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Poster Full Abstracts - Cell Cycle and Checkpoints
Poster board number is above title. The first author is the presenter
209
Characterization of Caprin Phosphorylation at the mid-blastula transition.
Xi Chen, Ophelia Papoulas. The Section of Molecular Cell and
Developmental Biology, The University of Texas at Austin, Austin, TX.
The molecular signals driving the developmental shift referred to as the mid-blastula transition (MBT) remain mysterious. In the syncytial
Drosophila
embryo, rapid synchronous nuclear divisions cease at the MBT to permit membrane invagination and formation of a cellular blastoderm. We have reported
that the RNA-binding translational regulators Fragile X mental retardation protein (FMRP) and Cytoplasmic activation/proliferation-associated protein
(Caprin) act at the MBT to modulate levels of cell cycle regulators necessary for this prolonged interphase. FMRP has been most extensively studied in the
nervous system because loss of FMRP causes Fragile X Syndrome, the most common form of heritable human mental retardation and autism. However both
FMRP and Caprin (CAPR) are believed to mediate rapid local changes in translation in response to synaptic signals in neuronal dendrites. We find that both
proteins are present throughout early embryogenesis but act specifically at the MBT suggesting they may be responding to specific signaling at that time.
The nucleo-cytoplasmic ratio has long been viewed as a key signal triggering events of the MBT, but the molecular nature of the signal is unknown. Through
immunoblotting of precisely staged embryos we have found that CAPR becomes phosphorylated specifically at the MBT. We are characterizing the timing
and sites of phosphorylation using GST-fusions comprising thirds of the CAPR protein and a staged embryo cell extract-based in vitro kinase assay.
Preliminary data suggest that the middle portion of CAPR, containing the conserved G3BP/Rasputin binding domain, is specifically phosphorylated. Current
work is aimed at characterizing the functional significance of this modification, and identifying the specific residues modified and the kinase responsible.
Through these studies we hope to better understand signal-responsive control of translation and the signaling mechanisms underlying the MBT.
289A
mu2
affects mitosis and meiosis by regulating BubR1 expression in
Drosophila melanogaster
.
James M. Mason
1
, Raghuvar Dronamraju
1,2
. 1) Laboratory
of Molecular Genetics, NIH/NIEHS, Research Triangle Park, NC; 2) Department of Biochemistry and Biophysics, UNC, Chapel Hill, NC.
The molecular components that decide the development of an oocyte are largely uncharacterized. The
mu2
gene of
Drosophila melanogaster
encodes a
chromatin protein found in the oocyte nucleus that acts as a scaffold during DNA repair to elicit a DNA damage response and meiotic recombination in
oocytes.
mu2
a
mutant females delay the repair of radiation induced chromosome breaks in oocytes. Accurate segregation of chromosomes during cell
division requires organized centromeres and telomeres, which when defective activate the spindle assembly checkpoint. Using immunohistochemistry we
show that
mu2
a
mutants exhibit defective inner and outer kinetochore components, such as CIN and BubR1, during oocyte development. These defects may
lead to other observed phenotypes, such as delayed maturation of the pro-oocyte produced by a mutant mother, aneuploidy in the resulting zygote,
asynchronous mitosis in the early embryo, and an increase in the number of pole cells later in embryogenesis. MU2 is likely a non-essential downstream
effecter protein in cell cycle control, checkpoint activation and DNA repair processes.
290B
Distinct roles for multiple translesion polymerases during DNA double-strand break repair.
Mitch McVey
1
, Daniel P Kane
1
, Michael Shusterman
1
,
Kelly Beagan
1
, Yikang Rong
2
. 1) Biology, Tufts University, Medford, MA; 2) Laboratory of Biochemistry and Molecular Biology, National Cancer
Institute, Bethesda, MD.
DNA double-strand breaks, when repaired inaccurately, can promote mutagenesis in the form of point mutations, deletions, and genome rearrangements.
Historically, DNA translesion polymerases have been associated with mutagenesis during lesion bypass and postreplication repair. However, their role(s)
during DNA double-strand break repair are poorly defined, particularly in metazoans. To address this, we carried out a systematic genetic analysis of DNA
polymerase mutants in
Drosophila melanogaster
. We generated stocks with null mutations in genes encoding polymerases eta, zeta, theta, Rev1, and the
nonessential Pol32 subunit of polymerase delta. Using mutagen sensitivity analysis and two independent site-specific break repair assays, we showed that
translesion DNA polymerases eta and zeta are both involved in homologous recombination repair of DNA breaks. Furthermore, Pol32 is required for
extensive DNA synthesis during double-strand gap repair. Flies lacking both Pol32 and polymerase zeta have extreme defects in repair synthesis, indicating
that these polymerases may operate in separate stages of gap repair. Interestingly, rev1 mutants display an enhanced ability to carry out gap repair and have
increased repair synthesis tract lengths. In cases where homologous recombination aborts prematurely, polymerase theta functions in an alternative end
joining repair mechanism, independent of DNA ligase 4.
Based on these findings, we propose a model in which replicative and translesion polymerases compete for access to D-loop intermediates during
homologous recombination repair. Rev1 appears to be an important mediator during gap repair and may promote the recruitment of translesion polymerases
during the early stages of repair synthesis. Together, our results suggest surprising complexity in the enzymology of DNA synthesis during double-strand
break repair.
291C
Establishing linkage between GINS complex sub-unit Sld5 and checkpoint protein Chk2
(loki)
using
Drosophila melanogaster
as the model
organism.
Divya Devadasan, Tim Christensen. East Carolina University, Greenville, NC.
Eukaryotic DNA replication is controlled by a number of proteins that ensures the process takes place accurately. GINS, a hetero-tetrameric protein
complex is known to be essential for the initiation and progression of eukaryotic DNA replication. The GINS complex constitutes four subunits; Sld5, Psf1,
Psf2, Psf3. The Sld5 subunit of GINS is an evolutionarily conserved protein. Previous research from our lab shows that Sld5 is required for normal cell cycle
progression and the maintenance of genomic integrity. In addition, the depletion of other GINS sub-units Psf1 and Psf2 by siRNA in human fibroblasts lead
to genomic instability and activation of Chk2. Preliminary results in Drosophila show that there are mitotic phase delays in the Sld5 mutant lines compared
to wild-type. To further investigate the role of Sld5 in checkpoint signaling, a multifaceted approach is being used. First, Sld5-Chk2 double mutants were
generated to check for replication defects and cell cycle progression. The mitotic delay observed in Sld5 mutants were found to be mediated through Chk2.
Interestingly enough, the S-phase delay observed in Sld5 mutants did not appear to be mediated through Chk2, though there was an S-phase delay observed
endogenously in the loki/loki;Sld5/+ mutants. Evidence of endo-replication defects and differences in the packaging ratio of DNA between wild-type and
mutants was investigated in salivary glands. Sld5 mutants showed significantly larger amounts of DNA per nuclei although the nuclei were packaged similar
to wild-type. Levels of apoptotic cells in single and double mutants is also being investigated to determine if cell death caused due to Sld5 depletion is
regulated by Chk2. Errors due to under-replication or over-replication can lead to disastrous consequences leading to several genetic diseases like cancer,