Chapter 4 – Nucleic Acids (part 2)

Werner’s Syndrome

• There are multiple different progeria (early onset aging) diseases

• Of these, Werner’s Syndrome is probably the most well known

• Autosomal recessive disease (both parents need at least one copy)

Werner’s Syndrome basics

• Patients develop rapid aging, beginning at approximately early adolescence / young adulthood.

• Occurs rarely, much like many of the progeria diseases ◦ Ex: Hutchinson-Gilford progeria syndrome (premature aging in children)

• Patients typically pass due to diseases thought to affect the elderly preferentially

Etiology of Werners Syndrome

• Approximately 1:100000 are diagnosed with this disease

• Disease more common in Japan, as well as Sardinia (a small Italian island)

• Three quarters of all patients diagnosed there (Japan)

• The pathology of this disease can be pinpointed to the disruption of a single gene: wrn

The wrn gene and its mutations

The gene products accomplish multiple tasks:

- Helps break down polynucleotides (exonuclease)

Help remodel and repair DNA (helicase)

- Aid in the interaction of DNA and proteins (RQC and HRDC domains)

Werners syndrome Pathology

Can be summarized as “pro-aging” “Pre-clinically”, patients tend to have shortened stature as well as a lack of a growth spurt as adolescents.

However, in the patient’s early 20’s, they begin to develop the hallmark signs of the disease:

◦ Mild cognitive impairment

◦ Cataracts

◦ Early greying of hair

◦Skin changes (scleroderma)

Clinical signs / progression of Werners syndrome

Develops in childhood / early adolescence

◦ Average age of diagnosis in late teens – early 20s, though could be later Later in life, develop a smattering of diseases associated with old age:

◦ Osteoporosis

◦ Brain atrophy(partial or complete wasting away) and dementia

◦ Cancers / neoplasms(tumor)

◦ Includes a 50-fold (!) increase in melanoma (a tumor of melanin-forming cells) development

◦ Atherosclerosis (disease of the arteries)

◦ Patients typically pass from these complications

What does the regular functioning werner gene do?

Major function: DNA repair ◦

Without the proper functioning of the gene, DNA repair does not get corrected

◦ Leads to instability of the genome, affecting the proper expression of other proteins

Though there are no good treatments for the disease, it has lead to much information about what occurs during the aging process.

Secondary structure of DNA – The 3D structure of the double helix

1. The primary structure of genetic inheritance was found to be nucleic acids, specifically DNA i. This information was found from two separate experiments in bacteria

2. This molecule’s structure has a specific 3D shape in an aqueous environment, which is highly regular and symmetrical

So how do we know the shape of the helix?

Preliminary experiments: An attempt to know the chemical composition

◦ We knew of the phosphate and sugars, but what of the bases

◦ Using a number of acid degradation techniques, it was determined that:

◦ The ratio of A – T was always the same and

◦ The ratio of G – C was always the same

However, the defining experiments came from DNA x-ray crystallography

DNA x-ray crystallography:

◦ Form a crystal of a substance, any substance

◦ Send a beam of x-rays (photons) at the crystal

◦ The pattern of the diffraction (scattering) of the rays gives clues about the structure

DNA-b structure in three dimensions

Major vs. minor groove

Right-handed and clockwise direction

•Another kind of DNA (DNA-a) works using the same principle

How does DNA transfer from parent to daughter cells?

We were unsure of this particular mechanism until a specific experiment elucidated it

Was it ◦ Conservatively? – DNA always stays together

◦ Semi-conservatively? – DNA mixes one old strand with a new one

◦ Dispersive? – DNA intermixes, both strands break apart and remix together

Experimental procedure to find out how DNA transfers from parent to daughter cells:

Manipulation of the isotopes – once again ◦

No radioactivity this time

◦ Grew one population of E. coli in media containing nitrogen 15

◦ A heavier atom – produced heavier DNA at a specific density

◦ Grew another population of E. coli in media with nitrogen 14

◦ Produced lighter DNA

◦ Then, another new of E. coli in media with N15, then the next generation onto N14

result of first portion of experiment:

• Bottom pane reveals result of first portion of experiment:15N in one strand of DNA and 14N in the other- this result is expected if the newly replicated DNA is a hybrid molecular species, consisting one one half parental material and one half new DNA

• Clearly the DNA hybridizes. If it didn’t, there would be two populations.

• But, does it always hybridize?

Let’s keep growing onto N14 media, what happens?

If DNA was truly dispersed, it would continue to have one peak.

What we see is two peaks, indicating that individual strands, once replicated, stay together.

Therefore dna replication is Semi-conservative

What other shapes can DNA be found in:

DNA in the body is mostly found in the B-form, so we’ll only briefly cover others DNA can also be found in something called the A-form. Similar to b-form

A-form of DNA. Similar to b-form… but: Key differences:

◦ More compact: base pairs are closer together

◦ Slightly wider double helix

◦ Angle of base pairs is tilted compared to b-form

Why the B-form of dna over the A-form?

More stable in water, forms a more stable bond with h-bonding in the minor groove of the molecule

Dna Denaturation

We are not simply concerned with DNA staying together, but also being able to come apart ◦ This is denaturation

◦ Also referred to as “melting”, but not strictly a true melting, as it does not turn to liquid

◦ The molecule “melts” along the h-bonds

When native double stranded DNA is heated above its “melting” temperature, it becomes denatured, separating into single strands. The two random-coil strands have higher energy then the double helix

Tertiary Structure of nucleic acids

Tertiary=Folding of DNA / RNA in on itself

◦ In DNA, this is found mostly in bacteria. Why?

◦ The DNA is mostly circular. No chromosome

◦ Used a mechanism to supercoil and compress the DNA

Also found in mitochondria. Why? Think of the origins of mitochondria…

More important for eukaryotes and RNA rather than DNA,

Quaternary Structure of nucleic acids

Combination of DNA with histone proteins ◦ Helps to form chromosomes and other tightly wound structures

Tertiary structures

• Strained circle: double-stranded circular DNA

• SuperCoil: Double stranded DNA

Single Stranded nucleic acids

• Typically, this is RNA rather than DNA

• “Harpin” structures formed by self-complimentary sequences; the chain folds back on itself to make a stem-loop sequence

A hairpin loop

• A hairpin loop in action: tRNA

• Notice what it has on the acceptor stem? An amino acid

what does the “melting” of DNA depend on?

This “melting” depends on the composition of the material More G-C bonding = higher melting point More A-T bonding = lower melting point Also occurs at a very small temperature range.

RNA Polymerase

An RNA Polymerase (of which there are a few), creating a polynucleotide from ribonucleotides Notice the “melting” between the DNA strands

The process is complicated slightly by chromatin