#9708 DNA replication initiation - improve def

[Midori Harris]

Hello lucky GO people,

In SF 3549889, we realized that the start and end of initiation could do to be much more precisely
specified in the 'DNA replication initiation' (GO:0006270) def.

It's fairly straightforward in bacteria, but unfortunately for eukaryotes it may not be easy to pin down the start. (This is all based on skimming through papers from a 'dna replication initiation review' PubMed search and a small amount of googling.)

In bacteria, initiation starts when the initiator protein (DnaA; most or all species use this gene name) binds to the origin and melts the DNA. DnaA then loads a helicase, the helicase melts origin DNA, DNA polymerase and some other proteins come along, and Bob's your uncle. Initiation includes primer synthesis* and ends when DNA polymerases start to extend the primers (i.e. when elongation starts).

*but not the synthesis of Okazaki fragment primers; that's lagging strand elongation

For euks, it's well established that these these steps have to happen:

- ORC binds to replication origins;

- MCM proteins and a few others, e.g. Cdt1 & Cdc6 (budding yeast names) join ORC at origins to form the prereplicative complex (pre-RC);

- Yet more proteins (the GINS complex and Cdc45 in fission yeast) join the pre-RC to form the DNA replication preinitiation complex (pre-IC) -- a name that may or may not be accurate; also, Cdt1 & Cdc6 leave;

- Protein phosphorylation happens;

- The helicase (MCM hexamer) melts DNA;

- DNA polymerases and other proteins join the fun and the first primers are synthesized.

The exact order of some of the protein phosphorylation events with respect to some of the complex assembly events isn't well established.

Some sources seem to say that initiation starts with pre-IC assembly (despite its name); others count pre-RC formation as an early initiation event; still others (tho apparently not many) say DNA melting is the beginning of initiation proper.

Most, however, are woollier and don't explicitly peg any of the steps as Step 1 of Initiation. As a result, the only things I'm really confident of are that (a) ORC binding is not the first step -- ORC is bound to origins throughout the cell cycle -- and (b) initiation doesn't start any later than DNA melting.

I think the end of initiation is basically the same as in bacteria, i.e. primer extension/beginning of elongation.

Sorry for all the parentheticals and weasel words, but I'm having trouble extracting anything firmer. There's a lot of consistency regarding what events happen, and to the effect that all of the above events are somehow very relevant to initiation, but not about precisely where the line between 'precedes, and required for, initiation' and 'part of initiation' belongs.

Oh, and just to make it even more complicated, the connections with S phase of the cell cycle may have to be evaluated too, because the pre-RC forms during G1. So if pre-RC assembly counts as part of initiation, then not all of the entire replication process takes place during S phase. Ow my head.

A specific note: depending on where GO puts the beginning of initiation, the text def of GO:0071163 may have to be adjusted.

have fun,
m

[Valerie Wood]

Message from Nick Rhind
---

Midori's description is pretty spot on. I would make the following minor changes.

Cdt1 is associate with MCM and is required for ORC/Cdc6-dependent MCM loading, but Cdt1 does not appear to be a stable part of the preRC.

preRC phosphorylation (MCM by DDK? Sld2 and Sld3 by CDK?) is required for stable Cdc45 and GINS binding.

[Valerie Wood]

and (also from Nick in answer to "where does DNA replication iniitiation begin"?)

I would say initiation begins with the phosphorylation of preRC by CDK and DDK leading to the recruitment of Cdc45 and GINS, which I think is synonymous with preRC -> preIC.

[Midori Harris]

Re:
> preRC phosphorylation is required for stable Cdc45 and GINS binding.

One of the main questions in SF 3549889 is which comes first, Cdc45 + GINS binding or phosphorylation. I found a review from 2007 that said it wasn't known at the time, so if Nick knows of more recent evidence that settles the question, that will help with both SFs.

[Jane Lomax]

So the definitions of these terms need to be refined:

DNA replication initiation ; GO:0006270
DNA replication preinitiation complex assembly ; GO:0071163
DNA-dependent DNA replication ; GO:0006261 (?)

The definitions should probably state the initiation events in both proks and euks.

So the first event for euks is the the phosphorylation of the preRC which leads to the formation of the preIC?

Do you want to take a stab at the new definitions, Midori, or shall I?

[Midori Harris]

I doubt I'd get to it any time soon ...

[Midori Harris]

the other ticket referenced is now [ontology-requests:#9634]