Download Ionizing radiation And Double

Ionizing radiation and
double-strand break repair
Kevin D. Brown, Ph.D.
Electromagnetic Spectrum
Ionizing
radiation
Gamma
Rays
0.01 nm
X-Rays
1 nm
Ultra
Violet
100 nm
Visible Spectrum Infrared
400-700 nm
1 mm
Radio Waves
(Microwave, TV, Radio)
1 meter
1 km
Sources of Ionizing Radiation
Ionizing Radiation as a Genotoxin
IR exposure can damage DNA due to both
direct and indirect effects. Direct effects = DNA damage due to bombardment
with γ-rays.
Indirect effects = IR causes the formation of reactive species (e.g. H2O2, OH-)
IR exposure results in a myriad of damage to bases
Walker page 21
Deoxyribose damage
Damaged bases can lead to destabilization of glycosidic bonds leading
to abasic sites within the DNA
• OH radicals can lead to destabilization of ribose ring structures
Generation
of abasic site
Phosphodiester backbone damage
(strand breaks)
ss breaks result in damage that cannot be repaired via simple re-ligation
(while 5ʼ PO4 groups are usually present, the 3ʼ end is not generally a simple OH group; rather phosphate and phosphoglycolate groups are present as well as
damaged sugars and completely missing bases (nucleotide gaps)
The exact mechanisms that give rise to ds breaks is unknown
(two ss breaks?, different mechanism than ss breaks?)
1 Gy (100 Rad) of γ-rays results in 600-1000 ss breaks, 16-40 ds breaks and
~250 damaged bases (primarily thymidine)
DSB repair - historical perspective
Krasin and Hutchinson (1977) found that survival
following IR exposure in E. coli was enhanced in bacteria
containing replicated genomes.
Pollard, Fluke and Kazanis (1981) observed that bacteria
that carry multiple copies of the genome exhibit
increased resistance to IR. These observations, as well as similar
findings in yeast, suggest that alternate
copies of the genome are used in repair
mechanisms
Homologous Recombination-based DSB repair
MRN-dependent
Rad51-dependent
(RPA, Rad51, Rad52)
Mitotic
recombination
Bacteria and yeast, homologous recombination DSB repair is the principal mechanism of DSB repair.
In higher eucaryotes, non-homologous end joining (NHEJ)
or single-strand annealing (SSA) are the primary mechanisms for DSB repair.
Single-strand annealing
3ʼ
Direct repeats
IR
5ʼ to 3ʼ
endonucleases
Single
Strand
alignment
3ʼ
Removal of
Non-homologous
3ʼ ends DNA synthesis
Ligation
Molecules involved in SSA
SSA occurs independent of Rad51 -- no strand invasion
Because no homologous chromosome used in repair!
However, Rad52 is required because there is strand annealing
MRN is required for strand resection
The mismatch repair proteins Msh2 and Msh3 as well as Rad1
and Rad10 are required for efficient SSA and appear to be
needed to remove the non-homologous 3' tails from the annealed
intermediate.
Non-Homologous End Joining
MRN, WRN,
Artemis
Molecules involved in NHEJ
Ku70/Ku80 heterodimer: Avidly binds to DNA ends.
The regulatory subunits of DNA-PKcs
DNA-PKcs: Protein kinase, activity activated by free
DNA ends. Relevant substrates are unknown.
MRN complex, WRN helicase, Artemis endonuclease:
Strand resection and end processing.
XRCC4/Ligase IV complex: Required for ligation
Repair factors are recruited to sites of damage
Kim et al (2005) Independent and sequential recruitment of NHEJ and HR factors to DNA damage
sites in mammalian cells. JCB, 170:341-347.
DSB repair and genetic fidelity
HR-based DSB repair: Because homologous
chromosome is used, no loss of genetic information
SSA: Loss of information that lies between sites of
homology used in repair.
NHEJ: Loss of genetic information due to strand resection.
Because both SSA and NHEJ do not utilize homologous
chromosomes for repair, it is possible that repair could result
in translocation of genetic material.
Surviving Fraction (%)
DSB repair: It’s all about survival
100
10
1
0.1
0.01
0.001
0
NHF
GM5849C (A-T)
RKO
LoVo
HCT-116
2
5
10
IR Dose (Gy)
15
A single unrejoined DSB per cell per lethal event in yeast, suggesting that as
little as 1 to 2.5% of the double strand breaks produced by ionizing radiation
are never rejoined and become lethal.
RPA
H2AX
MRN complex
ATM
ATR
Chk1
Chk2
C-Abl
The MRN complex localizes to the sites of DNA damage
The proteins MRE11, Rad50, and Nbs-1 (XRS2 in yeast) form a complex termed MRN (MRX in yeast) Human and yeast MRE11 homologs have Mn-dependent nuclease activity in vitro.
Predominantly, MRE11 displays 3’ - 5’ exonuclease activity and MRN is thought to act in strand
resection during meiotic recombination and DSB repair.
The pig pile at the DSB
The pig pile is catalyzed by ATM-dependent phosphorylation
reactions and binding of BRCT domain-containing proteins
(53BP1, MDC1, BRCA1, NBS1 (the “N” of MRN))
Dysregulated coordination
between lagging/leading
strand synthesis
RPA-bound
Single stranded DNA
The dance at the stalled fork
1)  The kinase ATR binds to RPA coated ssDNA through its binding partner ATRIP
2)  The 9-1-1 complex (Rad9/Hus1/Rad1), a PCNA-like clamp is loaded on the DNA at
the stalled fork. The clamp loader is RFC where the largest of the subunits (RFC-1)
is substituted with Rad17.
3)  Rad9 is phosphorylated by ATR
4)  BRCT domains on TopBP1 (topoisomerase-binding protein 1) associate with the
phosphorylated sites on Rad9.
5)  The completion of the 9-1-1/TopBP1/ATR complex fully activates ATR which then
subsequently activates the kinase Chk1
ATM and ATR activate overlapping pathways that trigger cell
cycle arrest, apoptosis, DNA repair and other DNA damage
response (DDR) mechanisms