Download Chapter 10 – DNA Replication

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 5′→3′ direction
• Old 3′→5′ template strand can
be replicated continuously
– Leading strand
• Old 5′→3′ 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 5′→3′ 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 can’t
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
5′→3′ 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 2nd strand replication
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