Download Molecular Biology of the Cell

Alberts • Johnson • Lewis • Morgan • Raff • Roberts • Walter
Molecular Biology
of the Cell
Sixth Edition
Chapter 5
DNA Replication, Repair,
and Recombination
Copyright © Garland Science 2015
”…short-term survival of cell can depend on
preventing changes in its DNA, the long-term
survival of a species requires that DNA
sequences be changeable over many
generations…”
Topics
• DNA replication mechanisms
• The initiation and completion of DNA
replication in chromosomes
• DNA repair
• Homologous recombination
• Pages: 237-287
Terms to know
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DNA helicase
DNA ligase
DNA polymerase
DNA primase
DNA topoisomerase
Lagging and leading strand
Replication fork
RNA primer
Single strand DNA binding protein
Strand directed mismatch repair
Chemistry of DNA synthesis
• Base pairing between
incoming nucleotide and
existing strand of DNA
guides formation of new
strand
• Incoming nucleotide
provides energy for
reaction
• What would happen if ddCTP were added to a DNA
replication reaction in large excess over dCTP
• What would happen if ddCTP were added to 10% of
the concentration of dCTP?
• What would happen if ddCMP were added to 10% of
the concentration of dCTP or in large excess?
Current view of the events at the
bacterial replication fork
Proofreading mechanisms of DNA
replication
• Overall fidelity of replication is about 1 in 1010
• Initial base-pairing is not sufficient to maintain
fidelity of DNA replication
– Besides the standard complementary base
pairs also alternative base pairing is
possible
– With slight changes in helix geometry two
H-bonds can be formed between G and T
– Tautomeric forms of the four base pairs,
e.g. C pairs with A, G with T
DNA synthesis – effect of correct
base pairing
• Correct nucleotide has higher affinity for the moving polymerase
as correct base pairing is energetically more favorable
• Conformational change of polymerase occurs more readily with
correct base pairing
Exonucleolytic proofreading by DNA
polymerase
• C accidentally incorporated at
the end of the growing end
• For further elongation a basepaired 3´-OH end needs to be
available, unpaired ends not
readily extended
• 3’ to 5’-exonucleolytic
proofreading activity removes
unpaired and also mispaired
residues
Strand distinction mechanisms
• How to differentiate between newly synthesized and
template DNA?
• E. coli/prokaryotes
– Methylation of A residue in the sequence GATC
– Methylation occurs some time after DNA
replication and allows distinguishing new from old
strand
• Eukaryotes:
– Recognition is based on nicks on the DNA
– On the lagging strand nicks are a result from
discontinous DNA synthesis, leading strand nicks
are introduced by unknown mechanism
Strand-directed mismatch repair
• Error rate after replication is
about 1 error in 107 bases
• Mismatch repair detects
distortions in the DNA helix
• MutS binds to mismatch
• MutL scans for nick and
triggers degradation of nicked
strand
• In bacteria MutH introduces
nicks at unmethylated GATC
Pro- and eukaryotic DNA replication
• Fundamental features of pro- and eukaryotic
DNA replication are conserved
• More components in eukayotic replication
machines, e.g. different polymerases for
leading and lagging strand
• Eukaryotic replication machinery must
replicate through nucleosomes
• Eukaryotic DNA replication tighly coordinated
with mitosis
Initiation of replication – a replication
bubble formed by replication fork initiation
• The position at which DNA is
first opened is called
replication origin
• DNA around replication
origin are normally rich in AT base pairs
• Two replication forks are
finally formed and move in
opposite directions
Initiation of DNA replication in
bacteria
• Binding of initiator proteins to DNA
destabilizes DNA double helix
• Helicases (DnaB) are brought in by
Helicase loading proteins
preventing binding to other ssDNA.
• Helicase opens up the single
stranded region enabling primases
(DnaG) to bind
• Completion of repliation fork
DnaA
DnaB
DnaG
+SSBproteins
Control of replication initiation
• Replication and cell divison are not necessarily synchronized but
cell must ensure that only one round of replication occurs per cell
cycle
• Premature onset of next replication rounds must be prevented
• Seq A protein binds to hemimethylated origin blocking initiator
proteins
• Origin becomes replication competent after methylation and
dissociation of Seq A (in E.coli Dam methylase is responsible)
Multiple origins of replication exist in
mammals
• Replication rate: 50 nucleotides per second
• Average length of human chromosome: 150*106 bp
– 30000 – 50000 origins of replication present on single
chromosome
DNA replication initiation in
eukaryotes
• Mechanism must ensure that each ori
is only activated once per cell cycle
• Prereplicative complex forms during
G1 phase
• Phosphorylation activates helicase
and leads to displacement of origin
recognition complex (ORC)
• Replication takes places during S
phase
• ORC rebinds in phosporylated form
and cannot accept helicase unless it
becomes dephosphorlyated in G1
phase
Chromosome structure and replication
• Chromosome replication requires
synthesis and assembly of new
chromosomal proteins
• Histone octamer broken into H3-H4
tetramer and H2A-H2B dimer
• H3-H4 tetramer stays loosely
attached to DNA, randomly
distributed between both strands
• Nucleosomes complemented with
newly synthesized histone proteins
• Lenght of Okazaki fragments
restricted by positions of
nucleosomes
Displacement requires also
chromatin remodeling complex
Replication of DNA ends: Telomer
replication
• The final RNA primer on the
lagging-strand template cannot
be replaced by DNA because
there is no free 3’-OH end
available
• Telomere DNA is recogized by
sequence-specific DNA-binding
proteins
• Telomerases recognize tip of
telomere DNA and elongates it in
the 5’-3’ direction using an RNA
template
Terms to know
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Base excision repair
DNA repair
Nonhomologous end joining
Nucleotide excision repair
Homologous recombination
DNA repair
• DNA is highly stable material
• Susceptible under physiological conditions to
spontaneous changes
– Hydrolysis of N-glycosyl linkages to deoxyribose,
depurination & depyrimidation
– Deamination of cytosine
– Encounter with reactive metabolites, oxygen, Sadenosylmethionine
– UV-radiation
• DNA is under constant surveillance for damage
Deamination =
hydrolysis
methylation
oxidation
Hydrolysis
Hydrolysis
• These DNA lesions are result
of normal chemical reactions
that take place in a cell.
• Exposure to external
chemicals increases
formation and greater variety
modifications occurs
Base excision repair
• Mediated by set of DNA
glycosylases, recognizing a
specific type of altered base
• Glycosylases move along DNA
and flip bases out of the helix
• Glycosylases probe all faces of
the base and recognize
modifications
• Damaged base is removed
Recognition of unusual nucleotide in
DNA by base-flipping
Nucleotide excision repair
• Targets bulky lesions such
as pyrimidine dimers,
chemical modifications
• Enzyme complex scans
DNA for distortions in the
helix
• Phosphodiester backbone is
cleaved on both side of
lesion and single-stranded
oligonucleotide is removed
Repair of double-strand breaks
• DNA double-helix breaks occur due to
ionizing radiation, replication errors, oxidizing
agents and other metabolites
• Two distinct mechanisms exist
– Nonhomologous end joining, quick and
dirty solution by joining two ends by DNA
ligase
– Homologous recombination, accurate
repair mechanism
• Nonhomologous end joining creates deletion
• Homologous recombination restores the original DNA sequence
CRISPR/Cas9 based genome
engineering – a case for cellular DNA
repair
Shao et al., Nature Protocols 9:2493–2512 (2014)
Non-homologous end joining
• Initial degradation of the broken
DNA ends is important because
nucleotides at the site of initial
break are often damaged.
• Central role is played by the Ku
protein, a heterodimer that
grasps broken chromosome
ends
• Often creates scars in the
genome
• Any two free ends can be joined,
e.g. leading to chromosome
rearrangments
Homologous recombination
• Heteroduplex formation is an essential
feature of HR
Probing for
complementary
bases
• For HR to occur the broken DNA has to be
broughy into proximity with the homologous,
but unbroken DNA
Homologous recombination
• Basic steps:
– Creating 3’ overhang
– Strand exchange by
complementary base
pairing
– DNA synthesis and
ligation of nicks
• Exact pathways of HR
differ from one case to the
next
Critical step: Strand invasion by
RecA
• ATP-bound RecA binds single
stranded DNA holding it in an
elongated form (groups of
nucleotide triplets separated)
• Protein-DNA filaments binds to
dsDNA and stretches it
destabilizing the helix
• Invading strand probes the DNA
for complementary regions using
triplets, if match, adjacent
tripletes are probed
• If extensive match is found, ATP
is hydrolysed and RecA
dissociates
Spotlight: lnactivation of NHEJ
improves genetic engineering
PLoS ONE 7(6): e39720. doi:10.1371/journal.pone.0039720