ssb1 query

[ValWood]

ssb1 has DNA unwinding involved in DNA replication

is that the correct term (wouldn't that imply it was a helicase?)

It seems that it is more "While Ssb1 is known to stabilize DNA strand separation, the Cut14 hinge promotes DNA annealing. PMID:22645654" i.e opposing annealing?

This is a CC annotation... @mah11 what do you think?

[mah11]

RPA definitely has a connection to the process of unwinding, in that once a helicase unwinds some duplex DNA, RPA binding to the single-stranded regions keeps it unwound. I suppose one could argue that that's regulating unwinding rather than direct participation. I don't have strong feelings either way ...

(how is this a cellular component annotation?)

[ValWood]

Community Curation ...I need a new abbreviation don't I?

I see unwinding is the opposite of annealing isn't it, because 'unwinding' is only defined as strand separation.... its probably OK, but it is regulating rather than doing any unwinding. I think of it more as negatively regulating reannealing...

[ValWood]

I'm not sure what happens here, but there is another term

GO:0036310 annealing helicase activity Catalysis of the ATP-dependent rewinding of single-stranded DNA (ssDNA) to reform base pairs between strands. Often acts on ssDNA bubbles bound by replication protein A (RPA).

which has 11 proteins annotated by IDA....

[ValWood]

Woah I am so confused by this. What is annealing helicase activity It has 14 experimental annotations, one is a SWI/SNF subunit which seemed odd so I followed this up to here http://www.uniprot.org/citations/22705370

Proteins with annealing activity are newly identified ATP-dependent motors that can rewind RPA-coated complementary single-stranded DNA bubbles. AH2 (annealing helicase 2, also named as ZRANB3) is the second protein with annealing activity, the function of which is still unknown. Here, we report that AH2 is recruited to stalled replication forks and that cells depleted of AH2 are hypersensitive to replication stresses. Furthermore, AH2 binds to PCNA, which is crucial for its function at stalled replication forks. Interestingly, we identified a HARP-like (HPL) domain in AH2 that is indispensible for its annealing activity in vitro and its function in vivo. Moreover, searching of HPL domain in SNF2 family of proteins led to the identification of SMARCA1 and RAD54L, both of which possess annealing activity. Thus, this study not only demonstrates the in vivo functions of AH2, but also reveals a common feature of this new subfamily of proteins with annealing activity.

but why would these need to possess "ATPase rewinding activity". Surely you don't need any energy to rewind a complementary bubble once you have removed the single stranded binding proteins preventing the reannealing?

[ValWood]

OK this is a rewinding helicase http://www.uniprot.org/uniprot/Q9NZC9#family_and_domains

[mah11]

Community Curation ...I need a new abbreviation don't I?

heh, yeah, cellular component got there first

'unwinding' is only defined as strand separation

Yep, it's not a very process-y definition. I kind of think it only starts feeling process-ish when I also take regulation (context 'n whatnot) into account ...

I think of it more as negatively regulating reannealing

That sounds reasonable.

I'll have a look at the annealing helicase business tomorrow (FYPO tickets beckon ...)

[ValWood]

I just got a response from Norihiko,

Thank you for your mail. We think the former understanding is true, condensin causes renaturation of ssDNA to dsDNA resulting in RPA dissociation from ssDNA.

Many thanks, Norihiko

so in this session curs/aedeeafb353d00c4/ro/

i will move his suggestions to refine his annotations to GO:0000733 DNA strand renaturation

with 2 child terms Removal of RPA proteins associated with ssDNA and Removal of RNA associated with ssDNA to comments...can always revisit if things change.....

Does that make sense? No hurry, I can stick this one through now....

[ValWood]

Norihiko said in his feedback that he couldn't capture the AFM data. This refers to the above so I suspect he was referring to the term specificity. This is the experiment.

To visualize the removal of RPA from hdDNA, atomic force microscopy (AFM) was used [4,21](figure 6d–f). Highly puri- fied bacterial single-strand DNA binding protein (SSB; purchased from Promega)-coated hdDNA was incubated with the S. pombe SMC dimer. Within 10 min, a sharp dsDNA band was formed (figure 6d). AFM images of hdDNA before and after the addition of SSB, followed by the addition of the SMC dimer, are shown in figure 6e. Beaded ssDNA coated with SSB was observed for hdDNA mixed with SSB (middle), while dsDNA was plentifully produced 30 min after the incu- bation with SMC dimer (right; control hdDNA and dsDNA images, left top and bottom, respectively). Purified S. pombe RPA was also examined by AFM (figure 6f, left). Coated ssDNA similar to that produced by bacterial SSB was observed. Thirty minutes after the incu- bation with SMC dimer, dsDNA was plentifully observed (figure 6f, right). Taken together, our data show that conden- sin SMC promotes DNA reannealing and releases bacterial SSB or S. pombe RPA bound to complementary ssDNA.

It seems a shame that there isn't a way to capture that RPA is 'displaced' during the annealing, but I ca't think of a way to do it elegantly.....

[mah11]

I haven't got very far with annealing helicases other than becoming convinced that, yep, there is such a beast, and some are ATP-dependent.

As for RPA displacement, does anything (e.g. SMC) do it (catalyze/speed up removal)? If so, GO could add a term analogous to 'histone displacement':

[Term]
id: GO:0001207
name: histone displacement
namespace: biological_process
def: "The removal of histones, including histone dimers, from nucleosomes
within chromatin." [GOC:krc, PMID:15525516, PMID:17496903, PMID:21807128]
is_a: GO:0034728 ! nucleosome organization

I don't know whether specific ('RPA displacement') or generic ('protein displacement from single-stranded DNA'? or some such?) or both would be best. Also not sure what the is_a parent might be ...

[ValWood]

I'm confused by this...

negative regulation of annealing? does that make sense? rather than positive regulation of rewinding? Should I just close this one ;)

[mah11]

I'm confused by this...

me too! because ...

negative regulation of annealing? rather than positive regulation of rewinding?

I'm really not sure how I would tell these two things apart. Not at all sure I can help any more (if I have at all). :P

[ValWood]

I think they are slightly different (sometimes).

David and I had a similar issue this week with the regulation cohesin loading and unloading. The inverse +/-vs sound like they are the same thing, but they weren't. We had examples where the loading was clearly being negatively regulated, and examples where the unloading was being positively regulated.

Actually here, based on Norihiko's comment We think the former understanding is true, condensin causes renaturation of ssDNA to dsDNA resulting in RPA dissociation from ssDNA.

I'd say the ssb was 'negatively regulating (actually inhibiting?) annealing', and the thing removing the ssb was positively regulating rewinding/annealing?

[mah11]

I don't know anything to contradict that. That's as far as I can go ...

[ValWood]

I'm closing this...