Lecture 6 - Chromatin and DNA Damage Repair

ONCOL 335 - Radiobiology. University of Alberta

What are the two roles of chromatin

it is used as a signalling and docking platform

what is the general definition of a nucleosome

DNA wrapped around histones

how many histones make up a nucleosome

8 histones, thus nucleosomes are also called histone octamers

what histones make up the nucleosome

2 x H2A

2x H2B

2x H3

2x H4

What is the DNA between nucleosome beads called?

Linker DNA

what components make up a nucleosome bead

8 histone molecules and 146 base pairs of DNA

What is the affect of nucelosomes on ionizing radiation damage

clustered lesions

• spurs and blobs of ionization events have increased probability of having DNA damage if it wrapped around a nucleosome

what is heterochromatin

tightly wrapped chromatin, so tight that transcription factors cannot get to the DNA, so these sequences are not transcribed

What is Euchromatin?

loosly wrapped chromatin, transcription factors can reach DNA and attach machinery

How does DSB repair differ between euchromatin and heterochromatin?

DSB in heterochromatin are repaired slower and involve different signalling

what effect does heterochromatin have on radiation damage, particularly for high LET radiation?

heterochromatin will have more damage clusters than euchromatin

How can euchromatin be converted into heterochromatin?

Via covalent modifications

• when DNA is methylated (add CH₃), HP1 protein compacts DNA down

How are tumor supressors inactivated?

Accidental methylation

• if a methyl group is added to an euchromatin DNA section that contains a tumor supressor, it is coiled up into heterochromatin and inactivated

how can the nucleosome be remodeled if DNA damage is identified in a dense compact section?

1. Sliding

   • slide nucleosome away to make DNA accessible

2. Ejection

   • kick nucleosome off of DNA strand

3. H2A/H2B ejection

   • kick certain portions off of nucleosome

4. H2A/H2B dimer replacement

   • adding new dimer may make it easier to reach certain DNA section

what is epigenetics

a field of study focused on changes in DNA that do not involve alterations to the underlying sequence, but via chemical modifications that change the degrees to which genes are turned on and off.

• such as DNA methylation of nucelosomes

what are some other modifications that can be done to chromatin/histones?

covalent modifications of histone tails or DNA

Nucelosome remodeling

Histone variants

Non-Coding RNAs

what are the 4 types of histone tail modifications

1. Acetylation

2. Phosphorylation

3. Methylation

4. Ubiquitination

what amino acids are responsibile for phosphorylation

serine and thereonine

what amino acids are responsible for methylation

arginine and lysine

what amino acids is responsible for acetylation and ubiquitination

lysine

how does nucleosome remodling work

DNA around nucleosome is unravelled to be transcribed, and then ravelled back up again

how does non-coding RNA work?

RNA covers the chromatin/nucleosomes which inactivates it

what are histone variants?

histones like H2A and H3 for example are made up of different genes

• so an H2A gene can be made with different genes, resulting in variants

what is the most important histone variant?

H2Ax

why is the H2Ax variant important?

when DSB damage is detected, ATM will actually phosphorylate the S139 section of H2Ax which can then be used for signaling and docking

• activated H2Ax = gamma-H2Ax

Describe the activation process of H2Ax

At a DSB, MDC1 recognizes H2Ax and recruits ATM to phosphorylate H2Ax to become gamma-H2Ax

What is H2Ax focus formation?

H2Ax creates a platform for repair proteins and antibodies to attach to around the double strand break

What else does gamma-H2Ax do?

it acts a signal amplifier

• kinda acts like nodes of ranvier i think

How can chromatin foci formation be used to show DNA damage in microscopy?

attach fluorescent IgG’s to the H2Ax focus, allowing us to see the amount of DNA DSB in a cell after irradiation