Chapter 12 Flashcards
Replication has to be extremely accurate.
– Human zygote – contains 6.4 billion bp of DNA
– One error/million bp leads to 6400 mistakes every
time a cell divides, which would be catastrophic.
Replication also takes place at high speed.
– E. coli replicates its DNA at a rate of 1000
nucleotides/second.
– Human DNA - 50 nucleotides/second
Conservative replication model
Entirely new template
Dispersive replication model
Little pieces both broke down into fragments
Semiconservative replication model
Strands separate and new strand Synthesis
semiconservative replication depends on
Circular vs. linear DNA
replicon
A segment of DNA that undergoes replication
Origin of replication
What each replicon has
Bacteria VS. Eukaryote origin of replication
Bacteria: 1
Eukaryotes-many
Theta replication
common mode of replication in circular DNA
Rolling-circle replication
takes place in some viruses and in the F factor of E. Coli
conclusion of rolling-circle
multiple circular DNA molecules
Linear Eukaryotic Replication
Slower than bacteria
Too much DNA for single origin
Linear eukaryotic replication requirements
A template strand consisting of single stranded DNA
Raw material: Nucleotides
Enzymes and other proteins that read the template and assemble the substrate into A DNA molecule
Direction of replication
DNA polymerase adds nucleotides only to 3’ end of growing strand
Anti parallel nature of the double stranded DNA means
- one template is exposed in the 5’ to 3’ direction
vise versa
Continuous replication
occurring on the leading strand
Dissentious replication
Occurs on lagging strand
Synthesis is proceeding in the direction of the unwinding
Discontinuous replication is a result of Which property of DNA?
Anti Parallel nucleotide Strands
Replication taxes place in four stages
- Initiation
- unwinding
3.Elongation - Termination
Bacterial DNA Replication: Initiation
single origin of replication
(oriC)
- Initiator proteins bind and
cause short section of DNA to
unwind, allowing other single-strand-binding proteins to
attach to the polynucleotide
strand
Bacterial DNA Replication: Unwinding
The cell relies on several proteins and enzymes for unwinding
DNA helicase
Single-strand-binding proteins (SSBs)
DNA gyrase
Single-strand-binding proteins (SSBs)
- – attach to the exposed
single-stranded DNA after helicase unwinds DNA and prevent unnecessary binding and hairpins
DNA helicase
– breaks hydrogen bonds between strands, taking between nucleotides and breaking them
DNA gyrase
– a type II topoisomerase that reduces strain
ahead of the replication fork by making double-strand breaks
and then resealing the broken ends of DNA
relieves the tension
Bacterial DNA Replication: Elongation
Single-stranded DNA is used as a template for the synthesis of DNA!
DNA Polymerase
Primase
DNA Polymerase
– enzymes which elongate the new polynucleotide strand.
All DNA polymerases require a nucleotide with a 3’-OH group in order to add
the new nucleotides.
So, it needs primase to synthesize short stretches of
RNA nucleotides (primers).
Primase
– enzyme responsible for synthesizing short stretches
(~10-12) of RNA nucleotides (termed primers) which provide the
needed 3’-OH group for DNA polymerase to attach DNA nucleotide!
DNA Polymerase III
duplication of the chromosomal DNA
DNA Polymerase I
DNA polymerase I functions to fill DNA gaps that arise during DNA replication, repair, and recombination
5’ to 3’ polymerase activity
each polymerase can do the following
P1: Removes and
replaces primers
P2:DNA repair; restarts
replication DNA
halts synthesis
P3:Elongates DNA
P4:DNA repair
P5:DNA repair;
translesion DNA
synthesi
3’ to 5’ exonuclease activity
remove in 3’ to 5’ direction
P1: Removes and
replaces primers
P2:DNA repair; restarts
replication DNA
halts synthesis
P3:Elongates DNA
5’ to 3’ exonuclease activity
P1: Removes and
replaces primers
DNA ligase
Connecting nicks after RNA
primers are removed
DNA ligase seals the nicks
with a phosphodiester bond!
Elongation at replication
fork requires:
1.Helicase to unwind DNA
2.SSB to protect single nucleotide strands and
prevent secondary structures.
3.DNA gyrase to remove strain
- Primase to synthesize primers with a 3’-OH group
- DNA polymerase to synthesize the nucleotide
strands.
Bacterial DNA Replication: Termination
In some DNA molecules, termination occurs
whenever two replication forks meet.
In others, specific termination
sequences (Ter sites) block
further replication.
Replication at the Ends
of Chromosomes
single-celled eukaryotes and early embryonic cells –chromosomes do not shorten!
ends of linear chromosome replicated by telomerase
Nucleosomes assembly
Nucleosomes reassemble quickly following replication.
- Creation of nucleosomes requires:
‒ Disruption of original nucleosomes on the parental DNA
‒ Redistribution of preexisting histones on the new DNA
‒ The addition of newly synthesized histones to complete the
formation of new nucleosomes
Initiator protein
Binds to origin and separates strands of DNA to initiate
replication
DNA helicase
Unwinds DNA at replication fork
Single-strand-binding
proteins
Attach to single-stranded DNA and prevent secondary
structures from forming
DNA gyrase
Moves ahead of the replication fork, making and resealing
breaks in the double-helical DNA to release the torque that
builds up as a result of unwinding at the replication fork
DNA primase
Synthesizes a short RNA primer to provide a 3’-OH group
for the attachment of DNA nucleotides
DNA polymerase III
Elongates a new nucleotide strand from the 3’
DNA polymerase I
Removes RNA primers and replaces them with DNA
DNA ligase
Joins Okazaki fragments by sealing breaks in the sugar–
phosphate backbone of newly synthesized DNA
The Fidelity of DNA replication
Most of the errors that do arise are corrected in a
proofreading process.
– DNA polymerase I: 3" to 5' exonuclease activity removes the incorrectly paired nucleotide
Mismatch repair:
corrects errors after replication is
complete (will discuss more in Ch. 18).
Eukaryotic DNA Replication Differs in
Several Aspects
- Larger size of eukaryotic genomes requires
that replication be initiated at multiple origins - Eukaryotic chromosomes are linear
- DNA template is associated with histone
proteins in the form of nucleosomes, and
nucleosome assembly must immediately follow
DNA replication
Eukaryotic Origins of Replication
Origins of replication vary greatly in sequence
Research suggests origins in eukaryotes are
defined by modifications to the chromatin structure
Origin-recognition complex (ORC);
a multiprotein
complex, serves as initiator
Initiator ORC recruits and loads the helicase
right to the double-stranded DNA at the origin!
In eukaryotic cells – replication is
coordinated with
the cell cycle.
G1/S checkpoint holds cell in G1 until the DNA is
ready to be replicated!
Cell then enters S phase and DNA is replicated.
-This system ensures that the DNA is not replicated
again until the cell passes through mitosis.
The licensing of DNA Replication
Thousands of origins of replication allows entire
eukaryotic genome to be replicated in a timely
manner.
The precise replication is accomplished by separation of
the initiation of replication into two distinct steps
- Origins are first licensed – approved for replication
This takes place early in the cell cycle, when replication
licensing factors attach to each origin. - Replication machinery initiates replication at each licensed
origin.
Eukaryotic Unwinding
Helicases that separate double-stranded DNA have been
isolated from eukaryotic cells.
Enzymes and proteins are assumed to function in
unwinding of eukaryotic DNA in a similar manner as their
bacterial counterparts!
Eukaryotic cells contain many more different DNA polymerases
than bacteria.
Three DNA polymerases carry out most of the nuclear DNA
synthesis during replication
alpha, delta, and epsilon
