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Chapter 12 DNA Replication

Replication models for DNA


Conservative
DNA molecule remains intact and entire molecule serves as a
template
Results in one completely old molecule, and one completely new
molecule

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

Meselson and Stahl experiment


Equilibrium density
gradient centrifugation
Heavy salt solution used to
measure substances
densities
Denser molecules are
lower in tube

Radioactively labeled
DNA with 15N heavier
than DNA with 14N

Meselson and Stahl cont

Grew E. coli in 15N, then


swtiched to media with 14N

First replication
intermediate band
Rules out conservative
method

Second replication one


14N band (gets progressively
darker with each division)
and one intermediate band
(gets progressively lighter
with each division)

Semi-conservative method

Modes of Replication
Replication origin
Starting point of replication
A-T rich regions

Bacterial chromosomes have one; eukaryotic


chromosomes have many
Replicon individual unit of replication

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

Rolling Circle Replication


Viruses and certain
plasmids (F factor)
One strand breaks, new
nucleotides are added to
3 end using intact strand
as template
New strand displaces old
strand; old strand can
become double-stranded
based on complementarity

Only one strand serves


as a template

Linear Replication
Eukaryotic
chromosomes
Each has many origins
of replication
Each replicon is
smaller than
prokaryotic
chromosome

Replication forms
eventually meet and
replicons fuse

Requirements for replication


Single-stranded
template DNA
dNTPs
Deoxyribonucleoside
triphosphates (2
phosphates are
removed)

Enzymes and other


proteins

DNA polymerase
Can only add new nucleotides
to the 3 end
Replication in 53 direction

Old 35 template strand can


be replicated continuously
Leading strand

Old 53 template strand is


replicated in small fragments
Okazaki fragments
Lagging strand
As DNA unwinds, another
fragment is produced
Rolling-circle method does
NOT have lagging strand

Bacterial DNA Replication


4 general stages

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

Has high processivity


Stays attached to template for long time

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

Fidelity of DNA replication


Complementary base pairing
Proofreading
Incorrect alignment causes DNA poly III to backtrack and
remove incorrect base

Mismatch repair
Causes deformity in double strand
Old strand is methylated; new strand is not
Distinguishes strands

Incorrect nucleotide is excised out and replaced

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

Possible 2 strand replication


nd

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