Pich

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DDB G0288873 may be an ortholog of human ercc6-like, also known as PICH (polo-interacting checkpoint helicase). The assignment is tricky, however. When DDB_G0288873 is blasted to nonredundant protein sequences, a number of proteins are hit with very low e-value. When these proteins are blasted back to Dicty, they generally hit both DDB_G0288873 and ercc6. Sometimes ercc6 is the first hit, sometimes DDB_G0288873 is hit first. The differences may be considerable. Thus ercc-like from the zebra finch Taeniopygia guttata hits DDB_G0288873 at 1e-129 but ercc6 at 4e-86. Human ercc6-like, however, hits Dicty ercc6 before DDB_G0288873.

A tree constructed with several of these sequences puts ercc6 and ercc6-like in separate groups. DDB_G0288873 looks rather like a form ancestral to both groups, but it is closer to ercc6-like.

Image:ercc6 and ercc6-like.tif

Expression data make a perhaps more convincing case. DDB_G0288873, but not ercc6, is strongly (6x; p<e-95) overexpressed in a Dicty strain in which the retinoblastoma-like gene rblA has been disrupted. Most cells involved in S-phase or mitosis are overexpressed in this strain.

Both ercc6 and ercc6-like are DNA helicases. Ercc6 is involved in DNA repair, more particularly in replication-independent repair of UV-induced thymidine dimers. One would not particularly expect ercc6 to be overexpressed in the rblA disruptant. In fact, none of the genes involved in this pathway are stongly overexpressed:

ercc1 1x
ercc4 1.4x
ercc6 0.9x
ercc8 0.9x
repB 0.9x
repD 1.7x
rad4 1.5x
lig3 1.6x
xrcc1 1.3x

Ercc6-like is an essential component of the spindle checkpoint, which localizes to the kinetochore; in prometaphase it is found on thin threads that stretch between sister kinetochores. It is thought to function as part of the tension sensor, the functional core of the spindle checkpoint [1].

Other spindle checkpoint components are strongly overexpressed in the rblA null. These include:

mad2 4x
bub1 4x
bub3 6x
cdc20 11x
ube2c 7x
mps1 13x
anapc3 11x
anapc5 22x
anapc7 7x
anapc8 4x
anapc10 12x

This suggests that DDB_G0288873 may actually code for the spindle tension sensor in Dicty.

Harry MacWilliams May 2010


ADDENDUM:

The association of PICH with the spindle assembly checkpoint was originally suggested on the basis of RNAi experients in HeLa cells, which showed an abrogation of the SAC upon PICH knockdown. New results convincingly show that this effect is due to off-target RNAi effects, which lead, probably indirectly, to a co-depletion of Mad2[2].

Other work suggests that the above-mentioned threads connecting sister kinetochores are PICH-coated, catenated (entangled) centromeric DNA . These tangles are normally resolved during anaphase by topoisomerase II [3], and PICH may also interact with topoisomerase II in the resolution of tangles along the chromosome arms. The exact cause-and-effect relationships are at present obscure. Thus PICH is recruited to chromosome arms during prophase. PICH normally recruits polo-like kinase 1, and PLK1 phosphorylates cohesin, leading to the separation of chromosome arms. PICH knockdown leads to disorganization of chromosome architecture which can be prevented by concomitant treatment with a topII inhibitor [4]. A speculative hypothesis might be that PICH binds to stretched (catenated) DNA, and thus recruits PLK1 to sites of DNA catenation, but blocks topII decatenating activity until PLK1 has locally destabilized cohesin. PLK1 ultimately phosphorylates PICH, perhaps releasing topII.

In Dictyostelium, a PICH-gfp fusion lights up centrosomal and nuclear structures, but is toxic when expressed under an A15 promoter, even in a single-copy vector (Müller-Taubenberger, personal communication). In human cells, high-level expression of PICH is also toxic, apparently because it sequesters PLK1 (Hübner, personal communication).

Harry MacWilliams, September 2010

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