Biochemistry Midterm 2.2 - DNA Metabolism Flashcards
dynamic nature of DNA structure achieved by DNA metabolism
high fidelity DNA replication
constant check and repair of errors
segment rearrangement within chromosome or recombination between DNA molecules
Meselson-Stahl Experiment
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
what would conservative replication have looked like?
the original parent strands would not be present after the first generation
1:4 heavy to light density bands
Cairn’s experiment showed
radiolabeled DNA showed replication fork and bidirectional replication in circular bacterial DNA
Inman’s experiment
DNA slightly denatured and always opened up into bubbles at the same AT rich locations
loops initiate at unique origins
who discovered Pol I
what does Pol I look like
Arthur Kornberg
like a hand with a: palm, thumb and fingers
DNA polymerase uses what kind of catalysis
metal ion catalysis with Mg 2+
Mg 2+ makes the nucleophilic 3’ OH more powerful on growing end of strand
substrate used by DNA polymerase
what molecule is release?
nucleoside triphosphate
pyrophosphate is released (2 phosphates linked)
Direction of DNA polymerase
synthesizes 5’ to 3’
template strand is read 3’ to 5’
DNA primer is
movement steps
short complementary strand with a 3’ OH and can be DNA or RNA
insertion and translocation
DNA polymerase regions
insertion - nucleotide binds
post insertion - newly made base pair
how base pairing is so accurate
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)
proofreading mechanism in DNA polymerase I
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
Klenow fragment
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
DNA polymerase III (E. coli)
primary replication polymerase
polC gene
3’ to 5’ exonuclease
> 500,000 nucleotides before dissociating, 250-1000 nucleotides/s
Pol I (E. coli)
polA gene
3’ to 5’ and 5’ to 3’ exonuclease activity
3-200 nucleotides, 10-20 nucleotides/s
removes primers on lagging strand
Anatomy of Pol III
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)
replisome includes
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
new nucleotides are added to the
3’ end where the OH is
lagging strand is made
discontinuously, in short Okazaki fragments because for this strand helicase (replication fork) is moving backwards
Initiation phase in E coli
- DnaA recognizes oriC site (10 different proteins used)
- DNA wraps around protein complex which creates strain, and denaturation at DUE site
- DnaA binds to beta subunits of Pol III and hydrolyzes ATP
- DnaA and ADP dissociate
- New ATP binds and process restarts
20-40 minute process
DNA replication initiation at oriC
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
Dam methylase
methylates to mark which sequence is kept and which is passed to daughter cell
DnaA protein
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
Elongation phase in E coli
- Primase makes primer
- Pol III adds nucleotides to 3’ end (strand elongates with replication fork in leading strand and away from replication fork in lagging strand)
- core subunits of Pol III dissociate from 1 beta clamp to bind to a new one using 3 atp every time
- DNA Pol I/or RNase removes primer, fills gap (nick) and DNA ligase seals backbone
DnaB protein
helicase, breaks H bonds between DNA strands for replication
tethered to Pol III on the opposite strand
loaded onto DNA by DnaC protein
DnaC protein
helicase loader protein, helps helicase (DnaB) bind to DUE
DNA gyrase
DNA topoisomerase II in bacteria, relieves torsional strain generated by DNA unwinding
DnaG protein
primase, synthesizes RNA primers
DNA ligase mechanism
- 5’ phosphate is activated by attaching AMP
- 3’ OH attacks phosphate and seals nick and displacing AMP
requires ATP
Termination of DNA replication in E coli
- replication forks meet at Ter region
- Tus protein binds at Ter which causes replication fork to stop
- topoisomerase separates catenanes intertwined DNA for transient break in DNA
- unknown how the gap is filled
catenanes
chromosomes topologically intertwined circles separated by topoisomerase
DUE stands for and does
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
SSBP stands for and does
single stranded DNA binding protein
stabilizes separated DNA strands
what is nick translation?
clipping and adding a radioactive phosphate tag on 3’ end to signal for translation in experimental purposes
5’ –> 3’ exonuclease activity in Pol I is
involved in removal of RNA primers by nick translation
FIS and and IHF
factor for inversion stimulation - negatively regulates
integration host factor - enhances initiation
both at oriC, together they enhance replication when bound
TER sites
permissive and non-permissive sides,
