Lecture 1 DNA Structure

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Lecture 1 - DNA Structure

• DNA composition
• DNA forms
• Chromosomes

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All nucleotides have a common structure
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The five principal bases in nucleic acids
A, G, T, C are present in DNA A, G, U, C are
present in RNA
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The phosphodiester backbone of DNA
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The double helix
Hydrogen bonding between complementary base pairs
(A-T or G-C) holds the two strands together A-T
2 H bonds G-C 3 H bonds
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DNA can adopt different conformations
B-DNA Most common, 10 bp per helical
turn. A-DNA 11 bp per turn, RNA/DNA and RNA/RNA
helices. Z-DNA Left-handed helix. Triple-helix
Poly Pu/poly Py/poly Py All can exist in a
chromosome simultaneously.
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DNA forms
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Disease associations with Z DNA

• Rheumatoid arthritis
• Multiple sclerosis
• Type 1 Diabetes
• Infectious disease susceptibility
• Chronic hepatitis C
• Leukemia
• Lymphoma

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Z-DNA and Disease?
NRAMP1 -- metal transporter on macrophage
surface. NRampI effects macrophage function ?
Expression of chemokine KC, TNf-a, iNOS, MHC
class II expression Hyper-expression --
autoimmune disease? Reduced expression --
infectious disease susceptibility J Med Genet
(1999)36(4)295-9
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NRAMP1-gene promoter

• The promoter contains a Z-DNA stretch with 4
  alleles
• Allele 2 - t(gt)5ac(gt)5ac(gt)10g
• Allele 3 - t(gt)5ac(gt)5ac(gt)9g
• Allelles differ in expression to external stimuli
• LPS ? expression driven by allele 2
• LPS ? espression driven by allele 3

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Conclusions

• Chronic activation of allele 3 driven expression
  of NRAMP linked to autoimmune susceptibility
• Increased promoter activity
• Low level expression driven by allele 2
  contributes to infectious disease susceptibility
• Lower promoter activity
• Allele 3 protects against infectious disease
• Allele 2 protects against autoimmune disease

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Polymorphisms in the Z-DNA-forming motif of the
promoter and disease

• NRAMP1 also referred to as SCL11A1 gene
• Different populations have different frequencies
  of alleles
• Identification of association with disease
• PCR amplification of promoter regions
• Sequence analysis
• Comparison of diseased population with control

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Role of NRAMP1 gene in MS

• Role of Z-DNA forming repeat polymorphisms
• Comparison of allelic distribution in MS
  patients and controls in different populations
• Association of allele 5 in comparison of
  population matched controls
• Populations with low MS allele 5 not detected
• Populations with high prevalence allele 5 more
  frequent
• Kotze et al 2001 Blood, Cells, Molec Dis 2744

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SLC11A1 promoter and type 1 diabetes

• SCLC11A1 found in region of chromosome important
  in susceptibility to Type 1 diabetes
• Protective effect of allele 2
• Low expression of SCL11A1 protein
• Allele 3 more frequent in Type 1 diabetes (NS)

Nishino et al. 2005. Metabolism54628
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Z- DNA and disease

• Alzheimers disease
• Severely infected Z DNA conformation
• Moderate - probable B-Z intermediate form
• Normal - B-DNA conformation
• Amyloid Beta and aluminum (an etiologic factor)
  can modulate helical alterations in vivo

Suram et al. Neuromolecular Medicine
20022289-97 Hegde ML et al. J. Mol. Neurosci.
2004 2219-31.
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A role of Z-DNA binding in poxvirus pathogenesis

• E3L gene product is essential for virulence
• N-terminal domain has sequence similarity to Za
  family (binds Z-DNA)
• Tested virulence of E3L with decreased Z-DNA
  binding and chimeric viruses with different Z-DNA
  binding abilities
• Kim et al. 2003. PNAS. 1006974

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Fig. 2. Kim et al. 2003. PNAS. 1006974
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Fig. 3a. Kim et al. 2003. PNAS. 1006974
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Other ramifications of Z-DNA

• Z-DNA forming sequences induce genetic
  instability
• In mammalian cells
• Z-DNA induces nearby double-stranded breaks over
  regions consistent with dsb in human disease
• Can result in large scale deletions or
  rearrangements
• Replication independent and due to repair
  mechanisms
• Relevancy to carcinogenesis
• ds breaks and gene translocations map near Z-DNA
  forming sequences in leukemia and lymphomas

Wang et al. 2006. PNAS 1031677
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DNA can undergo reversible strand separation
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UV absorption and DNA denaturation
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GC content and DNA denaturation
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Genome complexity and Cot½
Cot½ (Concentration)(time for one-half
reassociation)
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Simple-sequence DNAs are concentrated in specific
chromosomal locations
Human metaphase chromosomes
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Simple sequence DNA and neurodegenerative disease
(CCG)n fragile X syndrome. (CAG)n
Huntingtons/Kennedys diseases. Myotonic
dystrophy spinocerebellar ataxia (AAG)n
Friedrichs ataxia. (CGA)n -- 4 different
conformations (CAG)n -- exclusively
Z-DNA Biochim Biophys Acta (2001) 1527
73-80
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DNA supercoiling
supercoiled
relaxed
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DNA forms separate on agarose gels
Your typical plasmid prep...
UNCUT LINEAR
KB
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FORM
6
5
II
III
4
3
I
2
1
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Types of cellular DNA
Plasmid 1 - 200 kb. Replicon (origin of
replicationORI) defines copy and
incompatibility. Narrow host range spread within
that range. Usually circular Chromosome (more
later)
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Organizing cellular DNA into chromosomes

• Most bacterial chromosomes are circular with one
  replication origin.
• But Borrelia, Rhodococcus have multiple, linear
  chromosomes.
• Eukaryotic chromosomes each contain one linear
  DNA molecule and multiple origins of replication.
• But the S. pombe genome may be circular.
• Bacterial DNA is usually associated with
  polyamines.
• Eukaryotic DNA usually associates with histones
  to form chromatin.

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Chromatin exists in extended and condensed forms
Nucleosome, beads on a string
30 nm fibers
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The solenoid model of condensed chromatin
Histones H2A, H2B, H3, H4
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Core histones are extensively modified
P phosphorylation Ac acetylation Me
methylation U ubiquitination
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The histone code hypothesis
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Nonhistone proteins provide a structural scaffold
for long chromatin loops
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Chromatin packing in metaphase chromosomes
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Chromosome number, size and shape at metaphase
are species-specific
Indian muntjac
Reeves muntjac
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Heterochromatin chromosome regions that do not
uncoil
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Three functional elements for stable inheritance
of eukaryotic chromosomes

• Origin for initiation of DNA replication.
• The centromere (point of spindle fiber
  attachment).
• The two ends (telomeres).

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Experimental evidence for the importance of the
origin of replication
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Experimental evidence for the importance of the
centromere
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Experimental evidence for the importance of
telomeres