Biochemistry Midterm 2.2 - DNA Metabolism

Question 1

dynamic nature of DNA structure achieved by DNA metabolism

Answer 1

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

Question 2

Meselson-Stahl Experiment

Answer 2

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

Question 3

what would conservative replication have looked like?

Answer 3

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

Question 4

Cairn’s experiment showed

Answer 4

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

Question 5

Inman’s experiment

Answer 5

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

Question 6

who discovered Pol I
what does Pol I look like

Answer 6

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

Question 7

DNA polymerase uses what kind of catalysis

Answer 7

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

Question 8

substrate used by DNA polymerase
what molecule is release?

Answer 8

nucleoside triphosphate
pyrophosphate is released (2 phosphates linked)

Question 9

Direction of DNA polymerase

Answer 9

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

Question 9

DNA primer is
movement steps

Answer 9

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

Question 10

DNA polymerase regions

Answer 10

insertion - nucleotide binds
post insertion - newly made base pair

Question 11

how base pairing is so accurate

Answer 11

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)

Question 12

proofreading mechanism in DNA polymerase I

Answer 12

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

Question 13

Klenow fragment

Answer 13

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

Question 14

DNA polymerase III (E. coli)

Answer 14

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

Question 15

Pol I (E. coli)

Answer 15

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

Question 16

Anatomy of Pol III

Answer 16

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)

Question 17

replisome includes

Answer 17

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

Question 18

new nucleotides are added to the

Answer 18

3’ end where the OH is

Question 19

lagging strand is made

Answer 19

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

Question 20

Initiation phase in E coli

Answer 20

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

Question 21

DNA replication initiation at oriC

Answer 21

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

Question 22

Dam methylase

Answer 22

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

Question 23

DnaA protein

Answer 23

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

Question 24

Elongation phase in E coli

Answer 24

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

Question 25

DnaB protein

Answer 25

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

Question 26

DnaC protein

Answer 26

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

Question 27

DNA gyrase

Answer 27

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

Question 28

DnaG protein

Answer 28

primase, synthesizes RNA primers

Question 29

DNA ligase mechanism

Answer 29

1. 5' phosphate is activated by attaching AMP 2. 3' OH attacks phosphate and seals nick and displacing AMP requires ATP

Question 30

Termination of DNA replication in E coli

Answer 30

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 3. unknown how the gap is filled

Question 31

catenanes

Answer 31

chromosomes topologically intertwined circles separated by topoisomerase

Question 32

DUE stands for and does

Answer 32

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

Question 33

SSBP stands for and does

Answer 33

single stranded DNA binding protein stabilizes separated DNA strands

Question 34

what is nick translation?

Answer 34

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

Question 35

5' --> 3' exonuclease activity in Pol I is

Answer 35

involved in removal of RNA primers by nick translation

Question 36

FIS and and IHF

Answer 36

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

Question 37

TER sites

Answer 37

permissive and non-permissive sides,