DNA replication arrest in XP variant cells after UV exposure
is diverted into an Mre11-dependent recombination pathway by the kinase
inhibitor wortmannin.
Limoli CL, Laposa R, Cleaver JE.
Department of Radiation Oncology, University of California, San Francisco, CA
94103-0806, USA.
Ultraviolet (UV) irradiation produces DNA photoproducts that are blocks to DNA
replication by normal replicative polymerases. A specialized, damage-specific,
distributive polymerase, Pol H or Pol h, that is the product of the hRad30A
gene, is required for replication past these photoproducts. This polymerase is
absent from XP variant (XP-V) cells that must employ other mechanisms to
negotiate blocks to DNA replication. These mechanisms include the use of
alternative polymerases or recombination between sister chromatids. Replication
forks arrested by UV damage in virus transformed XP-V cells degrade into DNA
double strand breaks that are sites for recombination, but in normal cells
arrested forks may be protected from degradation by p53 protein. These breaks
are sites for binding a protein complex, hMre11/hRad50/Nbs1, that colocalizes
with H2AX and PCNA, and can be visualized as immunofluorescent foci. The protein
complexes need phosphorylation to activate their DNA binding capacity.
Incubation of UV irradiated XP-V cells with the irreversible kinase inhibitor
wortmannin, however, increased the yield of Mre11 focus-positive cells. One
interpretation of this observation is that two classes of kinases are involved
after UV irradiation. One would be a wortmannin-resistant kinase that
phosphorylates the Mre11 complex. The other would be a wortmannin-sensitive
kinase that phosphorylates and activates the p53/large T in SV40 transformed
XP-V cells. The sensitive class corresponds to the PI3-kinases of ATM, ATR, and
DNA-PK, but the resistant class remains to be identified. Alternatively, the
elevated yield of Mre11 foci positive cells following wortmannin treatment may
reflect an overall perturbation to the signaling cascades regulated by
wortmannin-sensitive PI3 related kinases. In this scenario, wortmannin could
compromise damage inducible-signaling pathways that maintain the stability of
stalled forks, resulting in a further destabilization of stalled forks that then
degrade, with the formation of DNA double strand breaks.
