Growth, Differentiation and Sexuality

46 S.D. Harris
entry, and the CDK NimX accumulates in an inactive,
tyrosine-15 phosphorylated form (Fig. 3.6).
By contrast, NimA activity is not affected. This
mechanism is sufficient to block entry into mitosis,
asthearrestisabrogatedbymutationsthateliminate
the AnkA kinase or change tyrosine-15 to
a residue that cannot be phosphorylated (Ye et al.
1997b; Kraus and Harris 2001). Accordingly, as established
in animal cells and S. pombe, DNA damage
presumably alters the balance of AnkA (Wee1)
and NimT (Cdc25) activity (Elledge 1996), leading
to increased tyrosine-15 phosphorylation of NimX
and inhibition of mitotic entry.
TheCDKtyrosine-15regulatorymodulealso
prevents inappropriate mitosis when DNA replication
is slowed in A. nidulans (i.e., using 5 mM
hydroxyurea; Ye et al. 1996). However, when replication
is completely blocked (i.e., using 100 mM
hydroxyurea), a functional APC is also required to
inhibit mitotic entry. Presumably, this additional
level of regulation ensures that NimA kinase remains
inactive and cannot initiate premature mitosis
(Fig. 3.6; Ye et al. 1996).
The DNA damage signaling pathway that leads
to mitotic checkpoint activation has been exten-
Fig. 3.6. Model for checkpoint regulation of mitosis in Aspergillus
nidulans. The UvsB/UvsD and Mre11 complexes
are activated in response to DNA damage or replication
stress. In analogy to yeast and animal cells, these complexes
presumably regulate the activity of both AnkA (upregulated)
and NimT (down-regulated), thereby preventing
activation of the NimXCDK.Under severe replication stress,
NimA activity is also inhibited, presumably via the APC. See
text for additional details
sively characterized in yeast (Zhou and Elledge
2000). Components of this pathway, including
UvsB (=Mec1), UvsD (=Rad26), ScaA (=Nbs1),
and MreA (=Mre11), have also been identified in A.
nidulans, where they appear to perform functions
similar to the yeast homologues (Fig. 3.6; de Souza
et al. 1999; Hofmann and Harris 2000; Bruschi
et al. 2001; Semighini et al. 2003). Nevertheless,
it is not clear how this pathway regulates CDK
tyrosine-15 phosphorylation or NimA activity to
arrest mitosis. For example, does this step require
homologues of the Chk1 and/or Chk2 protein
kinases, as it does in yeast and animal cells?
Moreover, genetic studies clearly show that the
fungal DNA damage signaling pathway regulates
additional functions that contribute to viability
when genome integrity has been compromised
(Ye et al. 1997b; Hofmann and Harris 2000).
These functions likely include the activation
of DNA repair pathways (Hofmann and Harris
2000; Semighini and Harris, unpublished data)
and the inhibition of DNA re-replication (de
Souza et al. 1999). Further studies also revealed
a novel post-mitotic function for the DNA damage
signaling pathway. In particular, low levels of
damage that are not sufficient to block mitosis
prevent septum formation in pre-divisional hyphae
(Harris and Kraus 1998). This response is
mediated by the DNA damage signaling pathway
and NimX tyrosine-15 phosphorylation (Harris
and Kraus 1998; Kraus and Harris 2001), though
the underlying mechanism remains unknown.
The observation that DNA damage triggers the
checkpoint-mediated arrest of mitotic entry raises
two important questions in the context of fungal
biology. First, how does a single nucleus respond
to DNA damage within a multinucleate hyphal cell?
In yeast and animal cells, the DNA damage checkpoint
signal is nuclear autonomous (Sluder et al.
1995; Demeter et al. 2000). It is not clear if this is
also true for filamentous fungi, particularly those
that display a “parasynchronous” mitotic wave. If
so, damaged nuclei must possess a mechanism that
makes them refractory to a passing wave. Alternatively,
the presence of a threshold level of damaged
nucleimayabolishthewaveandrevertthecellto
an asynchronous pattern of mitosis. Second, how is
the mitotic checkpoint modulated during development?
In some filamentous fungi (i.e., A. nidulans;
Timberlake 1990), the cell cycle appears to accelerate
during asexual development to permit the rapid
production of spores. This may imply that mitotic
checkpoints are eliminated, such that, like the syn-