DLA 3: DNA Packaging/Supercoiling and Telomerase

How does DNA packaging occur in Prokaryotes?

DNA Supercoiling

Prokaryotic DNA supercoiling is accomplished by the action of what 2 enzymes and association with what proteins?

1- DNA Topoisomerase I

2- DNA gyrase

Hu proteins

What is the structure of a Bacterial nucleoid?

Supercoiled DNA loops are attached to a protein core

the protein core contains: Topoisomerase I, DNA gyrase and Hu proteins

What are the main function of Supercoiling of DNA?

Compacts the DNA so that it can be packaged into the cell

What does Negative supercoiling promote?

Strand separation ie DNA replication

When are Positive supercoils introduced? What effect do they have?

During DNA replication and transcription

Positive supercoiling makes DNA strand separation more difficult

image: a) Positive supercoils (the front segment of a DNA molecule cross over the back segment from left to right). (b) Negative supercoils

Where are Positive supercoils introduced? Negative supercoils? What corrects these supercoilings?

Ahead of the protein

Behind the protein

Topoisomerase I and DNA gyrase

What is an example of a drug that inhibits DNA gyrase?

Cirprofloxacin

prescribed as an antibiotic

Supercoiling can be relieved in a DNA double helix containing what? What is the issue with this?

A free end by rotation of the entire molecule

most DNA molecules do not contain a freely rotating end as DNA molecules in the cell are often associated and bound to various matrix or scaffold proteins

As a result DNA supercoiling is a common phenomenon

What is the MOA of Topoisomerase I ?

1- Forms a single covalent bond with the DNA and breaking a phosphodiester bonds in one DNA strand

2- The two ends of the DNA double helix can now rotate relative to each other, relieving accumulated strain

When is DNA Gyrase (Topoisomerase II) activated? Does it require energy?

When 2 double helices cross over each other

Yes, this reaction requires ATP hydrolysis

What is the MOA of DNA gyrase in relieving strain?

1- DNA gyrase makes a covalent linkage to both strands of one DNA helix and making a double stranded break

2- The second DNA helix is passed through the break

3- The break is resealed and the enzyme dissociates

What are the 3 main functions of Chromatin?

1- Packaging of DNA to allow the genome to fit inside the eukaryote

2- Maintain the structure of chromosome during metaphase and protect it from damage

3- Regulate gene expression by making regions of the genome accessible to transcription (uncondensed DNA) or repressing transcription by forming a condensed DNA structure and making that region of the genome inaccessible to the transcriptional machinery.

What are the 2 main types of chromosomes based on chromosome staining properties?

1- Heterochromatin

- Condensed

- stains dark

- transcriptionally inactive

2- Euchromatin

- Uncondensed

- stains light

-transcriptionally active

What components make up the structure of the Nucleosome? What is the Nucleosome core made up of?

Histone protiens + DNA

Octamer: 2 H2A, 2 H2B, 2 H3 and 2 H4 histone proteins

What are Nucleosomes connected together by? What structure do they produce?

Linker DNA and H1 protein

Beads on a string (11nm)- more condensed chromatin

Nucleosomes can associate closer to each other and form what? This structure results from interactions between?

30 nm fiber

Adjacent histone H1 proteins

What is one of the main steps involved in formaiton of Heterochromatin?

DNA Methylation

DNA methylation tags a region of the genome for histone modification that causes the formation of heterochromatin.

What are the steps involved in the formation of Heterochromatin?

1- Dnmt3 (DNA methyltransferase) methylates (adds methyl groups) DNA

2- MeCP2 protein recruites HDAC1 enzyme complex

3- HDAC1 deacetylates histones

4- Heterochromatin is formed

What effect does Histone Acetylation have ?

Reduces affinity between histone and DNA by removing positive charges and thus increases transcription

What is the difference between Deacetylnucleosome and Acetylated nucleosome?

Nucleosome is more tightly associated with the DNA reducing access to the DNA (heterochromatin = notranscription

A more 'open' conformation

is achieved, facilitating access

to DNA (euchromatin = potential for transcription)

What is Telomerase? Function?

An enzyme that adds bases to the ends of telomeres; abundant in young cells but eventually runs out.

What is a telomere and what is its function?

Repetitive DNA at the end of a eukaryotic chromosome

Protects the messages of the DNA from being destroyed during DNA replication.

What is Telomerase?

Enzyme responsible for maintenance of the length of telomeres by addition of guanine-rich repetitive sequences

What enzyme has the ability to synthesize DNA using RNA as a template?

Telomerase

since it has reverse transcripase (retrovirus) activity