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Dispersive
DNA molecule breaks into fragments, which serve as template and
then reassemble
Each molecule a mixture of old and new
Semi-conservative
DNA separates into two strands, and each serves as a template
Each molecule consists of one old strand and one new strand
Radioactively labeled
DNA with 15N heavier
than DNA with 14N
First replication
intermediate band
Rules out conservative
method
Semi-conservative method
Modes of Replication
Replication origin
Starting point of replication
A-T rich regions
3 types of replication
Theta replication
Rolling circle replication
Linear replication
Theta Replication
Circular DNA in bacteria
Replication bubble
formed from DNA
unwinding and strands
separating
Replication fork point
where two strands
separate
Continues bi-directionally
until they meet
Linear Replication
Eukaryotic
chromosomes
Each has many origins
of replication
Each replicon is
smaller than
prokaryotic
chromosome
Replication forms
eventually meet and
replicons fuse
DNA polymerase
Can only add new nucleotides
to the 3 end
Replication in 53 direction
Initiation
Unwinding
Elongation
Termination
Initiation
Initiator proteins bind to Ori
and unwind small segment
Allows other molecules to
bind to DNA
Unwinding
DNA helicases
Break hydrogen bonds
between 2 strands
Move in 53 direction
Single-strand binding
proteins
Prevents reannealing
DNA gyrase
In front of replication fork
Unwinding causes
supercoiling
Is a topoisomerase
makes double-stranded
break, and then reseals
break behind it
Releases tension
Unwinding cont
Primers
DNA polymerase cant
initiate a new strand
it can only elongate an
existing strand
Primase
RNA polymerase
Does not require a
primer
Adds short stretch of
RNA nucleotides
which is later replaced
by DNA nucleotides
and ligated together
Leading strand
requires one primer;
lagging requires many
Elongation
DNA polymerase III
Adds nucleotides to 3 end
Has 3 5 exonuclease activity
Can backtrack and replace an incorrect nucleotide
DNA polymerase I
Has same direction abilities as III; in addition has 53
exonuclease activity
Removes RNA primer and replaces nucleotides with DNA
nucleotides
Elongation cont
Phosphodiester bonds
Covalent bond formed
between 5 phosphate
group of new nucleotide to
3 -OH group of last
nucleotide
DNA ligase
DNA poly I replaces primer
leaves nick between last
replaced nucleotide and 1st
original DNA nucleotide
Ligase creates
phosphodiester bond to
form continuous strand
Termination
When 2 replication forks meet or specific
DNA sequence is encountered
Termination protein binds to sequence and
blocks helicase binding
Mismatch repair
Causes deformity in double strand
Old strand is methylated; new strand is not
Distinguishes strands
Eukaryotic replication
Have multiple polymerases (greek letters)
alpha and delta are major ones
Nucleosome
DNA coiled around 8 histone proteins
Newly synthesized DNA molecules are quickly
re-associated with histones (a mix of old and
newly made)
Linear chromosomes
Circular DNA has a free
OH group in front of
primer for new nucleotide
to attach to
Linear chromosomes
After primer is removed at
the end of the
chromosome, there is no
free OH group
Chromosome would
shorten with each
replication, removing
telomeres and destabilize
chromosome
Telomerase
Telomeres are short
repeating sequences
Telomerase is a
ribonucleoprotein
RNA portion 12-22
complementary
nucleotides
Protein portion acts as
an enzyme to extend 3
end with complementary
DNA
2nd strand replication
unknown mechanism
Telomerase cont
Activity decreases/stops in most mature
cells
May lead to cellular aging due to destabilization of chromosomes
Normal cells have a limited number of
replications
Telomerase activity has been shown to
continue in cancer cells immortal cells