Eukaryotic Nuclear DNA
· Combined length of human metaphase chromosomes is 2.6 meters (7.8e9 BP)
· These are packaged into 46 chromosomes that are 200 µm
o 10000 (104) fold condensation
· Packaged as chromatin which contains nucleic acids and protein (nucleoprotein)
· Bases are stacked like coins. This stacking gives the stability (not the hydrogen bonds, the hydrogen bonds neutralize each other allowing for the hydrophobic interactions of the base pairs.
· Chains run anti parallel to each other
Chromatin
· Chromatin is DNA that is associated with a group of small proteins called histones
o Histones are highly conserved evolutionarily
o Small proteins
o Core Histones are H2a, H2b, H3 and H4. They exist in octamers consisting of two each of the four core histones
o Linker Histone is H1. It exists between each nucleosome
o A nucleosome is the core histone octamer wrapped with DNA (two rotations—approx 146 bp)
· Two types
o Heterochromatin—Transcriptionally inactive, more tightly packed
o Euchromatin—Transcriptionally ACTIVE
· Levels of structure of Chromatin
o DNA is wrapped around histones to form nucleosomes (beads on a string)
o 6 nucleosomes coil to form solenoids
o Solenoids loop onto a scaffold protein that coils to form mini-bands
o Mini-bands stack to from the chromosome
Major Components of DNA structure
· Deoxyribose
· Nitrogenous bases
Cytosine Guanine Adenine Thymine
· Double stranded DNA has specific base pairing. The distance between base pairs can vary based on secondary structure, but is usually around 11 Å
o Guanine:Cytosine (three hydrogen bonds)
o Adenine:Thymine (two hydrogen bonds)
· Chargoff’s Rule based on specific base pairing in double stranded DNA
o The amount of Cytosine will equal the amount of Guanine
o The amount of Adenine will equal the amound to Thymine
· The structure of DNA gives it a polarity. DNA has a 5’à3’ polarity Each new nucleotided added is added to the 3’ end because the new nucleotide provides the phophate in the sugar phosphate backbone (3’ end is extended)
· DNA is anti-parallel, which means that the strands are opposite in direction. The 5’ end of one strand will be next to the 3’ end of the other strand
· DNA is a double helix
o Major Groove—Identify by looking for entire base pairing in field of view, minor groove does not show an entire base pair as it is partially obscured by the sugars
o Minor Groove
o Three forms
§ A Form-right handed, really condensed
§ B Form-right handed, most common
§ Z Form-left handed, sugar phosphate backbone forms a zig-zag pattern.
· Duplex B-form DNA
o Two RIGHT handed helices
o Cannot be separated unless unwound
o The two strands are anti-parallel and complimentary
o Base pairs are planar and perpendicular to the sugar phosphate backbone
o 3.4 Å per base pair, 34 Å per turn, therefore 10 bp per turn
o Approximate width of helix is 20Å
· The Base Pairs are some what hydrophobic and the sugar phosphate backbone is hydrophilic. The helix stability is helped maintained by the hydrophobic interactions
Recognition elements of DNA
· Bases can directly interact with compounds. This is called direct read out but is more like fitting a hand in a glover rather than reading a book.
· Phophates can interact in a charge-charge fashion. Almost all DNA binding proteins have some sort of charge-charge interaction involved with binding
· Structure patterns
o Double stranded DNA
o Single Stranded DNA (also formed during replication)
o Nicks/gaps
o Bent DNA
o Tetramers, A form, B form, Z form
Why is knowing DNA structure important?
· Drug Action
o Intercalating agents—compound slides between sets of base pairs and causes the DNA strand to stretch out and unwind. This causes instability and inactivation
§ Ethidium
§ Doxorubicin/Daunorubicin-two parts of interest
ú Intercalating region (anthracyclene ring) slides between bases
ú Sugar-rests in the minor groove
§ Bisdaunomycin-two daunomycin linked by two benzene rings.
ú The two anthracyclene rings intercalate into the DNA
ú The benzene rings rest in the minor groove.
ú Bisdaunomycin binds with 100x the affinity of daunomycin
§ Actinomycin D
ú Intercalates between guanine and cytosine base pairs
o Minor Groove Binding Agents
§ Netropsin-antimicrobial
§ Mitomycin C—anti-neoplastic
§ Hoechst 33258-DNA stain
§ TATA Binding Protein
ú Transcription Factor
§ Cro (from the lambda phage)
ú Also binds major grooves
o Major Groove Binding Agents
§ Cro (from the lambda phage)
ú Also binds minor grooves
· Structure Changes Expression—heterochromatin, euchromatin, and helicase all represent some change in the structure
o Sequence specific recognition proteins must have access to the DNA to bind to it.
· If we know the correct structure, we can identify unusual structures (especially during replication, recombination and in telomeres)
o Triplex DNA
o Tetraplex DNA
o Parallel Strand DNA
o Hairpin structures
o Three/fourway junctions-During recombination
CLINICAL RELEVENCE
o Bloom’s syndrome
· Defective Blooms Helicase that undoes tetraplex DNA
o Höogsteen base pairing (H-Bond donors and receivers are different than in Watson and Crick Base Pairs
o N7 is used rather than N1 which allows for base pairing with an additional nitrogenous base.
o In Bloom’s syndrome, there is no helicase (BLM) to break up a G4 tetraplex and thus DNA cannot replicate.
§ BLM is a family member of the RecQ helicase family
· Presents with:
o pre/post natal growth retardation
o Sensitivity to sunlight
o Impaired fertility
o Immunodeficiency
o High incidence of leukemia and solid tumor
o Propensity toward chromosome damage
o Werner’s Syndrome
· Autosomal Recessive inheritance
· Accelerated aging
· Helicase Deficiency
o Different from helicase involved in Bloom Syndrome (same family of RecQ helicase)
§ Helicases are job specific and cannot compensate for other helicases
o Rothmund-Thomson Syndrome
· Defective RecQ5 Helicase
· Genomic Instability (just like the other helicase defects)
· Increased Cancer
· Accelerated aging
BACK TO STRUCTURE AND PHYSICAL PROPERTIES
DNA may exist in several forms
· Double stranded
o Linear
o Circular
· Single stranded
o Linear
o Circular
· Supercoiled
o Superwound or slightly unwound double helix
o Superwinding happens as a consequence of DNA replication
o Super coils inhibits replication
Useful Properties of DNA
· UV Absorbance at 260nm (due to bases, not backbone)
o This can be used to determine the amount of DNA in a solution by comparison to standard curve
o Can also be used to monitor conformational changes (denaturation, annealing) by way of the hyper and hypochromic effect
§ Hyperchromic effect-As DNA denatures, the absorbance will increase
§ Hypochromic effect-As DNA reanneals, the absorbance will decreased
· Buoyant Density—centrifugation techniques
o GC rich DNA is heavier than AT rich DNA
o Can be used for purification of DNA segments
· DNA can denature
o Denaturation with heat, extreme pH, alcohols, ketones, urea and foramide (similar to agents that denature proteins)(list is not comprehensive)
o Denaturation causes an decrease in viscosity
o Denaturation can be measured by using the hyper(hypo)chromic effect.
§ Measure A260 as you increase temperature. Point at which 50% is denatured is called the melting temperature (Tm)
· DNA can Reanneal
o Reannealing Rate is controlled by concentration
o If the gene is more prevalent, it will reanneal quicker
o Single copy genes take the longest to anneal, as they have a low concentration (number of copies of the gene of interest compared to total number of genes)
· The Tm is the midpoint of the transition phase
o Dependent on base composition
§ Increased GC content increases Tm by increasing stability
o Dependent on Ionic Strength
§ Higher concentrations of monovalent cations will increase the Tm by stabilizing the duplex. They stabilize the negative charge of the phosphates
o Dependent on sequence
§ Same composition, different sequenceàdifferent Tm
o GENERAL RULE—Increase stability, increase Tm
The rate of Reannealing gives rise to several classes of DNA
· Single Copy Genes (aka Unique Sequence)-Reanneal SLOWLY
o Protein Coding Genes
o Duplicated Genes
· Middle Repetitive Genes
o tRNA and rRNA genes
o Pseudogenes
§ Product of the integration of mRNA into the gene, duplication or transposition
§ Have become non functional as a result of losing or gaining something.
§ Often identifiable due to the appearance of a poly-A tail
· Repetitive DNA
o Simple-Sequence DNA
§ Satellite Sequences—sequences that consist of MANY short sequences (3-10 nucleotides long)
§ Alu Sequences—family of 300 bp sequences that occur over 1 million times in the genome and usually contain the AluI restriction endonuclease
o Dispersed Repetitive DNA (Mobile Genetic Elements)
Gene Families
25-50% of protein coding genes are solitary, meaning most protein coding genes have a duplicate or triplicate (or alot-icate). Most are just duplicate.
· Duplicated Genes are usually found clustered together
· Duplications due to unequal cross-over (can help explain why duplicated genes are usually found clusters
CLINICAL RELEVENCE
o Fragile X syndrome
· Most common form of inherited mental retardation
o Incidence increases through out generations due to accumulation of extra repeats
· Caused by a CGG repeated motif in the 5’ untranlated region of the FMR1 gene.
· FMR1 is inactivated when there are excessive repeats
· Causes
o Unequal cross-over during recombination—one chromosome has more repeats and the other has less
o Amplification during replication that causes more CGG repeats to be replicated.
o Huntington’s Disease
· Polyglutaminylation of proteins is a result of a CAG repeat in the Huntingtinin gene.
· Polygutamination results in neurotoxic proteins
Restriction Endonucleases
· Restriction endonucleases are enzymes that bind specific sequences of nucleic acids and cleave that sequence, either bluntly or staggered.
· They do not progressively degrade DNA, they just cleave that specific sequence
· Thought to be a defense mechanism against phage infection. Host DNA will have methylated Adenosine residues that are not recognized by the restriction endonuclease and are therefore not degraded. The phage DNA will be cleaved though.
· A restriction map identifies linear series of sequeces affected by different endonucleases
· Restriction endonuclease sequences give measurement to polymorphisms by differences in restriction maps of a gene, chromosome, or locus.
o RFLP (Restriction Fragment Length Polymorphism) occur frequently enough to be used as genetic markers
o Changes in allelic sequences occur at a frequency of 1/1000 bp (relatively frequent)
§ Changes in restriction endonuclease sites can be detected by eletrophoresis (changes in banding on gel)
· Clinical Uses
o Detection of mutated genes
§ Hemoglobin A DNA has several restriction endonuclease sites in the sequence. Sickle Cell mutation causes one site to no longer be recognized.
o Paternity testing
§ Look for bands in common with the child
Blotting
· Technique
- Cut DNA samples with a restriction endonuclease and run though an agarose eletrophoresis gel.
- Place nitrocellulose paper wit absorbent napkins (paper) on top
- Contents of gel will transfer to the nitrocellulose paper
- Hybridize nitrocellulose paper with a radioactive probe, rinse and expose nitrocellulose paper to x-ray film
· Types
o Southern-DNA
§ Specific seqence of DNA from within a mix of DNA. Probe is a complementary nucleic acid sequence with a radioactive tag
§ Location of mark on X-ray film will show where the DNA is on the gel
o Northern-RNA
§ Same idea as southern, but with RNA
o Western-Protein
§ Proteins are detected by using a labeled antibody
o SouthWestern-Protein bound to DNA
§ Complementary DNA probe
o NorthWestern-Protein bound to RNA
§ Complementary DNA probe
o FarWestern-Protein bound to a Protein
§ Antibody
RNA
Major components of RNA are very similar to those of DNA with two exceptions
· RNA uses Ribose instead of deoxyribose, which allows RNA to fold into its unusual patterns
· RNA uses Uracil instead of Thymidine
Ribose Uracil
RNA Structure
· Single stranded is most common, but duplex structures are possible
o Duplex structure most related to A-Form DNA
· Single Stranded RNA will form secondary structures with the lowes energy content
o Translation-Any complementary portions will anneal and form double stranded RNA
· RNA does demonstrate some Höosteen base pairing
Folding patterns of large RNAs are complex
· Segments with A Form DNA like structure
· Hairpin
· Unusual Base pairing (höogsteen bp, reverse Höogsteen bp, base triples
· Tertiary interactions between different domains are common
· RNAs can form structures not seen in DNA of similar sequence do to the ribose in place of the deoxyribose
· 2’ hydroxy can form complex H bonding patterns
Uses of modified Nucleosides
· Drugs
o Acycloguanosine is a chain terminator and prevents DNA replication. Antiviral
o Cytosine Arabinoside is incorporated into the chain, but causes Topoisomerase I to disassociate and will not replicate the DNA. Effective against Acute Lymphocytic Leukemia (ALL)
o Azidothymidine is a chain terminator. The C3 hydroxyl is replaced with a azido group (N3). Treatment for AIDS
· Sequencing
o Dideoxy DNA Sequencing
§ Dideoxynucleotide triphosphate is incorporated but is a chain breaker. If you add a small amount of ddNTP to a test tube with all other deoxyNTP, template, radioactively labeled primers, and polymerase, multiple strands of varying length replicas of the template will be made. If you use four tubes, each with a different ddNTP, and you electrophorese all of these on the same gel, you can get a sequence
CYSTIC FIBROSIS QUESTION PG 151 WHAT IS THE DEFECT?
Frame shift mutation caused by deletion of 3 nucleotides (1 A.A.àLeu)
5’ TATCA TCTTT GGTGTT 3’ N-Tyr-His-Leu-Trp-Cys-C
5’ TATCA TxxxT GGTGTT TCC 3’ N-Tyr-His-Trp-Cys-Cys-C
Polymerase Chain Reaction
· Allows for the amplification of genes greater than 1 billion times
· Requirements
o A knowledge of the sequence must be known to create primers
o Taq Polymerase is used because it is derived from a thermophilic organism (Thermus aquaticus)