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

*GATC normally methylated on the ACTAG *• 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 system2. Recognized by the Mismatch Repair System

Fig. 20.39

Mismatch repairin 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.

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 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

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: and is gapped on L strand.

5 L strand finishes replicating, and then both and are converted to supercoiled forms.

Mammalian (mouse) mtDNA Replication

Condensing and Packaging of DNA into a small space is a universal feature

of cells and other genetic systems.

Lengths of various genomes

Organism or organelle Type Approximate Length

TMV 1 single-stranded RNA 2 um 6.4 kb

Adenovirus 1 double-stranded DNA 11 um 35 kb

Bacteriophage T4 1 double-stranded DNA 55 um 170 kb

E. coli 1 double-stranded DNA 1.3 mm 4.2 X 103 kb

Human mitochondria about 10 identical double-

stranded DNAs

5 um each 16 kb each

Human nucleus 46 chromosomes of double-

stranded DNA

1.8 m 6X 106 kb

from Genes IV Benjamin Lewin pg 390

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.

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. Histones2. Non-histone chromosomal proteins

• Two main types of chromatin:1. Euchromatin - dispersed appearance by

TEM, transcriptionally active2. 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

Electron microscopy of a “chromatin spread”.

A.k.a. a “Miller Spread”, after Oscar Miller, the inventor.

Nucleosomal “beads-on-a-string” structure.

Eukaryotic Chromatin

21,000 Daltons

15,00011,000

Nucleosomes (beads) contain Histones

core

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

H4

H3

H1

H2B

H2A

DNA

Packing ratio ~5 (DNA is condensed ~5-fold by forming it into nucleosomes)

Nucleosome core = octamer of histones (2 each of H2A, H2B, H3, and H4) + 2 wraps (145 bp) of DNA

Condensation of SV40 DNA into nucleosomes

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

100 mM NaCl

Chromatin condenses further into a 30 nM (diameter) Fiber when made up to near-physiological ionic strength.

Packing ratio ~6-8-fold for this step

Similar to Fig. 13.10e-g

H1 histone

DNA

Nucleosomecore

27Å

110Å

57Å

30 nM Fiber is a Solenoid with 6 nucleosomes per turn

Side view End view

Histone H1 links nucleosomes together in the solenoid.

Packing ratio ~ 25 for this step = 1000 overall

Solenoid attaches to Scaffold, generating Loops

Fig. 13.11

DoubleHelix

Beads-on-a-string

Solenoid (condensed fiber)

Loops“Snaking the Solenoid”

700 nm fiber

Probably involve scaffold attachment regions (SARs) in the DNA being packaged.

sc

Packing DNA in a Eukaryotic Nucleus