Lecture 4 - DNA Damage and it's Repair - Part II

ONCOL 335 - Radiobiology. University of Alberta

Three types of DNA damage caused after ionizing radiation

damaged bases, single strand breaks, DSB (most lethal)

an average cell per day accumulates ____ lesions of DNA damage, while a cell receives ____ after 2 Gy of low LET ionizing radiation?

50 000, 3-4000

why are the 4000 lesions caused by radiation therapy more lethal than the 50 000 lesions caused normally

endogenous lesions are isolated and distributed anywhere in the body since they are random, but in radiaiton, the electron tracks deposity energy in clusters

• also time span as huge impact: endogenous lesions happen over a long period of time while RT damage happens within microseconds

what type LET has a higher concentration cluster of ionization events?

high LET

in low LET, what percent of damage is isolated DNA lesions, and what percent is damage clusters?

70% isolated DNA lesions

30% damage clusters

in high LET, what percent of damage is isolated DNA lesions, and what percent is damage clusters?

10% isolated DNA lesions

90% damage clusters

• due to high amount of energy released in short time periods

Do DSB breaks have ‘blunt’ perfect ends that we see in cartoon depictions?

no, rarely ever. instead they often have overhangs, that look like single strand breaks in opposite directions

What leads to strongly modified base pairs as shown in the image below

radical chemistry that occurs to ionizing radiation

what are the two main DNA repair mechanisms?

1. Homologous recombination

2. Non-homologus end-joining

non-homologous end joining

the primary repair mechanism that is extremly fast and may lead to errors

• cells clean up the break and put it back together

in addition to DNA damage repair, what else is NHEJ used for?

VDJ recombination —> leads to IgG diversity

when is NHEJ used?

before DNA replication, especially in G1 phase

What genes of the PIKK family are important for DNA damage signalling?

ATR, ATM, DNAPK, mTOR

NHEJ Step 1 - Recognition

Damage is recognized by the Ku 70/80 dimer which forms a ring on the double stranded break

Ku 70/80 then recruits a DNAPK enzyme to phosphorylate the proteins for step 2

NHEJ Step 2 - Remove Damaged Nucleotides

DNA-protoein kinase activates (via phosphorylation) Artemis, which is a nuclease which cuts out nucleotides not needed in DNA damage repair

NHEJ Step 3 - Gap Filling/DNA synthesis

DNA polymerase mu or lambda are recruited to add base pairs back

NHEJ Step 4 - Seal Gaps

Ku 70/80 recruit XRCC4 and XLF to form bridge and hold the ends of the DNA together

XRCC4 then recruits ligase IV to seal the nucleotides together

what happens when a person has an Artemis deficiency (the NHEJ nuclease)

they have SCID

• severe combined immunodeficiency

Homologous Recombination

uses a sister chromatid template and is error free

when does HR repair occur?

must be after DNA synthesis: mid-S to G2

are DNA repair mechanisms active in M-phase?

no, both NHEJ and HR are shut down in mitosis

HR - Pre Step 1

Strand Resection: damaged strand searches for the complementary strand on the undamaged chromosome

HR Step 1 - recognize DNA damage

The MRN complex recognizes the double stranded breaks and then recruit the next set of proteins via activating the kinase ATM

What are the three proteins that make up the MRN complex

MRE11

RAD50

NBS1

HR Step 2 - removal of damage nuclides

ATM then activates EXO1 nuclease and helicase to resect the damaged nuclides

HR Pre Step 3 - RPA coating

RPA comes and bins to ssDNA to protect it so cell doesn’t attack it

HR Step 3 - forming of Holliday Structure

BRCA2 and Rad 51 remove the RPA coating

resolvase and Rad 54 then allow for strand invasion leading to a holliday structure

HR Step 4 - Resolution of Holliday structure

holliday structure is cleaved, resulting in corrected DNA

When is NHEJ dominant over HR?

G1

When is HR dominant over NHEJ?

HR dominates in Mid-S to G2 phase

are NHEJ and HR mutually exclusive?

no, theoretically NHEJ can happen in S and G2 but it is not common

What is step that determines that HR will be done over NHEJ

if part of the DNA is resected, it must do HR

what controls the competition between NHEJ and HR?

cell cycle controls

• end resection is stimulated by CDK activity, which mediates phosphorylation of substrates

what gene mutaton causes ataxia telangectasia

ATM

What gene mutaton causes Nijmegen breakage syndrome

NBS1

What gene mutaton causes AT- like disorder?

MRE11

What gene mutaton causes RAD50 deficiency?

Rad50

What gene mutatons may cause SCID?

Artemis

Ligase IV

XLF

What gene mutaton causes fanconi anemia

FANC2, BRCA2

What gene mutaton causes familial breast, ovarian carcinoma syndrom? (Angelina Jolie gene)

BRCA1 and 2

What gene mutaton causes Li-Fraumeni syndrome

p53, CHK2