Download Laboratory Projects

Determine the repertoire of chromosome movement in
segregation, repair and recombination
Identify forces responsible for chromosome movement
Is chromosome movement integral for DNA repair?
What is the cellular response, including chromosome reorganization
to DNA damage?
KBY2501 (LacO integrated ~1.1Kb from CEN11)
~1.1 Kb
LacO Repeat CEN11
(32mer)
I
III
KBY2502 (LacO integrated ~12.7 Kb from CEN11)
~12 Kb
LacO Repeat
(32mer)
CEN11
I
III
Comparison Timecourse of Chromosome Markers 1Kb and 25Kb from CEN3
9
8
Spot Separation Distance (um )
7
6
5
CEN spot
separation
4
25Kb from CEN3
spot separation
3
2
1
0
0:00:00
0:14:24
0:28:48
0:43:12
Elapsed Tim e (hrs:m in:sec)
0:57:36
1:12:00
1:26:24
Yeast chromosomes oscillate from pole to pole prior to
anaphase onset.
Centromeres separate prior to the chromosome arms
Rate of separation of chromosome arms
exceeds rate of centromere separation
Mechanisms of cohesion and separation are
likely to differ along the chromatin fiber
Centromeres on the same chromatid attach to opposite poles:
Chromosome Breakage
Centromeres on the same chromatid attach to same poles:
NO Chromosome Breakage
Chromosome rearrangements visualized in time
course from galactose to glucose
Gal Glu
Gal Glu
1100bp
865bp
RAD+
Repair mutant
rad52- or rad1-
Single strand excision
Repair via Single strand annealing
Revealed by 1100 bp repair product
Activate Dicentric Chromosome
Does mid-anaphase delay precede break ?
or
mid-anaphase delay follows break
Mid-anaphase pause is dependent upon RAD9 checkpoint gene
Mid-anaphase pause is not dependent upon the
spindle assembly checkpoint genes, MAD2 or BUB1
RAD9 recognizes DNA damage and/or chromatin alterations
Does RAD9 “mobilize” chromosomes/repair machinery
in response to DNA damage
Viability %
80
70
60
50
40
30
20
10
0
ka
r9
p3
ki
dy
ne
in
u7
0
yk
ra
d1
ra
d5
2
ra
d9
W
ild
ty
pe
Viability %
Viability of cells upon activation of the dicentric chromosome
Repair mutants (rad1, rad52 and Yku70), rad9 checkpoint
and mt based motility mutants (dynein, kip3, kar9)


The mid-anaphase pause is “longer” in the absence of
cytoplasmic dynein or kip3
Cytoplasmic microtubule-based motor proteins
contribute to the fidelity of chromosome repair
Mt based motor proteins effect the DNA damage response
How do “repair mutants” effect chromosome organization
Ku- Non-homologous end-joining
Physiological
functions of Ku
• Ku70, Ku80 knockouts in mice have similar
phenotype to SCID
– V(D)J defects arrest lymphocyte development
• Ku70, Ku80 -/- mice are runts compared to +/littermates
– Number of cell divisions in development limited by
impaired ability to repair endogenously generated DNA
damage
– Ku-deficient cells might take longer to repair this
damage
• Ku80 -/- dams fail to nurture their pups
Yeast Ku in
telomere maintenance
• Disruption of yKu70p and yKu80p genes
affect telomeric silencing, telomere length
maintenance and viability of cells
containing dicentric chromosomes
– inactivate Ku, lose telomeric silencing
– inactivate Ku, shorten telomeres
– inactivate Ku, decrease viability of cells
containing dicentric plasmids
• Ku clusters yeast
telomeres to peripheral
sites in nucleus
– In diploids, telomeres
usually found in 6-7
clusters around nuclear
periphery
– In Ku subunit mutants,
more clusters in
random locations
Featherstone, C., and Jackson, S. Mutat Res. 1999 May 14;434(1):3-15. Review.
Activation of dicentric chromosome in Ku mutants
results in chromosome decondensation
Chromosome decondenses in stepwise fashion
Dicentric chromosome stretching in
metaphase in a stepwise fashion
and recoil upon spindle disassembly
Regularity of spacing upon chromatin decompaction
Chromosome breakage and retraction to spindle poles
Sir2p binds to stretched lacO DNA
MAT
Chromatin stretching is recognized in vivo
 Chromatin can act as an in vivo tensiometer
RAD9+
rad9-
Rad9 is required for chromosome unfolding in response to
activation of the dicentric chromosome
Laboratory Projects
Dale Beach- RNA localization
Paul Maddox-Mt polarity and dynamics
Elaine Yeh- Asymmetric protein determinants
THANKS !!!