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: 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

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.

Mammalian Mt DNA

Mt DNA replication

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.

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

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.

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 ?) 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

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

Narcissus flower cell with heterochromatin in the nucleus.

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.

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

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).

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

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 SV40 chromosome: nucleosomal DNA Naked (or Nekkid) SV40 DNA

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

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.

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

Fig. 13.11

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.

Packing DNA in a Eukaryotic Nucleus sc