It’s not in the comics per se, but when I think of Darkseid I always remember him in the animated series when he killed Dan Turpin. It was on tonight and it still makes me get misty. I know he’s done a lot, but that one just sticks with me, the petty evil of it. 
Joker’s signature, I’d say, would be either the murder of Jason Todd or the attempt to blow up the UN that immediately preceded it.
His attempt to blow up New York (which got him thrown into the Slab rather than Arkham, much to his distress) was pretty badass, too, but nobody ever seems to remember that one.
Mongul - destroying Coast City and leading to the Hal Jordan going mad and becoming Parallax (later adjusted by retcon, but Mongul still destroyed Coast City.)
Sinestro - Conquering and despotically ruling his home planet of Korugar.
Deathstroke - Engineering the introduction of Terra into the Teen Titans as a spy and traitor.
Captain Boomerang - Killing Jack Drake (Robin’s father)
Doomsday - Killing Superman
Aw man, is that from Start of Darkness? I haven’t read any of the OotS books yet. Also, I would argue that an act not depicted in the regular comic wouldn’t be very well known and not his most famous/notorious deed.
IIRC the 2nd time it wasn’t really Doom, but the kid he had brainwashed to replace him in case he met an early demise…Kristoff i think was his real name.
Kristoff not only launched the building into space, but once in orbit he blew it up.
<Adam West voice> …must not … make … political joke… must resist … would devastate … cafe society… can’t … mention … face-shooting… <\Adam West voice>
This twist has to be the most spoiled plot point on the entire Internet. People are always quoting it for some reason or another, completely ignoring the fact that Watchmen is not as mainstream as they think it is and tons of people who would otherwise read never do because the big twist is severely spoiled.
Well, to be honest anyone interested in comics would probably read it anyway, despite knowing the twist.
Agreed (although I still haven’t read it), but it’s such a good twist and why Watchmen fans think it’s OK to ruin it is beyond me.
Given this and other posts here, one of the mods should probably put a general spoiler warning in the thread title. Because I can’t see how we can avoid spoilers in this thread - Doctor Evil’s signature act of supervillainy was that time he did…you know…that supervillainy thing.
But to address your specific point, yes, it was in Start of Darkness. And even with all the evil deeds Xykon has done (and if you haven’t read SoD you’ve missed a lot of them) this was still the top one. Xykon himself said it was what seperated him from lesser evil beings like Red Cloak - they want to use evil means to advance some cause; he’s just Evil (with a capital E) for the sake of being evil.
Keiser Soze’s signature act was to convince the world that he doesn’t exist.
Professor Moriarty’s signature act was to come to a deadlock ending with both Holmes and himself plummeting to their watery graves (possibly.)
Salieri’s signature act was to have Mozart drink himself to death while trying to write a Requiem for the ghost of his father. (If you watch the play/movie.)
… I miss Christmas.
Snidely Whiplash tying Nell Fenwick to the railroad tracks.
Kid Miracleman’s utter obliteration of central London – 40,000 people brutally murdered by hand – is probably his signature crime.
It’s quite possibly the most horrific thing I’ve ever seen in a superhero comic book (albeit a pretty non-mainstream one). It’s made even more horrific by his amused greeting to the heroes as they come to stop him: