.................................................................................................................................................... ii
CoAuthourship .................................................................................................................................... iii
Acknowledgements.............................................................................................................................. iv
Table
of
Contents ..................................................................................................................................vi
List
of
Figures......................................................................................................................................... ix
List
of
Tables ..........................................................................................................................................xi
List
of
Abbreviations,
Symbols,
and
Chemical
Formulas .......................................................xii
Chapter
1:
General
Introduction ......................................................................................................1
1.1
The
molecular
basis
of
cancer.......................................................................................................................5
1.2
The
Rad9A
checkpoint
protein .....................................................................................................................9
Chapter
2:
Literature
Review ......................................................................................................... 10
2.1
Chromatin
structure....................................................................................................................................... 11
2.1.1
Histone
modifications................................................................................................................ 12
2.1.2
DNA
modifications ...................................................................................................................... 13
2.2
DNA
damage
detection ................................................................................................................................. 14
2.2.1
ATM.................................................................................................................................................... 14
2.2.2
ATR/ATRIP..................................................................................................................................... 18
2.2.3
DNA‐PK ............................................................................................................................................ 19
2.2.4
The
911
Complex
and
the
Rad17/RFC
clamp
loading
complex .............................. 20
2.2.5
The
MRN
complex ....................................................................................................................... 28
2.2.6
Summary
of
DNA
damage
sensors ....................................................................................... 30
2.3
Checkpoint
transducers ................................................................................................................................ 30
2.3.1
ATM,
ATR/ATRIP,
and
DNA‐PK............................................................................................. 31
2.3.2
γH2AX ............................................................................................................................................... 33
2.3.3
Claspin .............................................................................................................................................. 34
2.3.4
BRCA1
and
BARD1 ...................................................................................................................... 35
2.3.5
TopBP1............................................................................................................................................. 37
2.3.6
Summary
of
DNA
damage
transducers .............................................................................. 38
2.4
Checkpoint
effectors ....................................................................................................................................... 38
2.4.1
Chk1................................................................................................................................................... 39
2.4.2
Chk2................................................................................................................................................... 42
2.4.3
The
Cdc25
Phosphatases.......................................................................................................... 45
2.4.4
Wee1/Myt1 .................................................................................................................................... 47
2.4.5
Cyclin/CDK
complexes .............................................................................................................. 48
2.4.6
Summary
of
the
DNA
damage
effectors ............................................................................. 50
2.5
Doublestrand
break
repair ........................................................................................................................ 50
vii
2.5.1
Homologous
recombination.................................................................................................... 51
2.5.2
Non‐homologous
end
joining ................................................................................................. 54
2.5.3
Alternative
non‐homologous
end
joining ......................................................................... 57
2.6
Checkpoint
Release ......................................................................................................................................... 58
2.7
Hypotheses
and
specific
aims ..................................................................................................................... 59
Chapter
3:
Materials
and
Methods................................................................................................ 61
3.1
Cell
Lines
and
Culture
Conditions............................................................................................................. 61
3.2
Cell
Synchronization,
Transfections
and
Treatments...................................................................... 62
3.3
Immunofluorescence
and
Confocal
Microscopy ................................................................................. 65
3.4
Immunoprecipitations
and
Immunoblotting ...................................................................................... 68
3.5
mRNA
extraction,
cDNA
synthesis,
and
qRTPCR .............................................................................. 70
Chapter
4:
The
Rad9A
checkpoint
protein
is
required
for
nuclear
localization
of
the
Claspin
adaptor
protein ................................................................................................................... 71
4.1
Rad9A
and
Claspin
interact
constitutively ........................................................................................... 71
4.1.1
The
Rad9A‐Claspin
interaction
is
not
cell
cycle
dependent...................................... 71
4.1.2
Modification
of
the
Rad9A
protein
does
not
affect
the
interaction
with
Claspin ........................................................................................................................................................... 74
4.2
Rad9A
affects
the
nuclear
localization
of
Claspin............................................................................. 78
4.2.1
Over‐expression
of
a
non‐nuclear
form
of
Rad9A
alters
the
subcellular
localization
of
Claspin ............................................................................................................. 78
4.2.2
Extraction‐resistant
Rad9A
forms
large
nuclear
foci
with
Claspin........................ 85
4.2.3
Rad9A
molecules
are
able
to
multimerize ........................................................................ 90
4.3
Rad9A/B
are
responsible
for
Claspin
localization ............................................................................ 93
4.3.1
rad9Anull
mES
cells
express
Rad9B................................................................................... 93
4.3.2
Differentiation
of
Rad9A‐null
mES
cells
alters
the
subcellular
localization
of
Claspin
but
has
no
affect
on
WT
mES
cells..................................................................... 96
4.3.3
Conclusions
about
the
interaction
between
Rad9A
and
Claspin ............................ 96
Chapter
5:
The
identification
and
characterization
of
a
novel
nuclear
structure
containing
members
of
the
homologous
recombination
DNA
damage
response
pathway.................................................................................................................................................. 98
5.1
The
RDF
closely
associate
with
Xi............................................................................................................. 98
5.1.1
Rad9A
closely
associates
with
the
histone
variant
macroH2A1 ............................. 98
5.1.2
Rad9A
colocalizes
with
the
facultative
heterochromatin
marker
H3trimK9..101
5.1.3
RDF‐macroH2A1
foci
overlap
with
BRCA1
foci............................................................101
5.1.4
RDF
colocalize
with
γ‐H2AX
independent
of
exogenous
DNA
damage ..............104
5.2
The
RDF
colocalize
with
members
of
the
homologous
recombination
pathway...............113