Title: Repair of replication errors by the MisMatch Repair System: Marking newly synthesized DNA in E. coli
1Repair 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
2Mismatch 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
3Mismatch 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
4Mismatch 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
5Mammalian 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.
6Mammalian Mt DNA
7Mt DNA replication
8Mammalian (mouse) mtDNA Replication
- Two origins of replication H (for heavy strand)
and L (for light strand) that are used
sequentially for unidirectional replication
(from each origin). - Persistent D-loop at H ori, which is extended to
start replication of the H strand. - Once 2/3 of H strand is replicated, L ori is
exposed and replication of L strand starts. - The lagging L strand replication gives 2 type of
molecules a and b. b is gapped on L strand. - b L strand finishes replicating, and then both a
and b are converted to supercoiled forms.
9Condensing and Packaging of DNA into a small
space is a universal feature of cells and other
genetic systems.
10Genomic 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.
11(No Transcript)
12Structure of a Eukaryotic Nucleus
13Nuclear 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
14Nuclear 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
15Electron microscopic views of pores in the
nuclear envelope.
Freeze-fracture EM
Transmission EM (TEM)
16Model of a nuclear pore (A is top view)
Fig. 1.37, Buchanan et al.
17DNA 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
18Tobacco meristem cell Nucleus with large
Nucleolus, and Euchromatin. Stars indicate
heterogeneity in the nucleolus.
Euchromatin
19Narcissus flower cell with heterochromatin in the
nucleus.
Heterochromatin
20Eukaryotic Chromatin
Electron microscopy of a chromatin spread.
A.k.a. a Miller Spread, after Oscar Miller,
the inventor.
Nucleosomal beads-on-a-string structure.
21Nucleosomes (beads) contain Histones
21,000 Daltons 15,000 11,000
core
22(No Transcript)
23- 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
24Nucleosome 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)
25Condensation of SV40 DNA into nucleosomes
SV40 chromosome nucleosomal DNA
Naked (or Nekkid) SV40 DNA
26Chromatin 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
2730 nM Fiber is a Solenoid with 6 nucleosomes per
turn
Side view
End view
Histone H1 links nucleosomes together in the
solenoid.
28Solenoid attaches to Scaffold, generating Loops
Packing ratio 25 for this step 1000 overall
29Fig. 13.11
30700 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.
31Packing DNA in a Eukaryotic Nucleus
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