1 - Nuclear and DNA Structure and Function

Question 1

What is the difference between Heterochromatin and Euchromatin?

Nuclear/DNA Structure/Function

Answer 1

Packing density. Heterochromatin is densely packed -> less transcriptionally active. Euchromatin is more loosely packed -> higher transcriptional activity as compared to Heterochromatin.

Question 2

What structure facilitates the transport of macromolecules across the nuclear membrane? How? Explain the mechanism for bi-directional transport.

Nuclear/DNA Structure/Function

Answer 2

Nuclear Pore Complex. Importins (Nuclear localization) and Exportins (Nuclear Export). Nuclear Locatlization Sequences (NLS) and Nuclear Export Sequences (NES) facilitate binding of importins and exportins to “cargo” proteins. Importin binds NLS in absence of RanGTP. RanGTP stimulates binding of cargo to exportin. RanGAP (phosphorylase) hydrolyzed GTP to GDP causing dissociation of RanGDP/cargo/exportin complex.

RanGTP/RanGDP concentration gradient, RanGAP in cytosol facilitates this

Question 3

What is the difference between the effect of steroid hormones and post-translational modifications?

Nuclear/DNA Structure/Function

Answer 3

Steroid Hormones are nonpolar. This allows them to be transported to the Nucleus (with importin) and can act as a nuclear transcriptional factor to regulate gene expression. Post translational modifications can also affect presence of certain gene byproducts but, as they occur in the cytosol with polar (hydrophilic) molecules, these effects tend to be temporary/of shorter duration.

Question 4

What is the Nuclear Lamina made of? Medical relevance?

Nuclear/DNA Structure/Function

Answer 4

the nuclear lamina is a network of intermediate filaments (lamins). It is contiguous with the inner nuclear membrane and provides structural support. This knowledge is medically relevant because diseases that affect lamins (Laminopathies).

Question 5

What is Emery-Dreifuss Syndrome? What class of disease is it?

Nuclear/DNA Structure/Function

Answer 5

Muscular dystrophy. Emerin (EMD gene), a transmembrane protein necessary in muscle function, loses function in this laminopathy.

Question 6

What is Hutchinson-Gilford Syndrome? What class of disease is it?

Nuclear/DNA Structure/Function

Answer 6

aka progeria. Prelamin A becomes progerin because of a splicing error resulting in premature cell death and accelerated aging.

Question 7

What is a Barr body?

Nuclear/DNA Structure/Function

Answer 7

A barr body is an inactivated X Chromosome.

Chromatin states can be passed down through generations through this hig

Question 8

What is the nucleolus?

Nuclear/DNA Structure/Function

Answer 8

suborganelle in nucleus formed around squences of ribosomal DNA (Nuclear organizing center). synthesizes rRNAm, prominence of nucleolus proportionate to activity

Question 9

What are the types of nuclear injury?

Nuclear/DNA Structure/Function

Answer 9

A nuclear injury is irreversible. Pyknosis - shrinkage and condensation of nucleus into a basophilic mass. Karyorrhexis - fragmentation of nucleus. Karyolysis - dissolution of nucleus from DNAases and RNAases.

Question 10

Chromosome vs. Chromatin vs. Chromatid

Nuclear/DNA Structure/Function

Answer 10

Chromatin is DNA wrapped around histones and other structural proteins, one unit of which is called a nucleosome. Chromatin is compacted into structures known as homologous paird of chromosomes. The replicated chromosomes form pairs held togetjer by a centromere known as sister chromatids.

Question 11

What is a histone?

Nuclear/DNA Structure/Function

Answer 11

major structural proteins of chromosomes. DNA is wrapped twice around a histone octamer to make a nucleosome. Six nucleosomes are assembled into a solenoid to associate with H1 histones.

H3 Histone tails are prime for post translational modification

Question 12

Describe DNA replication simply.

Nuclear/DNA Structure/Function

Answer 12

nucleotides are added at the 3’ end in DNA replication. N to C for translation, 5 to 3 for replication. DNA synthesis begins at 2 replication origins cation. Replication forks form at each origin. The double helix is opened with helicase and and DNA polymerase binds. However, these single stranded DNA lengths can fold in on themselves into “hairpins” so they are stabilized by single-stranded binding proteins (on lagging strand especially). Unwinding double stranded DNA causes torsional strain that has to be stabilized by topoisomerases by creating transient ss breaks. Primase attaches at replication origins synthesizing a short RNA primer than Polymerase ⍺. Polymerase operates 5 to 3 on leading strand, but catches up to itself, creating okazaki fragments on the lagging strand (and also on leading strand). Rna primer is removed and DNA is filled in with polymerase 1. separations between okazaki fragments are sealed by ligase.

Type1 Isomerase cuts/seals ss. Type2 isomerase cuts both strands.

Question 13

What do Fluoroquinolones do?

Nuclear/DNA Structure/Function

Answer 13

Fluoroquinolones bind to two nuclear enzymes, inhibiting DNA replication.

Question 14

What is the purpose of telomeres? Why is this an issue? How is it resolved?

Nuclear/DNA Structure/Function

Answer 14

Nucleotides are lost when ligase seals the gaps between okazaki fragments. Chromosome ends have Telomeres - long competitive sequences that protect these ends. Thus, Telomere shortening occurs. Telomerase, an RNA-dependent DNA polymerase, maintains the length of telomeres, mitigating cell aging. Increased telomerase activity halts cell senescence and can cause cancer cell growth.

Question 15

Explain the DNA repair mechanisms.

Nuclear/DNA Structure/Function

Answer 15

DNA polymerase has proofreading activity.
Mismatch repair: a DNA mismatch is removed by repair proteins and DNA polymerase and ligase repair.

Deamination (changes nucleotides to uracil or related) and depurination (removes purine on nucleotide) cause DNA damage. Deamination can be solves by base excision repair: enxyme recognized uracil and removes it. Loss of pyrimidine recognized by endonuclease and polymerase and ligase replace the base and seal.

Nucleotide Excision repair: thymine dimers from UV radiation can be removed. A protein complex recognizes the thymine dimers and exinucleases cause nicks on the sides of the damage. Damage is removed and DNA polymerase and ligase replace and seal.

Xeroderma pigmentosum is an autosomal recessive genetic disorder of NE.

Multiple basal cell carcinomas (basaliomas) and other skin malignancies frequently occur at a young age in those with XP

Question 16

What is Nucleotide excision repair used for?

Nuclear/DNA Structure/Function

Answer 16

NER is used for repair of lesions that cause large distortions of the DNA structure (large DNA adducts).

Question 17

What is Base excision repair used for?

Nuclear/DNA Structure/Function

Answer 17

BER recognizes chemically altered bases (deamination,
dealkylation, depurination, ROS) that cause small DNA adducts (usually a single base)

Question 18

What is Mismatch repair used for?

Nuclear/DNA Structure/Function

Answer 18

MMR is used during replication to repair polymerase mistakes. Evidence supports association of MMR machinery with components of replication machinery. MMR uses nicks to identify newly synthesized strand to repair, so it makes sense that MMR would be closely associated with replication machinery. Adducts
often cause large kinks in DNA (~60 o toward narrow groove).

Question 19

How do you repair double stranded DNA breaks?

Nuclear/DNA Structure/Function

Answer 19

nonhomologous end joining: Ku70/80, procession of DNA end by nuclease and end joining by ligase. may lose some nucleotides and be deleterious.

homologous recombination: can only be used during S/G2 phase since homolog is present here during replication. broken ends are processed by recombination specific nuclease (MRN complex) and Rad51 and BRCA1 invade the strand and repair the double strand break. No loss of nucleotides at repair site.

BRCA2 binds the single strand damaged DNA and directly interacts with the recombinase RAD51 to stimulate a vital step of homologous recombination. By repairing DNA, BRCA2 plays a role in maintaining the stability of the human genome by preventing dangerous gene rearrangements that can lead to cancer.
In addition to breast cancer in men and women, mutations in BRCA2 also lead to an increased risk of ovarian, Fallopian tube, prostate, and pancreatic cancers, as well as malignant melanoma.