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
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
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
Mismatch Repair and Colon Cancer
•
•
•
•
Hereditary nonpolyposis colon cancer (HNPCC)
1/200 Americans is affected (15% of colon cancers)
Characterized by microsatellite instability:
1. Microsatellites are tandem repeats of 1-4 bp
sequences that change during lifetime of
HNPCC patients
2. 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
Mammalian Mitochondrial DNA (MtDNA)
1.
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 posttranscriptionally 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.
Mammalian Mt DNA
Mt DNA replication
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.
Condensing and Packaging of
DNA into a small space is a universal feature
of cells and other genetic systems.
Genomic DNAs are much longer than the cells or viruses
that contain them!
Lengths of various
Organism or organelle
TMV
Adenovirus
Bacteriophage T4
E. coli
genome s
Type
1 single-st randed RNA
1 doubl e-stranded DNA
1 doubl e-stranded DNA
1 doubl e-stranded DNA
Approximate Length
2 um
6.4 kb
11 um
35 kb
55 um
170 kb
1.3 mm
4.2 X 103 kb
Human mitochondria
about 10 ident ical doublest randed DNAs
46 chromosomes of doublest randed DNA
5 um each
16 kb each
1.8 m
6X 106 kb
Human nucleus
from Genes IV Benjam in Lewin pg 390
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.
Structure of
a Eukaryotic
Nucleus
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
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
Electron microscopic views of pores in the nuclear envelope.
Freeze-fracture EM
Transmission EM (TEM)
Model of a nuclear pore (A is top view)
Fig. 1.37, Buchanan et al.
DNA is in “Chromatin”
•
DNA + proteins (+ RNAs ?)
1. Histones
2. Non-histone chromosomal proteins
•
Two main types of chromatin:
1. Euchromatin - dispersed appearance by
TEM, transcriptionally active
2. Heterochromatin – dense appearance by
TEM, transcriptionally repressed, includes
highly repetitive regions such as
telomeres and centromeres
Tobacco meristem
cell : Nucleus with
large Nucleolus,
and Euchromatin.
Stars indicate
heterogeneity in the
nucleolus.
Euchromatin
Narcissus flower
cell with
heterochromatin
in the nucleus.
Heterochromatin
Eukaryotic Chromatin
Electron microscopy
of a “chromatin
spread”.
A.k.a. a “Miller
Spread”, after Oscar
Miller, the inventor.
Nucleosomal “beads-ona-string” structure.
Nucleosomes (beads) contain Histones
21,000 Daltons
core
15,000
11,000
Bacteria and organelles (in some
eukaryotes) don’t 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
H2B
H2 A H3
H1
H4
H2 B
DNA
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)
Condensation of SV40 DNA into nucleosomes
Naked (or Nekkid) SV40 DNA
SV40 chromosome:
nucleosomal DNA
Chromatin condenses further into a 30 nM
(diameter) Fiber when made up to nearphysiological ionic strength.
100 mM NaCl
Packing ratio ~6-8-fold for this step
Similar to Fig. 13.10e-g
30 nM Fiber is a Solenoid with 6 nucleosomes per turn
DNA
27Å
H1 hist one
110Å
Nucleosome
cor e
57Å
Side view
End view
Histone H1 links nucleosomes together in the solenoid.
Solenoid attaches to Scaffold, generating Loops
Packing ratio ~ 25 for this step = 1000 overall
Fig. 13.11
Double
Helix
Beadson-a-string
Solenoid
(condensed
fiber)
700 nm fiber
Loops
“Snaking
the Solenoid”
Probably involve scaffold
attachment regions (SARs)
in the DNA being packaged.
Packing DNA in a Eukaryotic Nucleus
sc