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RADIOBIOLOGY 105
NONHOMOLOGOUS END-JOINING DEFICIENCY OF L5178Y-S CELLS
IS NOT ASSOCIATED WITH MUTATION
IN THE ABCDE AUTOPHOSPHORYLATION CLUSTER
Kamil Brzóska, Marcin Kruszewski, Irena Szumiel
Recent studies by Block et al. [1] indicated that
cells expressing DNA-dependent protein kinase
(DNA-PK) catalytic subunit (DNA-PK cs ) with mutated
autophosphorylation sites in the so-called
ABCDE cluster of serine and threonine residues
in the central part of the molecule are defective in
the repair of ionising radiation-induced double
strand breaks (DSB). The purified mutated DNA-PK
proteins, however, had shown in an in vitro test
the same protein kinase activity as the wild type
enzyme and were able to undergo ATP-dependent
autophosphorylation and inactivation. Thus, the
properties of mutants corresponded with those of
L5178Y-S (LY-S) cells (review in [2,3]) which show
casette) in search for possible mutations that would
affect autophosphorylation the DNA-PK in LY-S
cells. LY-R cells served as reference cells. In spite
of phenotypic features suggesting the existence of
such mutations, no differences were found in size
of RT-PCR product (not shown). This, however,
does not exclude a point mutation that might cause
the loss of autophosphorylation sites. So, we sequenced
the region of interest. Again, no differences
were found in the sequence of RT-PCR product
obtained from LY-R and LY-S cells (Fig.).
These results indicate that the defect in NHEJ in
the latter cells is not related to the structure of the
ABCDE casette.
Fig. Nucleotide (A) and predicted amino acid (B) sequences of DNA-PK cs region containing ABCDE autophosphorylation
cluster of LY-S and LY-R cells compared with sequence of murine DNA-PK cs ABCDE autophosphorylation cluster
(GenBank accesion No. D87521). Exon 58 of DNA-PK cs is highlighted by black background colour, five phosphorylation
sites present in murine ABCDE cluster are shown in gray. Nucleotide sequences of DNA-PK cs region
containing ABCDE autophosphorylation cluster of LY-S and LY-R cells were deposited in GenBank under the
accesion Nos. DQ235257 and DQ235258, respectively.
both phenotypic features that are characteristic of
cells with a defective nonhomologous end-joining
(NHEJ): a slown down DSB repair and a pronounced
G1 phase radiosensitivity. However, in
spite of the functional impairment in LY-S cells,
the same level of DNA-PK activity is present in
total cell extracts of both LY cell lines [4].
It seemed plausible that such an autophosphorylation
defect which does not affect kinase activity
but is a cause of failure in DNA end-joining is
the cause of impaired NHEJ in LY-S cells. Therefore,
we examined the Prkdc gene fragment coding
the cluster of autophosphorylation sites (ABCDE
The work was supported by the Polish Ministry
of Scientific Research and Information Technology
statutory grant for the INCT.
References
[1]. Block W.D., Yu Y., Merkle D., Gifford J.L., Ding Q.,
Meek K., Lees-Miller S.P.: Nucleic Acids Res., 32,
4351-4357 (2004).
[2]. Szumiel I.: Int. J. Radiat. Biol., 81, 339-352 (2005).
[3]. Szumiel I.: Int. J. Radiat. Biol., 81, 353-365 (2005).
[4]. Wojewodzka M., Kruszewski M., Sochanowicz B.,
Szumiel I.: Int. J. Radiat. Biol., 80, 473-482 (2004).