1. 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

2. 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

3. 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

4. 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

5. 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.

6. Mammalian Mt DNA

7. Mt DNA replication

8. Mammalian (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.

9. Condensing and Packaging of DNA into a small space is a universal feature of cells and other genetic systems.

10. DNA Packaging Problem More Acute for Eukaryotes!
Genomic DNAs are much longer than the cells or viruses that contain them!
On average, eukaryotic cells are ~10X larger than prokaryotic cells, but nuclear DNA is ~1000X larger than bacterial DNA.
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.

12. Structure of a Eukaryotic Nucleus

13. 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

14. 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

15. Electron microscopic views of pores in the nuclear envelope.
Freeze-fracture EM
Transmission EM (TEM)

16. Model of a nuclear pore (A is top view)
Fig. 1.37, Buchanan et al.

17. 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
TEM – transmission electron microscopy

18. Tobacco meristem cell : Nucleus with large Nucleolus, and Euchromatin.
Stars indicate heterogeneity in the nucleolus.
Euchromatin

19. Narcissus flower cell with heterochromatin in the nucleus.

20. Electron microscopy of a “chromatin spread”.
Eukaryotic Chromatin
Electron microscopy of a “chromatin spread”.
A.k.a. a “Miller Spread”, after Oscar Miller, the inventor.
Oscar
Nucleosomal “beads-on-a-string” structure.

21. Nucleosomes (beads) contain Histones
21,000 Daltons
15,000
11,000
core

22. Bacteria and organelles (in some eukaryotes) have Hu (histone-like) protein that compacts DNA. They also have proteins that anchor the genomic DNA to membranes (thylakoid membranes in chloroplasts, inner membrane in mitochondria, and cytoplasmic membrane in E. coli).

23. 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

24. 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)

25. Condensation of SV40 DNA into nucleosomes
SV40 chromosome: nucleosomal DNA
Naked (or Nekkid) SV40 DNA

26. 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 e-g

27. 30 nM Fiber is a Solenoid with 6 nucleosomes per turn
H1 links nucleosomes together in the solenoid.
Side view
End view
Histone H1 links nucleosomes together in the solenoid.

28. Solenoid attaches to Scaffold, generating Loops
Packing ratio ~ 25 for this step = 1000 overall

29. Fig

30. Double
Helix
Solenoid (condensed fiber)
Loops
“Snaking
the Solenoid”
700 nm fiber
Beads-
on-a-string
Probably involve scaffold attachment regions (SARs) in the DNA being packaged.

31. Packing DNA in a Eukaryotic Nucleussc