Rolling circle replication-

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Koeng

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Feb 7, 2013, 8:48:45 PM2/7/13
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Hey I was wondering what exactly makes the DNA "nicked" in the origin. Is it just like that? Or is there other enzyme that makes it nicked? This question has just been like an itch in my mind

Nathan McCorkle

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Feb 7, 2013, 11:25:41 PM2/7/13
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Generally when I've heard about plasmids being nicked, it's referring
to damage encountered during an extraction process.

If you're asking how they coil and uncoil during/after replication,
you probably want to start with topoisomerase.

--
-Nathan

Cathal Garvey

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Feb 8, 2013, 12:42:17 AM2/8/13
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In RCM, there's a dedicated enzyme called "rep" that's encoded usually
next to the origin nick site. In at least some plasmids (those I used as
a baseline for my designs), rep actually covalently bonds to the 5' end
of the DNA as it nicks, "lifting" the DNA as it nicks it to clearly
expose a loose 3' end for extension.

Although rep initially works as a monomer I think, I recall that in
order to work it needs a pair of reps: possibly the nicking process
requires a dimer, but a monomer is bound covalently? But the partner
monomer resumes its role later in finishing synthesis in such a way that
it permanently inactivates both monomers, preventing catalysis of
additional plasmids. I guess that means it's not an "enzyme" as it isn't
a catalyst, but rather is consumed in the reaction.

I had a paper describing this neatly, but I seem to have lost it. Do
some searching on RCM and rep dimers and you should find some good info.

Mega

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Feb 8, 2013, 2:47:17 AM2/8/13
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But the partner 
monomer resumes its role later in finishing synthesis in such a way that 
it permanently inactivates both monomers, preventing catalysis of 
additional plasmids.

That sounds like you could engineer the monomer so that it doesn't inactivate them, thus getting an infinite plasmid  producing machine / bacteria. Without any primers. 

The bacterium should secrete the plasmids then, else it would burst some time :) 

Cathal Garvey

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Feb 8, 2013, 4:20:48 AM2/8/13
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Perhaps, but that would make the plasmid unstable; evolution would
quickly select for a pure culture of plasmid-free bacteria..
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Koeng

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Feb 8, 2013, 9:48:48 AM2/8/13
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If it bonds to the 5' DNA as it nicks, then what makes it not nick the host DNA?

I am going to do some searching on RCM and rep dimers :) thanks for the suggestion. I have always been interested in origin of replications!
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