PAINT conference call GO:0000725 recombinational repair PTHR16771 and PTHR23074 Monica Munoz-Torres, PhD | @monimunozto Berkeley Bioinformatics Open-Source Projects (BBOP)

Genomics Division, Lawrence Berkeley National Laboratory 24 June, 2014

UNIVERSITY OF CALIFORNIA

Outline 1.  Thought process 2.  GO Term: recombinational repair 3.  PTHR16771

• Proteasome Complex Subunit DSS.

4.  PTHR23074 • AAA ATPase.

PAINT Conf Call GO:0000725 recombinational repair

Outline 2

Thought Process

•  Find gene with annotation to GO term of interest

•  Inspect details at UniProt (FASTA, GO terms, etc.)

•  Link to PubMed, extract information

•  Inspect CDD (e.g. BLAST vs nr)

•  Inspect all annotations associated with gene in question reviewing both tree and MSA

•  Propagate as appropriate

Thought Process 3

GO Term

Accession: GO:0000725

Name: recombinational repair

Ontology: biological_process

Definition: A DNA repair process that involves the exchange, reciprocal or nonreciprocal, of genetic material between the broken DNA molecule and a homologous region of DNA. Source: GOC:elh

GO Term 4

PTHR16771

PTHR16771: Proteasome Complex Subunit DSS1

•  BRCA2 is a breast cancer susceptibility gene implicated in the repair of double-strand breaks by homologous recombination with RAD51.

•  DSS1 interacts with BRCA2 in a domain-specific way

•  DSS1 is required for the BRCA2-RAD51 complex to become associated with sites of DNA damage and for the maintenance of genomic stability, a function conserved from lower eukaryotes to humans.

•  DSS1 is involved in DNA damage repair via homologous recombination.

PTHR16771 6

DSS1_SEM1 [cd13768, pfam05160] proteasome complex subunit DSS1/SEM1 (s:CDD)

•  The evolutionarily conserved deleted in split hand/split foot protein 1 (DSS1)/Sem1 is a subunit of the regulatory particle (RP) of the proteasome.

PTHR16771 7

Pruned

•  Inspecting MSA and comparing with the Conserved domains of DSSm1/SEM1 family, I pruned the following species:

20140505: Pruned TETTS_TTHERM_00227230

20140505: Pruned ORYSJ_P0693B08.32

20140505: Pruned PUCGT_PGTG_08936

PTHR16771 8

Reasons for pruning

•  ORYSJ_P0693B08.32 and PUCGT_PGTG_08936 have long (20 - 40 aa) strings of residues interrupting the conserved domain of DSS1 (increasing the length of the peptide), which has been very well characterized to 71aa in length.

•  TETTS_TTHERM_00227230 has an extended string of ~40 aa at the beginning of the protein, and the second portion of its conserved domain is severely corrupted (degenerate) in comparison to all other proteins in the family

PTHR16771 9

Reasons for pruning

PTHR16771 10

Decision # cellular_component 20140513: Has function proteasome complex (GO:0000502) in

Eukaryota_PTN000423400 20140513: Colocalizes with nucleus (GO:0005634) in

Eukaryota_PTN000423400 # biological_process 20140513: Has function proteolysis (GO:0006508) in

Eukaryota_PTN000423400 20140509: Has function double-strand break repair via homologous

recombination (GO:0000724) in Eukaryota_PTN000423400 # Pruned from tree 20140513: Pruned ORYSJ_P0693B08.32 20140513: Pruned TETTS_TTHERM_00227230 20140513: Pruned PUCGT_PGTG_08936

PTHR16771 11

Questions

•  Should I annotate other terms? or only concentrate on the one I am reviewing?

•  I find myself hesitating about propagating due to degeneracy of conserved domains in MSA (thus, I pruned three species).

How much weight should MSA have on decisions? Does everyone else rely heavily on them?

PTHR16771 12

MF: Peptidase activity?

•  Although Human_SHFM1 has an annotation to peptidase activity GO:0008233, the paper reporting the IDA does not make any claims of peptidase activity, or any of its synonyms (i.e. hydrolase, acting on peptide bonds; peptide hydrolase activity; protease activity; proteinase activity).

•  “Yeast and mammalian two-hybrid assays showed that DSS1 can associate with BRCA2 in the region of amino acids 2472 to 2957 in the C terminus of the protein. Using coimmunoprecipitation of epitope-tagged BRCA2 and DSS1 cDNA constructs transiently expressed in COS cells, authors demonstrated an association. Furthermore, endogenous BRCA2 could be coimmunoprecipitated with endogenous DSS1 in MCF7 cells, demonstrating an in vivo association.”

•  No further details on the nature of the bond are discussed. Should this term be removed? or changed to 'catalytic activity' instead?

PTHR16771 13

Questions

From the "Evidence" tab:

•  What does "has function nucleus" mean? and "has function proteasome complex"? and how can I avoid that syntax? am I supposed to avoid the syntax? “has function” vs “contributes to”?

•  Warnings: (see next)

PTHR16771 14

Warnings that disappear… #WARNINGS

WARNING: Experimental data changed!

The following annotations are no longer supported by experimental evidence

and have been removed:

Opisthokonta_PTN000423402 to double-strand break repair via homologous recombination

Bilateria_PTN000423403 to double-strand break repair via homologous recombination

PTHR16771 15

•  I got a warning about experimental evidence being removed, but the experimental evidence is still being displayed. My initial thought was that the culprit was my "redundant" propagation of a process annotation to a less derived node, then a more ancestral one. Is this the cause? ---> Update: the errors disappeared. ???

Previously: Decision

#cellular component

20140513: Has function proteasome complex (GO:0000502) in Eukaryota_PTN000423400

20140513: Colocalizes with nucleus (GO:0005634) in Bilateria_PTN000423403

20140513: Colocalizes with nucleus (GO:0005634) in Eukaryota_PTN000423400

20140507: Has function nucleus (GO:0005634) in Bilateria_PTN000423403

#biological process

20140513: Has function proteolysis (GO:0006508) in Eukaryota_PTN000423400

20140509: Has function proteolysis (GO:0006508) in Opisthokonta_PTN000423402

20140509: Has function double-strand break repair via homologous recombination (GO:0000724) in Eukaryota_PTN000423400

#Pruned

20140513: Pruned ORYSJ_P0693B08.32

20140513: Pruned TETTS_TTHERM_00227230

20140513: Pruned PUCGT_PGTG_08936

PTHR16771 16

PTHR23074

PTHR23074: AAA ATPase •  AAA+ Superfamily: “ATPases Associated with a wide variety of

cellular Activities”

•  An ancient group of ATPases belonging to the ASCE (for additional strand, catalytic E) division of the P-loop NTPase fold.

•  Members function as molecular chaperons, ATPase subunits of proteases, helicases, or nucleic-acid stimulated ATPases.

•  AAA+ proteins contain several distinct features in addition to the conserved alpha-beta-alpha core domain structure and the Walker A and B motifs of the P-loop NTPases.

PTHR23074 18

FIGNL1: RAD51-binding protein fidgetin-like 1

•  RAD51 recombinase plays central role in homologous recombination (HR), which is critical for repair of DNA double-strand breaks.

•  FIGNL1 specifically interacts with RAD51 through its conserved RAD51 binding domain.

•  Forms part of protein complex FIGNL1 + KIAA0146/SPIDR, required for DNA repair

PTHR23074 19

PTHR23074: FIGNL1

PTHR23074 20

Inspecting Conserved Domains

PTHR23074 21

FIGNL1

KATNAL2

VPS4A

PTHR23074: KATNAL2

PTHR23074 22

Observations:

•  Paraphyletic

•  Plants + animals

•  No Experimental Annotations

•  AAA ATPase conserved domain

Decision

FIGNL1's RAD51 binding domain (FRBD) is required for "regulation of double-strand break repair via homologous recombination" (GO:0010569) activity.

FIGNL1 and its homologs contain also an AAA superfamily domain, and vsp4 domain. The required binding domain is only present in this family. Members from other clades were reviewed. Neither family had the required FRBD and one did not have a vsp4 domain either.

Decision to propagate GO:0010569 only to Eukaryota_PTN000553645 as the Most Recent Common Ancestor for this biological process for the FIGNL1 gene.

PTHR23074 23

Propagated term

Footer 24

Propagated term

PTHR23074 25

Evidence (text) # molecular_function

20140603: Contributes to ATPase activity (GO:0016887) in root_PTN000553509

# cellular_component

20140603: Has function nucleus (GO:0005634) in root_PTN000553509

# biological_process

20140603: Has function regulation of double-strand break repair via homologous recombination (GO:0010569) in Eukaryota_PTN000553645

# Notes

PTHR23074 26

Software Reports •  Space to enter PANTHER protein

family ID not visible

•  Arrow heads do not act as expected: i.e. do not move spaces along MSA

Software Reports 27

Software Reports

•  Large family eats up CPUs; PAINT freezes (tmp)

•  Is right-click on experimental annotation supposed to highlight the MRCA node for them? If so, not evident.

•  Permanent Tree ID (PANTREE) links broken / blank pages

•  Resizing a window takes several seconds

•  Challenging to propagate to “off-screen” node

Software Reports 28

UNIVERSITY OF CALIFORNIA