Exam 2: DNA & Replication Flashcards
3 parts of a nucleotide
sugar
phosphate
nitrogenous base
complementary base pairs
A—T with 2 bonds
G—C with 3 bonds
what is needed to make dsDNA
antiparallel strands with complementary base pairs
what stabilizes dsDNA
Mg2+ ions
Watson-Crick Model
2 antiparallel strands right-handed double helix phosphate backbones on the outside h-bonding from the bases on the inside complementary pairs Mg2+ ions to stabilize helix backbone
in vitro
in glass (test tube)
in vivo
in life
DNA replication in vitro is a ____
cell free system
you only use chemicals
5 things needed for in vitro DNA replication
DNA nucleotides in triphosphate form
a ssDNA template strand
a DNA primer
DNA polymerase enzyme
Mg2+ ions
what happens in in vitro DNA replication
DNA polymerase binds at the 3’ end of the primer
DNA polymerase extends the primer from 3’ to 5’
5’—3’(primer)-DNA polymerase—»
3’———–template——————–5’
what yields E for synthesis?
2 phosphates from the triphosphate nucleotides are lost in synthesis
Mg 2+
2 ions involved in each nucleotide addition (stabilization)
a cofactor of DNA polymerase
neutralizes charge repulsion of oxygens in DNA backbone
complications of in vivo DNA replication in E. coli
2 strands are wrapped around eachother and have to separate and replicate in opposite directions
unwinding can cause supercoiling
there is no primer when the two strands open up and separate
how many forks does in vivo DNA replication have?
two forks going in opposite directions at the same time
is there a primer in natural DNA replication?
no, there is a temporary short primer made of RNA
primase
an enzyme that makes short temporary primers from RNA
semidiscontinuous replication
differences in the way synthesis occours on the leading and lagging strand
the leading strand
is replicated continuously towards the fork
the lagging strand
is replicated discontinuously away from the fork
supercoil
when DNA unravels the strand uncoils and supercoils into loops
supercoils on short pieces of dsDNA
don’t last long, they spin out
supercoils on long linear DNA or circular DNA
accumulate
should supercoils be removed?
yes they must in both replication and transcription
how are supercoils removed?
with the enzyme topoisomerase
topoisomerase
2 types (1 & 2)
enzymes that cut dsDNA either in one strand or 2 strands
topoisomerase 1
cuts dsDNA on 1 strand
1) nick
2) rotate
3) ligate
topoisomerase 2
cuts dsDNA on 2 strands
1) double-strand break
2) pass through
3) rejoin
okazaki fragments
created during discontinuous synthesis on the lagging strand
contains an RNA primer on 5’ end and attached strand of ssDNA to 3’ end
5’-RRRRDDDD,RRRRDDDD-3’
DNA polymerase 1
connects Okazaki fragments by removing RNA nucleotides one by one from right of nick
attaches DNA nucleotides to left of the nick
RRRRDDDD
D,RRRDDDD
DD,RRDDDD
3 major differences in DNA replication in eukaryokes
chromosomes
packaging of DNA
rate of replication
chromosomes in eukaryote DNA replication
long, linear, end in telomeres
telomerase enzyme needed to restore chromosome ends
in eukaryotes, replication starts at…
replicon origins/bubbles that extend and meet each other
early and late replicating areas are organized locally into replicon families
DNA packaging in eukaryotes
chromatin has nucleosome (spooled) structure
histone proteins for nucleosomes are synthesized
nucleosome cores are replicated dispersively-old and new are divided randomly into new strands
eukaryoke DNA replication rate
movement of forks is slower
rate is 2000-10000 bp/min
~ 10,000 origins in mammalian genome
300,000 bp/replicon
what end does the primer go on?
3’
semiconservative replication
in every dsDNA one strand is the parent of the other
ori
single origin of replication in circular chromosome of a bacterium like E. coli
where on E. coli does replication begin and end
begins at ori and ends on other side of circular chromosome where the forks meet
