PMID:19033384 community curation

[fypoadmin]

1. advanced mitotic G1/S phase transition during cellular response to hydrogen peroxide
2. advanced mitotic G1/S phase transition during cellular response to MMS
3. normal DNA replication delay during cellular response to hydrogen peroxide
4. decreased protein phosphorylation during cellular response to MMS
5. normal DNA replication delay during cellular response to MMS

PMID:19033384

Original comment by: Antonialock

[fypoadmin]

Added the easy ones:

advanced mitotic G1/S phase transition during cellular response to hydrogen peroxide FYPO:0002991 advanced mitotic G1/S phase transition during cellular response to methyl methanesulfonate FYPO:0002992 decreased protein phosphorylation during cellular response to methyl methanesulfonate FYPO:0002993

For the other two, do you know whether the replication delay a) affects initiation? and/or b) refers to the G1/S transition? and/or c) is connected with a checkpoint (and if so, which one)?

I'm trying to figure out where to put the terms. From a quick look at the paper it seems that the delay is initiation occurring later with H2O2 or MMS than without in wild type cells, but I got confused trying to follow what's going on in the gcn2 mutant.

Original comment by: mah11

[fypoadmin]

Both H2O2 treated and MMs treated control cells arrested in G1. They used G1 synchronised cells however so these treatments may also cause delays in other phases (for instance, it looked like the gcn2 mutant was arrested intra-S if I remember right so that checkpoint is controlled by other proteins)

I annotated gcn2 to mitotic G1 DNA damage checkpoint - not saying it is not also mitotic G2 DNA damage checkpoint et cetera In the paper they show that this checkpoint is activated by some but not all DNA damage (it is activated by h2o2 and mms but not gamma irradiation for instance)

does that help?

Original comment by: Antonialock

[fypoadmin]

I wonder ... would it work if I name and define the terms as "normal negative regulation of DNA replication during X"?

Original comment by: mah11

[fypoadmin]

Yeah, I think that normal DNA replication delay during = normal negative regulation of DNA replication during

Original comment by: Antonialock

[fypoadmin]

ok, done

normal negative regulation of DNA replication during cellular response to hydrogen peroxide FYPO:0002996 normal negative regulation of DNA replication during cellular response to methyl methanesulfonate FYPO:0002997

Original comment by: mah11

[fypoadmin]

Hi Antonia,

Can you say anything about the G1/S transition delay? i.e. representing a G1 block? delaying initiation?

or an S-phase block representing delayed elongation or other aspect??

It would be useful to specify this in the FYPO term if poss as this is pone of the things I'll be looking at when I review cell cycle annotation

Original comment by: ValWood

[fypoadmin]

I think it is the "normal" DNA damage checkpoint - they use G1 as well as S phase markers to show that some mutants are "normal" i.e. they delay entry into S-phase when subjected to certain treatments.

Some mutants however don't sense the damage properly and don't bother arresting.

I don't understand how this could be refined further?

Original comment by: Antonialock

[fypoadmin]

which DNA damage checkpoint, this one? http://www.ebi.ac.uk/QuickGO/GTerm?id=GO:0031571

Original comment by: ValWood

[fypoadmin]

I'm querying this term:

normal DNA replication delay during cellular response to MMS

when the delay occurs. Does replication start? should it be

normal DNA replication initiation delay during cellular response to MMS

which places it at GO:0031571 (the terms don't have to link right now. we can see later if we can infer the specific checkpoint from the specific delay, but only if enough detail about when the delay occurs is captured).

I hope that makes sense?

Original comment by: ValWood

[fypoadmin]

that is what I annotated the gene to, but I cannot exclude that the gene products act at other cell cycle phases - they looked at cells synchronized in G1 only

it is for this session http://curation.pombase.org/pombe/curs/59469ef8c399b1f8

Original comment by: Antonialock

[fypoadmin]

You are right that phenotype cannot pin it down, but this one can?

Mcm6 loading was dramatically delayed in cells treated with H2O2, showing that the cell cycle of wild-type cells is delayed in G1 phase by H2O2.

Mcm loading must be a child of delayed inititation?

VAl

Original comment by: ValWood

[fypoadmin]

So you mean you'd rather have something along the lines of "normal G1 arrest during X"? or "normal delayed G1/S phase treansition during X"?

Original comment by: Antonialock

[fypoadmin]

This has got too confusing!

After looking at the paper, I think the existing phenotypes are OK based on the experiments, but there should be a phenotype annotation for "delayed mcm complex loading" (where mcmloading = localization of MCM complex to DNA replication origins?) This is the experiment which places the arrest in G1, and the block at the G1/S transition. v

Original comment by: ValWood

[fypoadmin]

localization of MCM complex to DNA replication origins is one of the key features of pre-replicative complex assembly (the other is that a couple of additional proteins also bind origins)

Original comment by: mah11

[fypoadmin]

yep...which places it in G1? http://www.ebi.ac.uk/QuickGO/GTerm?id=GO:0071163

so this would all be OK/correct?

You need this phenotype to infer the G1 checkpoint GO term. "normal DNA replication delay during cellular response to MMS" phenotype alone is not enough because a delay could occur in G1, or S

Original comment by: ValWood

[fypoadmin]

so could we just change the relevant terms to "normal G1 DNA damage checkpoint during cellular response to hydrogen peroxide/MMS"?

Original comment by: Antonialock

[fypoadmin]

I don't think so. I think the phenotype should be the delayed loading of MCM complex (which is what they observe). Then the GO annotation you have made are OK (they could be inferred from this phenotype, but not from the other ones on their own). If I understood the bits of the paper I read correctly.

Val

Original comment by: ValWood

[fypoadmin]

There's a catch: they only looked directly at Mcm6 loading (as a proxy for pre-RC assembly) in H2O2. In MMS they couldn't do the same assay (fig2 vs. fig. 3).

Original comment by: mah11

[fypoadmin]

So decreased pre-replicative complex assembly in response to H2O2 ?

This is also important to capture: We conclude that H2O2 induces a Gcn2-dependent delay in progression from G1 to S phase." It implies the G1/S transition delay is activated in response to oxidative stress also.

Original comment by: ValWood

[fypoadmin]

Antonia, if you make sure you capture all the phenotype details. Don't worry about the GO so much for this G1 stuff as we can figure out during review which combination of phenotypes can be used to make which checkpoint annotations.

v

Original comment by: ValWood

[fypoadmin]

no hold on, sorry, I just checked the curation session, I confused myself over which term refers to what:

For the gcn2 deletion, I requested: advanced mitotic G1/S phase transition during cellular response to hydrogen peroxide advanced mitotic G1/S phase transition during cellular response to MMS

to capture the fact that cells progress through G1/S despite the presence of substances that would make WT arrest. .

the terms normal "DNA replication delay during cellular response to hydrogen peroxide/MMS" refers to the fact that the mutant delays DNA replication in response to MMS (fig 4a) and h2o2 (fig2d) just like WT.

I.e. G1 DNA checkpoint = buggered S phase checkpoint= works fine

Original comment by: Antonialock

[fypoadmin]

never mind, deleted previous comment because I had it wrong (looking at wrong figure ...)

Original comment by: mah11

[fypoadmin]

so for gcn5delta, delayed pre-RC assembly in H2O2 fits ... right?

(Antonia's not the only one who's got herself confused ...)

Original comment by: mah11

[fypoadmin]

I think that "advanced mitotic G1/S phase transition during cellular response to hydrogen peroxide" is a "better" term than e.g. "advanced pre-RC loading during cellular response to h2o2"

because looking at pre-RC loading /phosphorylation status of cdc2 / presence of rum1 were only means to the end of finding out if cells traversed into S phase or not.

Original comment by: Antonialock

[fypoadmin]

argh, I've been getting advanced vs. delayed confused too ...

Original comment by: mah11

[fypoadmin]

Nope, it should be "advanced pre-RC assembly in H2O2 " which is why I requested "advanced mitotic G1/S phase transition during cellular response to hydrogen peroxide"

the mutant does however delay DNA replication in S-phase "normally" indicating tht the S-phase checkpoint works ok in this mutant, but the G1 checkpoint doesn't

Original comment by: Antonialock

[fypoadmin]

I'm all over it now :) I think if you want the term normal DNA replication delay during cellular response to hydrogen peroxide changed/more specific, then maybe add S-phase into it?

Original comment by: Antonialock

[fypoadmin]

OK I'm really, really, really confused.

I thought it was "delayed pre-RC loading during cellular response to h2o2"

and this could be linked somehow to mitotic G1/S phase transition during cellular response to hydrogen peroxide

i.e. mitotic G1/S phase transition during cellular response to hydrogen peroxide --delayed pre-RC loading during cellular response to h2o2

but delayed pre-RC loading was what was actually observed

Although now I feel I should read the paper in its entirety. No time today as I haven't finished the ISMB stuff of the talk for tomorrow (And I kind of forgot that I needed today it today because tomorrow I will be at thte meeting all day...arrrggg)

Original comment by: ValWood

[fypoadmin]

Re: I think if you want the term normal DNA replication delay during cellular response to hydrogen peroxide changed/more specific, then maybe add S-phase into it?

Noooo, this must precede S-phase...it's must happen in G1!

Original comment by: ValWood

[fypoadmin]

One more comment:

because looking at pre-RC loading /phosphorylation status of cdc2 / presence of rum1 >were only means to the end of finding out if cells traversed into S phase or not.

This is true, but shouldn't we be capturing the actual phenotype observed with the phenotype annotations, and not the inferences about the processes which these phenotypes indicate? We can capture the process with GO?

so for example phenotype = delayed pre-RC loading or whatever GO process = regulation of G1/S transition or G1 checkpoint or whatever.

If we make the 'inference' at the phenotype annotation stage, we can't fix easily later if a specific phenotype turns out to indicate something else, or more than one thing? or requires 2 observations simultaneously.

Although, these specific phenotypes could probably be has_output children of the process phenotypes?

like: delayed G1/S transition --has_output delayed pre-RC loading

We want to know which specific observation indicated a delayed G1/S transition

Maybe I am overthinking this? Perhaps we don't want to do this at all, but maybe we should discuss in the New Year?

Original comment by: ValWood

[fypoadmin]

Nah, it is not that complicated

-presence of Rum1 is a G1 marker -phosphorylated cdc2 is an S-phase marker

• loading of pre-RC is an "obligatory step in preparing for S-phase and this association stays until end of S" (paraphrased) so is also a G1/S marker

WT cells synchronized in G1 + treated to a bit of DNA damage:

• will have Rum1 hanging around for longer
• will delay pre-RC loading
• phosphorylated cdc2 will appear later

Gcn2 mutants however, when treated to the same DNA damage behave like WT + NO damage DURING G1 -> they just continue into S-phase without trying to respond to the damage.

Now, once in S-phase however they behave more like WT cells + damage in that they will still delay DNA replication - presumably the S-phase DNA damage checkpoint kicks in

Original comment by: Antonialock

[fypoadmin]

As far as I know, DNA replication happens during S-phase (I would feel really really bad if it didn't :) )

-gcn-delta cells do not arrest in G1 in response to DNA damage, they have an "advanced G1/S transition in response to DNA damage" -gcn-delta cells have a "normal DNA replication delay in response to DNA damage"

I think that "GO:0044783 G1 DNA damage checkpoint" is abnormal "GO:0031573 intra-S DNA damage checkpoint" is normal

Original comment by: Antonialock

[fypoadmin]

It's just because they used three diferent markers to achieve the same "aim" (determining an ADVANCED G1/S transition). I thought it would be a lot clearer since this is what they wanted to measure. Otherwise the terms will be "advanced pre-RC loading during hydrogen peroxide" "decreased protein level during celllar response to MMS assayed_using rum1" "increased protein phosphorylation during cellular response to MMS assayed_using(cdc2)

...I don't think these annotations would be as meaningful

Original comment by: Antonialock

[fypoadmin]

but to be fair, it is a nice paper, you should read it :p

Original comment by: Antonialock

[fypoadmin]

Re:

As far as I know, DNA replication happens during S-phase (I would feel really really bad if it didn't :) )

Yes DNA replication occurs during S-phase but the pre replicative complex assembly occurs during G1 phase. I'm not totally convinced that this is showing G1 _DNA damage_\ checkpoint is abnormal. That (is that your interpretation, or is that what the authors say? ) ;)

Isn't this just indicating that the G1/S transition is abnormal? and that there could be another checkpoint which is activated in response to stress?

I.e., some checkpoint is activated at G1, but is it the G1 DNA damage checkpoint or a new one? (we would expect DNA replication not to occur in either situation, because you need to cross the G1/S transition before DNA replication is initiated).

In the abstract the authors say: In fission yeast exposed to ultraviolet light the G1-S transition is regulated by a novel checkpoint that depends on the Gcn2 kinase.

Original comment by: ValWood

[fypoadmin]

I just don't feel that somehow we have got captured the novelty about what they are showing. I need to read the paper though. I might be over complicating it. Its on my list.

Original comment by: ValWood

[fypoadmin]

Well, they are exposing G1 synchronized cells to DNA damage and looking at the cells traversing into S-phase. I suppose it could be abnormal positive regulation of the transition? They seem pretty convinced it is a G1 dna damage checkpoint.

I think their "novel twist" is that this checkpoint is not activated in response to ionizing radition or UVA+PUVA treatment, and that therefore it is different from the G2 DNA damage checkpoint in what exact DNA damage it senses

Original comment by: Antonialock

[fypoadmin]

(this is not really within the scope of what we capture though? it feels like this should be inherent to the term i.e. the G1 DNA damage checkpoint is activated by the damage caused by H2O2, MMS etc but not by the types of damage caused by certain other chemicals)

Original comment by: Antonialock