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Biochemistry I > Biochemistry Midterm 2.2 - DNA Metabolism > Flashcards

Biochemistry Midterm 2.2 - DNA Metabolism Flashcards

1
Q

dynamic nature of DNA structure achieved by DNA metabolism

A

high fidelity DNA replication
constant check and repair of errors
segment rearrangement within chromosome or recombination between DNA molecules

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2
Q

Meselson-Stahl Experiment

A

cells grown in heavy N medium, then light N medium. Could track proportions of each to show that replication is using a template and is semi conservative

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3
Q

what would conservative replication have looked like?

A

the original parent strands would not be present after the first generation
1:4 heavy to light density bands

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4
Q

Cairn’s experiment showed

A

radiolabeled DNA showed replication fork and bidirectional replication in circular bacterial DNA

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5
Q

Inman’s experiment

A

DNA slightly denatured and always opened up into bubbles at the same AT rich locations
loops initiate at unique origins

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6
Q

who discovered Pol I
what does Pol I look like

A

Arthur Kornberg
like a hand with a: palm, thumb and fingers

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7
Q

DNA polymerase uses what kind of catalysis

A

metal ion catalysis with Mg 2+
Mg 2+ makes the nucleophilic 3’ OH more powerful on growing end of strand

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8
Q

substrate used by DNA polymerase
what molecule is release?

A

nucleoside triphosphate
pyrophosphate is released (2 phosphates linked)

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9
Q

Direction of DNA polymerase

A

synthesizes 5’ to 3’
template strand is read 3’ to 5’

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9
Q

DNA primer is
movement steps

A

short complementary strand with a 3’ OH and can be DNA or RNA
insertion and translocation

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10
Q

DNA polymerase regions

A

insertion - nucleotide binds
post insertion - newly made base pair

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11
Q

how base pairing is so accurate

A

DNA polymerase excludes base pairs with incorrect geometry
errors happen 1 in 10,000-100,000 base pairings
more accurate in E. coli ( 1 in 1-10 billion)

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12
Q

proofreading mechanism in DNA polymerase I

A

3’ to 5’ exonuclease activity in the exonuclease site fixes errors
by nick translation (also removes primers this way)
translocation is inhibited until fixed
where polymerase and 3’–> 5’ activity are in Pol I is called Klenow fragment

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13
Q

Klenow fragment

A

Pol I is proteolytically cleaved and the larger fragment containing 5’ to 3’ DNA polymerase activity and 3’ to 5’ exonuclease area is called Klenow fragment, used in experimental settings

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14
Q

DNA polymerase III (E. coli)

A

primary replication polymerase
polC gene
3’ to 5’ exonuclease
> 500,000 nucleotides before dissociating, 250-1000 nucleotides/s

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15
Q

Pol I (E. coli)

A

polA gene
3’ to 5’ and 5’ to 3’ exonuclease activity
3-200 nucleotides, 10-20 nucleotides/s
removes primers on lagging strand

16
Q

Anatomy of Pol III

A

9 subunits with 2 core domains
beta clamp subunits increase processivity
components: clamp loader (claw), 3 Pol III core complex (hook) connected to 3 beta sliding clamps (ring)

17
Q

replisome includes

A

over 20 proteins and enzymes
helicases - use ATP to unwind DNA strands (bubble)
topoisomerase - cuts strand to relieve tension and reattach
DNA binding proteins - stabiliize separated strands from reannealing
primase - to make RNA primer
DNA ligase - seal nicks between Okazaki fragments

18
Q

new nucleotides are added to the

A

3’ end where the OH is

19
Q

lagging strand is made

A

discontinuously, in short Okazaki fragments because for this strand helicase (replication fork) is moving backwards

20
Q

Initiation phase in E coli

A
  1. DnaA recognizes oriC site (10 different proteins used)
  2. DNA wraps around protein complex which creates strain, and denaturation at DUE site
  3. DnaA binds to beta subunits of Pol III and hydrolyzes ATP
  4. DnaA and ADP dissociate
  5. New ATP binds and process restarts
    20-40 minute process
21
Q

DNA replication initiation at oriC

A

initiation site for DNA replication in E coli
DNA proteins are ATPases, 8 bind to R/I sites
lots of A-T pairs
other sites for DNA binding proteins: IHF - integration host factor, FIS - factor for inversion stimulation

22
Q

Dam methylase

A

methylates to mark which sequence is kept and which is passed to daughter cell

23
Q

DnaA protein

A

protein that recognizes oriC and opens duplex by binding to R and I sites and coils DNA
promotes unwinding of DNA at oriC by inducing topological stress, causing denaturing at DUE site of AT rich sequences
has either ADP or ATP bound to supply energy

24
Q

Elongation phase in E coli

A
  1. Primase makes primer
  2. Pol III adds nucleotides to 3’ end (strand elongates with replication fork in leading strand and away from replication fork in lagging strand)
  3. core subunits of Pol III dissociate from 1 beta clamp to bind to a new one using 3 atp every time
  4. DNA Pol I/or RNase removes primer, fills gap (nick) and DNA ligase seals backbone
25
Q

DnaB protein

A

helicase, breaks H bonds between DNA strands for replication
tethered to Pol III on the opposite strand
loaded onto DNA by DnaC protein

26
Q

DnaC protein

A

helicase loader protein, helps helicase (DnaB) bind to DUE

27
Q

DNA gyrase

A

DNA topoisomerase II in bacteria, relieves torsional strain generated by DNA unwinding

28
Q

DnaG protein

A

primase, synthesizes RNA primers

29
Q

DNA ligase mechanism

A
  1. 5’ phosphate is activated by attaching AMP
  2. 3’ OH attacks phosphate and seals nick and displacing AMP
    requires ATP
30
Q

Termination of DNA replication in E coli

A
  1. replication forks meet at Ter region
  2. Tus protein binds at Ter which causes replication fork to stop
  3. topoisomerase separates catenanes intertwined DNA for transient break in DNA
  4. unknown how the gap is filled
31
Q

catenanes

A

chromosomes topologically intertwined circles separated by topoisomerase

32
Q

DUE stands for and does

A

DNA unwinding element, AKA 13 mer
first region in the origin of replication or oriC in prokaryotes
AKA 13 mer, 3 identical initiation sequences in tandem

33
Q

SSBP stands for and does

A

single stranded DNA binding protein
stabilizes separated DNA strands

34
Q

what is nick translation?

A

clipping and adding a radioactive phosphate tag on 3’ end to signal for translation in experimental purposes

35
Q

5’ –> 3’ exonuclease activity in Pol I is

A

involved in removal of RNA primers by nick translation

36
Q

FIS and and IHF

A

factor for inversion stimulation - negatively regulates
integration host factor - enhances initiation
both at oriC, together they enhance replication when bound

37
Q

TER sites

A

permissive and non-permissive sides,

Biochemistry I (12 decks)

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