Bacterial DNA replication Summary: What problems do these proteins solve? Tyr OH attacks PO4 and...
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Bacterial DNA replication
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Summary: What problems do these proteins solve?
Tyr OH attacks PO4 and forms a covalent intermediate
Structural changes in the protein open the gap by 20 Å!
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Function E. coli SV40 (simian virus 40)
Helicase DnaB T antigen
Primase
Primer removal
Primase (DnaG)
pol I’s 5’-3’exo
pol primase
FEN 1 (also RNaseH)
Polymerase
Core pol III (, , subunits) pol ,
Clamp loader complex RF-C
Sliding clamp PCNA
ssDNA binding SSB RF-A
Remove +sc at fork (swivel) gyrase topo I or topo II
Decatenation topo IV topo II
Ligase DNA ligase DNA ligase I
… other model systems include bacteriophage T4 and yeast
Summary: What problems do these proteins solve?
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The ends of (linear) eukaryotic chromosomes cannot be replicated by the replisome.
Not enough nucleotides for primase to start last lagging strand fragment
Chromosome ends shorten every generation!
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Telomere shortening signals trouble!
1. Telomere shortening releases telomere binding proteins (TBPs)
2. Further shortening affects expression of telomere-shortening sensitive genes
3. Further shortening leads to DNA damage and mutations.
Telomere binding proteins (TBPs)
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Telomerase replicates the ends (telomeres)
Telomere ssDNA
Telomerase extends the leading strand!Synthesis is in the 5’-->3’ direction.
Telomerase is a ribonucleoprotein (RNP). The enzyme contains RNA and proteins.
The RNA templates DNA synthesis. The proteins include the telomerase reverse transcriptase TERT.
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Telomerase cycles at the telomeres
Telomere ssDNA
TERT protein
TER RNA template
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Telomerase extends a chromosome 3’ overhang
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Conserved structures in TER and TERT
Core secondary structures shared in ciliate and vertebrate telomerase RNAs (TERs). (Sequences highly variable.)
148-209 nucleotides
1000s of nucleotides
TERT protein sequences conserved
1300 nucleotides
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Starting and stopping summary
1. DNA replication is controlled at the initiation step.
2. DNA replication starts at specific sites in E. coli and yeast.
3. In E. coli, DnaA recognizes OriC and promotes loading of the DnaB helicase by DnaC (helicase loader)
4. DnaA and DnaC reactions are coupled to ATP hydrolysis.
5. Bacterial chromosomes are circular, and termination occurs opposite OriC.
6. In E. coli, the helicase inhibitor protein, Tus, binds 10 ter DNA sites to trap the replisome at the end.
7. Eukaryotic chromosomes are linear, and the chromosome ends cannot be replicated by the replisome.
8. Telomerase extends the leading strand at the end.
9. Telomerase is a ribonucleoprotein (RNP) with RNA (template) and reverse-transcriptase subunits.
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DNA methods summary
1. Restriction enzymes cut at specific DNA sites. (N)2. Vectors allow genes to be “cloned” and proteins
“expressed”. (N)3. Gel electrophoresis separates DNA on the basis of
size.4. DNAs can be synthesized (up to ~100 bases
commercially). (N)5. PCR amplifies any target DNA sequence. (N)6. Genes and genomes can be sequenced by chain
termination. (N)7. Oligonucleotides can be used to change bases by
“site- directed mutagenesis”. (N)8. “Southern” blotting detects sequences by
hybridization.9. Microarrays detect gene expression patterns over the
genome.10.Genes can be knocked out (deleted) or replaced in
prokaryotes and eukaryotes. (N)
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Restriction enzymes cut DNA at specific sites
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Restriction enzymes cut DNA at specific sites
• 3 types of ends: 5’ overhang, blunt and 3’ overhang• Cognate methyl transferases protect host genome from digestion.
Restriction-modification systems degrade “foreign” DNA.
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Average frequency of restriction sites in “random” DNA sequences
The average occurrence of each sequence = 1/4n,where n = the site length and all bases are equally represented
Site size4
6
8
Average frequency 1/256
1/4,096
1/65,536
(1/4 x 1/4 x 1/4 x 1/4)
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Lots of different recognition sites known
Core four basesFlanking bases
None
A----T
C----G
G----C
T----A
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A simple cloning procedure
1. Cut “insert” and “vector” DNA with a restriction enzyme
2. Mix and join ends with DNA ligase. The ends should match for efficient ligation.
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Cloning without DNA ligase
1. Prepare open vector and insert with the same long “sticky” ends
+ Pol I Klenow fragment + dATP
1. Mix and let the ends anneal.
2. Transform the nicked plasmid. The plasmid is repaired in vivo.
1. Prepare an insert flanked by sites for a site-specific DNA recombinase.
2. Mix insert with the closed vector containing the recipient recombination site and recombinase enzyme.
+
3. (Have lunch.) Transform.
Ligation-independent cloning “Gateway” cloning
E +
No dT in template
TT
TTAA
T
ATA
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“Vectors” allow DNA sequences to be cloned - 1
Phage for cloning big (7-
25 kb) DNA pieces
Ori + selectable marker +
cloning site (polylinker)
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“Vectors” allow DNA sequences to be cloned - 2
“Reporter” genes:-gal, GFP . . .
Shuttle vectors: move genes
between organisms
Expression vectors:Make your favorite protein
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“Vectors” allow DNA sequences to be cloned -3
Transient transfection: eukaryotes
Stable transduction
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Gel electrophoresis separates DNA on the basis of size
Agarose: big fragments (>300 bp)Acrylamide: smaller fragments, higher resolution
Mobility proportional to log MW.
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Chemical DNA synthesis
Sequential rounds of coupling, oxidation and deprotection of the 5’ OH build up the oligonucleotide.
3’
5’
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Chemical DNA synthesis
Sequential rounds of coupling, oxidation and deprotection of the 5’ OH build up the oligonucleotide.
3’
5’
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Frontiers in DNA synthesis
Currently: 100-200 nucleotides routine (Assemble 5 kB) 10,000 = largest. Primer set for the human genome (30,000 genes) ~ $104
Goal 1: Make yeast chromosome 3: 300 kB without errors!
(Jeff Boeke)
Goal 2: Assemble 16 X 106 w/o errors for ~$1000 (George Church)
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DNA sequencing by partial chain termination
ddNTPs terminate the
chain
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DNA sequencing by partial chain termination
Small amount of ddGTP + excess dGTP partially
terminates chains at Cs in the template
ddNTPs terminate the
chain
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DNA sequencing by partial chain termination
1. All fragments start at the primer
2. All fragments ending in a particular base have a different length and a different color tag
3. Separating the mixture of products by size reveals the sequence.
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Two strategies for genome sequencingHierarchical Shotgun
SequencingSequencing
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PCR (Polymerase Chain Reaction): isolate and amplify any DNA sequence
N cycles amplifies the target sequence 2N-fold.
Copies: 1 2 4 8
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Site-directed mutagenesis
1. Anneal divergent mutagenic primers.
2. Replicate entire plasmid with a DNA pol lacking 5’-->3’ exonuclease.
3. Select against parental strands.
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Gene replacement in mice -- make donor cells
1. Insert drug markers into genome of ES cells
2. Select to enrich for homologous
recombinants
Check insertion site by Southern blotting
Neor confers resistance to G-418.
tkHSV confers sensitivity to ganciclovir.
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“Southern” blotting detects DNA sequences by hybridization
1. Digest DNA using restriction enzyme(s)
2. Run gel
3. Transfer DNA from gel to (nitrocellulose) paper.
4. Denature DNA, hybridize probe DNA, and wash off excess probe.
5. Detect the probe on the paper. E.g. by autoradiography.
“Northern” blotting detects RNA on the gel.
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Microarrays detect expressed genes by hybridization
1. Label cDNAs with red fluorophore in one condition and green fluorophore in another reference condition.
2. Mix red and green DNA and hybridize to a “microarray”.3. Relative to the reference, Red=enriched, yellow = =,
green = depleted.
Each spot has a different synthetic oligonucleotide complementary to a specific gene.
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Gene replacement in mice -- germline incorporationTransgenic mice express
a new geneWhich mouse
expresses extra copies of the
growth hormone gene?
1. Inject ES cells into early embryos, 2. Transfer embryos to foster mother,3. Breed chimeric mice and screen for progeny with mutant germ line,4. Screen progeny DNA for mutation, 5. Mate heterozygotes (X+/X-),6. Screen progeny DNA for KO genotype (X-/X-).
Entire process takes a year.
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DNA methods summary
1. Restriction enzymes cut at specific DNA sites. (N)2. Vectors allow genes to be “cloned” and proteins
“expressed”. (N)3. Gel electrophoresis separates DNA on the basis of
size.4. DNAs can be synthesized (up to ~100 bases
commercially). (N)5. PCR amplifies any target DNA sequence. (N)6. Genes and genomes can be sequenced by chain
termination. (N)7. Oligonucleotides can be used to change bases by
“site- directed mutagenesis”. (N)8. “Southern” blotting detects sequences by
hybridization.9. Microarrays detect gene expression patterns over the
genome.10.Genes can be knocked out (deleted) or replaced in
prokaryotes and eukaryotes. (N)