Repair of replication errors by the MisMatch Repair System: Marking newly synthesized DNA in E. coli

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Repair of replication errors by the MisMatch
Repair System Marking newly synthesized DNA in
E. coli
GATC normally methylated on the A CTAG

• Newly synthesized strands not methylated right
  away, delayed for 10 minutes gives
  hemi-methylated DNA
  
• GATC
• CTAG

Hemi-methylated DNA 1. Not recognized by the
oriC activation system 2. Recognized by the
Mismatch Repair System
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Mismatch repair in E. coli
MutL and mutS proteins recognize mismatch, and
activate mutH. mutH nicks strand across from
nearest methylated GATC. A helicase
exonuclease degrade from nick to beyond the
mismatch. DNA Pol III ligase do repair
synthesis.
Fig. 20.39
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Mismatch Repair

• Repairs replication errors that create mismatches
• In E. coli, new DNA not methylated right away
• mismatch recognized by mutS, then mutL binds and
  attracts mutH (endonuclease that cleaves nearest
  CTAG that is not methylated)
• Eucaryotes have mutS and mutL homologues, but no
  mutH
• also have the requisite exonuclease, but not
  clear how the strand specificity is determined

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Mismatch Repair and Colon Cancer

• Hereditary nonpolyposis colon cancer (HNPCC)
• 1/200 Americans is affected (15 of colon
  cancers)
• Characterized by microsatellite instability
• Microsatellites are tandem repeats of 1-4 bp
  sequences that change during lifetime of HNPCC
  patients
• Microsatellites are prone to replication slippage
  resulting in insertions or deletions, which are
  normally repaired by the Mismatch Repair (MMR)
  System
• Mutations in one of 5 mismatch repair (MMR)
  genes increase susceptibility to HNPCC

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Mammalian Mitochondrial DNA (MtDNA)

• Multi-copy, circular molecule of 16,000 bp.
  Uniparental-maternal inheritance.
• 2. Encodes genes for respiration (13 proteins)
  and translation (22 tRNAs, 2 rRNAs).
• 3. 2 promoters (1 on each strand) the STOP
  codons for the protein genes, UAA, created
  post-transcriptionally by polyadenylation
• 4. Some genetic diseases caused by mutations in
  mtDNA. Also, MtDNA mutations accumulate during
  aging.
• 5. MtDNA used to define phylogenetic
  relationships between species, subspecies, etc.,
  or define breeding populations.

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Mammalian Mt DNA
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Mt DNA replication
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Mammalian (mouse) mtDNA Replication

1. Two origins of replication H (for heavy strand)
   and L (for light strand) that are used
   sequentially for unidirectional replication
   (from each origin).
2. Persistent D-loop at H ori, which is extended to
   start replication of the H strand.
3. Once 2/3 of H strand is replicated, L ori is
   exposed and replication of L strand starts.
4. The lagging L strand replication gives 2 type of
   molecules a and b. b is gapped on L strand.
5. b L strand finishes replicating, and then both a
   and b are converted to supercoiled forms.

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Condensing and Packaging of DNA into a small
space is a universal feature of cells and other
genetic systems.
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Genomic DNAs are much longer than the cells or
viruses that contain them!
DNA Packaging Problem More Acute for
Eukaryotes! On average, eukaryotic cells are
10X larger than prokaryotic cells, but nuclear
DNA is 1000X larger than bacterial DNA.
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Structure of a Eukaryotic Nucleus
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Nuclear Architecture Overview

• Double-membrane envelope
• Has lumen that is continuous with ER
• Outer membrane also has ribosomes like ER
• Pores in nuclear envelope
• large, complex structures with octahedral
  geometry
• allow proteins and RNAs to pass
• transport of large proteins and RNAs requires
  energy
• Nuclear proteins have nuclear localization
  signals (NLS)
• short basic peptides, not always at N-terminus

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Nuclear architecture (cont.)

• nuclear skeleton (or lamina)
• intermediate filaments (lamins)
• anchor DNA and proteins (i.e., chromatin) to
  envelope
• Nucleolus
• site of pre-rRNA synthesis and ribosome assembly

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Electron microscopic views of pores in the
nuclear envelope.
Freeze-fracture EM
Transmission EM (TEM)
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Model of a nuclear pore (A is top view)
Fig. 1.37, Buchanan et al.
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DNA is in Chromatin

• DNA proteins ( RNAs ?)
• Histones
• Non-histone chromosomal proteins
• Two main types of chromatin
• Euchromatin - dispersed appearance by TEM,
  transcriptionally active
• Heterochromatin dense appearance by TEM,
  transcriptionally repressed, includes highly
  repetitive regions such as telomeres and
  centromeres

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Tobacco meristem cell Nucleus with large
Nucleolus, and Euchromatin. Stars indicate
heterogeneity in the nucleolus.
Euchromatin
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Narcissus flower cell with heterochromatin in the
nucleus.
Heterochromatin
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Eukaryotic Chromatin
Electron microscopy of a chromatin spread.
A.k.a. a Miller Spread, after Oscar Miller,
the inventor.
Nucleosomal beads-on-a-string structure.
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Nucleosomes (beads) contain Histones
21,000 Daltons 15,000 11,000
core
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• Bacteria and organelles (in some eukaryotes)
  dont have nucleosomes but do have a histone-like
  protein (Hu) that compacts DNA.
• They also have proteins that anchor the genomic
  DNA to these membranes
• thylakoid membrane in chloroplasts
• inner membrane in mitochondria
• cytoplasmic membrane in E. coli

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Nucleosome core octamer of histones (2 each of
H2A, H2B, H3, and H4) 2 wraps (145 bp) of DNA
Packing ratio 5 (DNA is condensed 5-fold by
forming it into nucleosomes)
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Condensation of SV40 DNA into nucleosomes
SV40 chromosome nucleosomal DNA
Naked (or Nekkid) SV40 DNA
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Chromatin condenses further into a 30 nM
(diameter) Fiber when made up to
near-physiological ionic strength.
100 mM NaCl
Packing ratio 6-8-fold for this step
Similar to Fig. 13.10e-g
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30 nM Fiber is a Solenoid with 6 nucleosomes per
turn
Side view
End view
Histone H1 links nucleosomes together in the
solenoid.
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Solenoid attaches to Scaffold, generating Loops
Packing ratio 25 for this step 1000 overall
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Fig. 13.11
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700 nm fiber
Solenoid (condensed fiber)
Loops Snaking the Solenoid
Double Helix
Beads- on-a-string
Probably involve scaffold attachment regions
(SARs) in the DNA being packaged.
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Packing DNA in a Eukaryotic Nucleus
sc