit in DNAPIII

A

beta subunit - functions as a clamp; associate with core at 3’ end of growing strand, increases processivity of polymerase (250-1000nt/sec)

39
Q

what is function of gamma complex in DNAPIII, parts, what proteins does it interact w/

A
  • multiple subunits
  • shared by two core polymerases
  • loads and unloads beta (clamp) onto DNA template (NEEDS ATP)
  • includes tau (2) to connect 2 pol III core (dimerization) -> leading/lagging are coordinated
40
Q

what direction is DNAPIII’s proofreading in?

A

3’-5’ exonucleolytic proofreading, high fidelity

41
Q
A

BIOL 308 Molecular Biology (14 decks)

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