Slides for PAINT Group Conference Call on 062414

1. 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

2. 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

3. 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

4. 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

5. PTHR16771

6. 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

7. 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

8. 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

9. 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

10. Reasons for pruning
PTHR16771 10

11. 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

12. 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

13. 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

14. 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

15. 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. ???

16. 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

17. PTHR23074

18. 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

19. 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

20. PTHR23074: FIGNL1
PTHR23074 20

21. Inspecting Conserved Domains
PTHR23074 21
FIGNL1
KATNAL2
VPS4A

22. PTHR23074: KATNAL2
PTHR23074 22
Observations:
• Paraphyletic
• Plants + animals
• No Experimental
Annotations
• AAA ATPase
conserved
domain

23. 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

24. Propagated term
Footer 24

25. Propagated term
PTHR23074 25

26. 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

27. 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

28. 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

29. UNIVERSITY OF
CALIFORNIA