HPS 420 Chapter 2&3

Describe the DNA structure

It is a helical structure that has 2 strands, the backbone has alt. sugars and bases, these two strands are joined by h-bonds between the pairs in the strands, they then twist and create a double helix.

Define Nucleotide

A chemical compound that has a sugar connected to a heterocyclic base and one or more phosphate group.

Define a Chromatin

Molecules of DNA wrapped around histones cores with linker DNA

Define nucleosomes

Appearance of a pearl necklace units

T/F
Each pearl has 4.5 turns of DNA wrapped around, corresponding to 150-200 bps

FALSE

It is 2.5 turns

Fill in the blank:

The linkers contain ___-__ BPS

The linkers contain 20-90 BPS

What are some types of DNA damage and many occur after 1 GY?

Base alterations: > 2000

Single Strand: 1000
Double strand: 40
Cross-links: 30
Alteration of sugars
Clustered Lessions

T/F

Strand breaks are the most common form of radiation induced dmg

T

T/F

Only DIRECT effects can cause strand breaks

FALSE

its direct and indirect

1 Gy of X rays produced how many single strand breaks/ double strand breaks during G1

Single: 1000
Double: 40

T/F

There are more DOUBLE strand breaks than Single

False

Single breaks are produced more for each double strand

Fill in the blanks:

1 Gy dose causes reprouctive death in _____ of mammalian cells

50%

Describe a DSB

They are a breakage of 2 strands of DNA less than 3 nucleotides apart

How are DSB produced?

By a single electron

OR

A combo of 2 SSBs in complementary strands by 2 electrons

Which strand break is considered a primary toxic lesions

Double strand brands because they are induced by ionizing radiation

Describe clustered Lesion

When energy from the radiation is not absorbed uniformly, which can then result in areas of DNA where there are some lesions

Who produced more clustered lesion

Densely ionizing radiation neurons/ alpha particles/ HZE particles)

Fill in the blanks:

___________ lesions within ___ turn of DNA is a ______ damage

two or more lesions within one helical turn of DNA is a clustered damage

What are some assays to detect DNA double strand damage?

Pulsed field gel electrophoresis

Single cell electrophoresis (comet assays)

Radiation-induced foci

There was a huge radiation in Nevada, a bunch of people were affected! What assay would be the most appropriate to use?

Pulsed field Gel electrophoresis

Describe pulsed field gel electrophoresis and what are their cons/pros?

Used for big outbreaks

The gold standard to detect DSB

Pros: reproducible, gold standard
Cons: Typically needs > 40 Gy, labor intensive

What are the steps to for pulsed field gel electrophoresis

1. Cells removed rom plate after radiation

2. Cells are mixed in melted agarose and put in a plug mold

3. Cells are lysed in agarose plugs

4. Plugs are then loaded into a gel and pulsed with an electrical field, which separates DNA fragments based on size

5. Radioactivity in each lane is quantified. More = more DSBS

Why is the gel pulsed with electricity during PFGE?

So we can denature the DNA to separate the DNA fragments based on

What are the 2 categories for single cell electrophoresis (comet assays)

Neutral comets

Alkaline comets

During a comet assay what would we for ONLY DSBs?

Neutral comets

During a comet assay what would we use to detect SSB and DSB

Alkaline comets

Why is it during comet assays for neutral comets it can only detect DBS

Because it won’t denature the dna, therefore only detecting doubles

Why is it during alkaline comets it detects SSB and DSB

Because the alkaline conditions denature the DNA, allowing detection of both single and double strand breaks.

T/F

During comet assays the longer , bigger the damage

T

Describe the comet assays procdeure

1. Cells mix with low melting agarose

2. Immobilize cells on the slide

3. Target cells with Lysis solution, which removes the membranes and histones from the DNA

4. Samples are treated with alkali which unwinds and denatures DNA

5. Samples are stained with intercalating dye and visualized by the epifluorescence microscopy following alkaline electrophoresis which shows DNA breaks

Describe the induced foci

They are detected by immunofluorescence and can be used to define kinetics of DSB induction and repair which are formed by the number of DSB-sensing and responding proteins

Fill in the blanks:

All DNA lesions are produced with a __________ response relationship up to a very ______ absorbed dose

All DNA lesions are produced with a linear dose response relationship up to a very high absorbed dose

T/F

DNA damage increases with dose, and that is called the linear dose response relationship

T

What can unpaired/mis-reparied dna lesions lead to?