Download DNA replication, repair, and recombination

• DNA Replication,
Repair, and
Recombination
DNA
Maintenance
• Mutation rate are
extremely low
• 1 mutation out of 109
nucleotides per
generation
DNA replication
Separation, Base pair
The Chemistry of
DNA replication
DNA Synthesis by DNA polymerase
DNA Polymerase
Nucleotide polymerizing enzyme, first discovered in 1957
DNA replication with
two forks
This Doesn’t Work!
DNA replication Fork
DNA Proofreading
Structures of DNA polymerase during polymerizing and editing
E: exonucleolytic; P: polymerization
Why 5’->3’?
The need for accuracy
Site-directed mismatch repair in eucaryotes
In procaryotes, old DNAs are usually methylated on A while newly
synthesized ones are not. So Cells can distinguish old and newly
synthesized DNAs and mutate mismatches on new ones.
DNA Proofreading
RNA usually doesn’t have this. Why?
• Pairing, correct nucleotide has higher
affinity binding to the moving polymerase
• Un-Paired nucleotide is easier to be off
before covalent ligation, even after binding.
• Exonucleotic proofreading
• Strand directed mismatch repair
DNA Primer synthesis
On Lagging strand
DNA Replication at the
Lagging strand
DNA Ligase
DNA Helicase
DNA double helix are tightly coupled.
High temperature is needed to break
them (95oC)
DNA Binding Protein
SSB: Single Strand DNA-binding Proteins, also called helix
destabilizing proteins
SSB Proteins
DNA
DNA Clamping Protein
Cycle of DNA Polymerase/Clamping Protein loading and unloading
At the lagging strand (how about leading strand?)
Protein machinery for DNA replication
A Moving
Replication
Structure of the Moving Complex
DNA winding
DNA topoisomerase I
DNA topoisomerase II
DNA topoisomerase II
Mammalian replication Fork
(eucaryote, DNA polymerase (primase) a synthesize RNA/DNA,
DNA polymerase delta is the real polymerase)
Summary
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DNA replication 5’->3’
DNA proof reading
Lagging strand, back-stitching, Okazaki
fragment
Proteins involved:
DNA polymerase, primase
DNA helicase and single-strand DNA-binding
protein (SSB)
DNA ligase, and enzyme to degrade RNA
DNA topoisomerases
DNA Replication in
Chromosome
DNA replication in Bacterial Genome
Initiating Proteins for DNA
replication
1. Initiator protein, 2. helicase
binding to initiator protein, 3.
helicase loading on DNA, 4.
helicase opens the DNA and
binds to primase, 5. RNA
primer synthesis, 6. DNA
polymerase binding and DNA
synthesis
Regulation for DNA replication
In Bacteria, hemimethylated origins are resistant to initiation,
delayed methylation leads to delayed initiation at the second phase
Dam methylase
DNA replication in eucaryotes
Multiple replication origin
50 nucleotides/second, autoradiography
The four standard phases of a eucaryotic cell
DNA replication occurring at S Phase (DNA synthesis phase)
G1 and G2, gap between S and M
Different regions of a chromosome are replicated at different times
Arrows point to the replicating regions at different times
Some facts about Replication in
eucaryotes
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Multiple replication origins occurring inclusters (20-80)
(replication units)
Replication units activated at different times
Within replication units, replication origins are separated
30,000-300,000 pairs apart.
Replication forks form in pairs and create a replication
bubbles moving in opposite directions
Different regions on the same chromosome are
replicated at distinct times in S phase
Condensed Chromatin replicates late, while less
condensed regions replicate earlier
The search and
identification of DNA
replication sequence
ARS: autonomously
replicating sequence
Only histidine
expression can help
cells to survive
The origins of DNA replication on chromosome III of the yeast S.
cerevisiae
A close look at an origin of replication in yeast
ORC: origin recognition complex
B1, B2, B3: other regions binding to required proteins
The replication origins of human genes are more complex
Even far distant DNA sequences could be important
Histone remains associated with DNA
In vitro experiments
DNAs with different sizes are replicated. Only the daughter
DNAs replicated from parental DNA with histones showed
histone binding
Addition of new histones
Chromatin assembly factors (CAFs)
help to add and assemble new
nucleosomes
Bacteria DNAs are circular, not a problem
There is a problem for eucaryote DNAs: ???
Hint: Telomere
Telomerase Structure
Reverse transcriptase
with RNA template to
bind to DNA strands
Telomerase and its function
T-loops at the end of
mammalian
chromosomes
Summary
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Specific DNA sequence determine replication
origin, recruiting proteins to form replication
machinery. relatively complex in eucaryotes
Bacteria has single replication origin.
Eucaryotes have multiple origins and less
defined.
Replication forks are activated at different times
in eucaryotes
Telomere and telomerase
DNA Repair
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Spontaneous DNA damage
Pathways to remove DNA damage
Damage detection
The repair of Double-strand break
DNA repair enzymes
Spontaneous Alterations of nucleotides
Red: oxidative damage; blue: hydrolytic attack; green:
uncontrolled methylation
Depurination and deamination
Thymine dimer
Mutation Generation passed on to
daughter DNAs
Mutation Generation passed on to
daughter DNAs
DNA Repair I
DNA Repair II
Recognition of unusual nucleotide
By base flipping recognized by DNA glycosylase family
Emergency DNA Repair for Double helix break
DNA Repair Summary
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Spontaneous DNA damage: spontaneous
alteration of bases, depurination and
deamination, thymine dimer
Pathways to remove DNA damage: base
excision repair, nucleotide excision repair
Damage detection: base flipping
The repair of Double-strand break:
nonhomolous end joining, homologous end
joining
DNA repair enzymes: heat shock proteins
DNA Recombination
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General recombination
Site specific recombination
General DNA Recombination
Heteroduplex joint
General Recombination
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Two homologous DNA molecules cross
over
The site of exchange can occur anywhere
A strand of one DNA molecule has
become base-paired to a strand of the
second DNA to create heteroduplex joint
No nucleotide sequences are altered
The procedure of general recombination
DNA synapsis: base pairs form between
complementary strands from the two
DNA molecules
DNA Hybridization
The initial step for DNA
recombination
RecA protein-mediated DNA synapsis
Rec A has multiple DNA binding sites, hence can
hold a single strand and a double helix together
Rec A is also a DNA-dependent ATPase
DNA Branch Migration
Holiday Junction for DNA recombination
Exchange of the first single strand between two different DNA
double helices is slow and difficult, then intermediate state Holiday
Junction, then complete exchange
Holiday Junction for
DNA recombination
and its resolution
Summary for General
Recombination
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General recombination allows large fraction of
genetic information to move from one
chromosome to another.
General recombination requires the breakage of
double helices, beginning with a single strand
breakage.
General recombination is facilitated by Rec A in
bacteria and its homologs in eucaryotes.
Holiday junction is the intermediate state of
general recombination
Site-specific recombination
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Moves specialized nucleotide sequence (mobile
genetic elements) between non-homologous sites
within a genome.
Transpositional site-specific recombination
Conservative site-specific recombinatinon
Transpositional site-specific
recombination
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Modest target site selectivity and insert mobile
genetic elements into many sites
Transposase enzyme cuts out mobile genetic
elements and insert them into specific sites.
Three of the many types of mobile genetic elements found in bacteria
Transposase gene: encoding enzymes for DNA breakage and joining
Red segments: DNA sequences as recognition sites for enzymes
Yellow segments: antibiotic genes
Cut and Paste Transposition
DNA-only
The structure of the central intermediate formed by transposase (integrase)
Replicative Transposition
Retrovirus-based Transposition
Retroviral-like retrotransposition
Reverse Transcriptase
RNA
Non-retroviral retrotransposition
Conservative Site Specific Recombination
Integration vs. inversion
Notice the arrows of directions
Bacteriophase Lambda
Genetic Engineering to control Gene expression
Summary
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DNA site-specific recombination
transpositional; conservative
Transposons: mobile genetic elements
Transpositional: DNA only transposons,
retroviral-like retrotransposons,
nonretroviral retrotransposons