Abstract: Neurons are highly vulnerable to genotoxic agents.
To restore genome integrity upon DNA lesions, neurons trigger
a DNA damage response (DDR) that requires chromatin
modifications and transcriptional silencing at DNA damage
sites. To study the reorganization of the active RNA polymerase
II (Pol II), which transcribes all mRNA-encoding genes,
and the participation of the ubiquitin-proteasome system
(UPS) in the neuronal DDR, we have used rat sensory ganglion
neurons exposed to X-rays (4 Gy) ionizing radiation (IR).
In control neurons, Pol II appears concentrated in numerous
chromatin microfoci identified as transcription factories by the
incorporation of 5?-fluorouridine into nascent RNA. Upon IR
treatment, numerous IR-induced foci (IRIF), which were immunoreactive
for ?H2AX and 53BP1, were observed as early
as 30 min post-IR; their number progressively reduced at 3 h,
1 day, and 3 days post-IR. The formation of IRIF was associated
with a decrease in Pol II levels by both immunofluorescence
and Western blotting. Treatment with the proteasome
inhibitor bortezomib strongly increased Pol II levels in both
control and irradiated neurons, suggesting that proteasome
plays a proteolytic role by clearing stalled Pol II complexes
at DNA damage sites, as a prelude to DNA repair. Neuronal
IRIF recruited ubiquitylated proteins, including ubiquitylated
histone H2A (Ub-H2A), and the catalytic proteasome 20S.
Ub-H2A has been associated with transcriptional silencing at
DNA damage sites. On the other hand, the participation of
UPS in neuronal DDR may be essential for the ubiquitylation
of Pol II and other proteasome substrates of the DNA repair
machinery and their subsequent proteasome-mediated
degradation.
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