Continuing with the theme of DNA repair via homologous recombination, I will discuss the following family during the PAINT call: PTHR13451 CLASS II CROSSOVER JUNCTION ENDONUCLEASE MUS81

1. PTHR13451
Class II crossover junction
endonuclease MUS81
Monica Munoz-Torres, PhD | @monimunozto
Berkeley Bioinformatics Open-Source Projects (BBOP)
Genomics Division, Lawrence Berkeley National Laboratory
Joint Genome Institute | University of California Berkeley | U.S. Department of Energy
PAINT Conference Call. 07 July, 2015

2. OUTLINE
PAINT
PTHR13451
-­‐
MUS81
2OUTLINE
• DNA
REPAIR
“chicken
feet”
&
resolvases
• MUS81
protein
&
endonuclease
complex
• CHALLENGES
and
thought
process
• EVIDENCE
summary

3. 3
COPING WITH BREAKUP
maintaining a stable structure
v During
DNA
replicaIon
the
cellular
DNA
is
especially
vulnerable
to
damage,
and
several
repair
pathways
or
read-­‐through
mechanisms
are
acIve.
v One
way
to
deal
with
DNA
damage
such
as
blocked
replicaIon
forks
is
regression
and
annealing
of
the
nascent
strands
to
form
a
Holliday
juncIon
(HJ)
structure.
PTHR13451 - MUS81

4. 4
REPLICATION FORK STALLING AND RESTART
repairing the damage
PTHR13451 - MUS81

5. 5
RESOLVASES
endonucleases for DNA repair
v The
Holliday
juncIon
(HJ)
is
a
central
intermediate
of
homologous
recombinaIon.
Its
cleavage
is
criIcal
for
the
formaIon
of
crossover
recombinants
during
meiosis,
which
in
turn
helps
to
establish
chiasmata
and
promote
geneIc
diversity.
v For
a
long
Ime
these
enzymes
that
cleave
HJs,
called
HJ
resolvases,
had
been
idenIfied
in
all
domains
of
life
except
eukaryoIc
nuclei.
*
PTHR13451 - MUS81

6. 6
MUS81
Methyl methansulfonate UV sensitive; clone 81
v MUS81
endonuclease
complex
has
been
shown
to
play
an
important
role
in
the
repair
of
stalled
or
blocked
replicaIon
forks
and
in
the
processing
of
meioIc
recombinaIon
intermediates
from
yeast
to
humans.
v This
endonuclease
complex
is
composed
of
two
subunits,
MUS81
and
EME1.
v The
MUS81
protein
is
conserved
throughout
all
analyzed
eukaryotes
but
not
eubacteria.
PTHR13451 - MUS81

7. 7
MUS81 & EME1 ARE NOT THE SAME GENE
challenge # 1
v PTHR13451
includes
both
subunits
of
the
endonuclease
complex
(MUS81
&
EME1)
required
for
the
repair
of
DSB
via
recombinaIon.
v There
is
not
significant
sequence
similarity
between
the
two,
when
aligning
EME1B
and
MUS81
from
Arabidopsis.
v “Significant
similarity”
between
the
two:
Range 1: 456 to 500GraphicsNext MatchPrevious Match
Alignment statistics for match #1
Score Expect Method Identities Positives Gaps
29.3 bits(64) 4e-04 Compositional matrix adjust. 14/45(31%) 24/45(53%)
0/45(0%)
Query 594 LMQVPQVTEEIAIAVLDMYPTLLSLASAYSHLEADVSAQEEMLRN 638
L+ +P+V A+AV YP++ SL Y V +E +L++
Sbjct 456 LVAIPKVQPRYALAVWKKYPSMKSLLKVYMDRNKSVHEKEFLLKD 500
PTHR13451 - MUS81

8. 8
PTHR13451
MSA
PTHR13451 - MUS81
EME1
MUS81

9. 9
PTHR13451
MSA
PTHR13451 - MUS81
MUS81
ERCC4
–
pfam02732

10. 10
PTHR13451
MSA
PTHR13451 - MUS81
EME1
ERCC4
–
pfam02732
EME1

11. 11
MUS81 & EME1
Why were these two genes brought together?
v Because
they
share
a
domain
architecture
common
to
crossover
juncIon
endonucleases
endonucleases,
in
this
case
ERCC4.
Since
both
EME1
and
MUS81
proteins
contain
ERCC4
domain,
PANTHER
grouped
them.
v ACTION:
“EME1”
porIon
of
the
tree
must
go
to
PTHR21077
v ERCC4
is
a
structural
domain
found
in
several
DNA
repair
nucleases
(e.g.
Rad1,
XPF)
and
crossover
juncIon
endonucleases
(e.g.
EME1
and
Mus81).
v references
for
ERCC4:
hbp://pfam.xfam.org/family/PF02732
hbp://smart.embl-­‐heidelberg.de/smart/do_annotaIon.pl?BLAST=DUMMY&DOMAIN=00891
PTHR13451 - MUS81

12. 12
MUS81 & EME1
conclusion
PTHR13451 - MUS81
ACTION:
“EME1”
porIon
of
the
tree
must
go
to
PTHR21077

13. 13
CELLULAR COMPONENT
challenge #2
v Mus81
Is
a
nuclear
protein
whose
abundance
increases
ajer
DNA
damage
or
inhibiIon
of
replicaIon,
and
there
are
a
number
of
references
in
support
of
this.
For
example,
literature
in
support
of
human
Mus81
at
hbp://www.uniprot.org/
uniprot/Q96NY9
v The
reference
available
in
PAINT
is
GO_REF:0000052
(Gene
Ontology
annotaIon
based
on
curaIon
of
immunofluorescence
data).
Although
“annotaIons
should
only
be
exported
to
the
GO
ConsorIum
if
the
localizaIons
are
supported
by
literature”,
there
is
no
addiIonal
informaIon
about
the
publicaIons
from
the
entries
in
PAINT.
v QuesEon:
how
do
we
interpret
this?
-­‐
perhaps
this
is
a
well
known
issue,
I
had
not
come
across
this
in
a
while
and
can't
remember
the
decision.
PTHR13451 - MUS81

14. 14
CELLULAR COMPONENT
challenge #2
PTHR13451 - MUS81

15. 15
CONDENSED NUCLEAR CHROMOSOME
challenge #3
v Propagated
annotaIon
to
nucleolus:
evidence
for
it
in
literature
for
thale
cress,
human,
and
yeast.
v What
to
do
with
"condensed
nuclear
chromosome",
available
only
from
Arabidopsis
paper?
PTHR13451 - MUS81

16. 16
CONDENSED NUCLEAR CHROMOSOME
challenge #3
PTHR13451 - MUS81

17. 17
CONDENSED NUCLEAR CHROMOSOME
challenge #3
Conclusion:
In
my
opinion:
propagate.
This
localizaIon
is
likely
to
be
spread
throughout
eukaryotes.
-­‐
Pascale
does
not
annotate
recombinaIon
proteins
to
anything
more
specific
than
“nucleus”.
-­‐
Paul:
how
important
is
this
annotaIon
to
understanding
the
biology?
PTHR13451 - MUS81

18. 18
CHIASMA ASSEMBLY
challenge #4
v Loss
of
MUS81:
sensiIvity
to
DNA
damaging
agents
in
yeast
and
mammals.
BUT,
its
role
in
meiosis
differs
drasIcally
between
eukaryotes.
v Sc.
pombe
MUS81
LOF
à
complete
sterility;
the
mutant
is
parIally
ferIle
in
Sa.
cerevisiae
and
fully
ferIle
in
mammals.
MUS81
mutants
in
Arabidopsis
showed
normal
ferIlity;
the
protein
is
not
essenIal
for
meiosis.
PTHR13451 - MUS81

19. 19
CHIASMA ASSEMBLY
challenge #4
v “Nevertheless,
observaIon
that
AtMUS81
inserIon
lines
have
no
detectable
meioIc
impairment
does
not
exclude
a
role
of
the
protein
in
a
minor
pathway
of
meioIc
recombinaIon”.
v Authors
concluded:
the
data
per
se
do
not
exclude
that
a
residual
chiasmata
frequency
in
Arabidopsis
depends
on
MUS81
acIon.
NOT
that
they
have
evidence
in
support
of
its
occurrence.
PTHR13451 - MUS81

20. 20
CHIASMA ASSEMBLY
challenge #4
v
Arabidopsis
thaliana:
annotaIon
to
"chiasma
assembly"
GO:0051026
(and
therefore
to
"reciprocal
meioIc
recombinaIon")
has
been
made.
I
believe
this
is
incorrect.
v My
conclusion:
Dispute!
PTHR13451 - MUS81

21. 21
SIGNALING or RESPONSE TO SIGNALING?
challenge #5
v The
following
annotaIons
are
present
in
the
family:
v "intra-­‐S
damage
checkpoint"
(GO:0031573;
annotated
in
fission
yeast)
v "response
to
intra-­‐S
damage
checkpoint
signaling"
(GO:0072429;
annotated
in
human):
v "intra-­‐S
damage
checkpoint"
GO:0031573:
is
a
mitoIc
cell
cycle
checkpoint
that
slows
DNA
synthesis
in
response
to
DNA
damage
by
the
prevenIon
of
new
origin
firing
and
the
stabilizaIon
of
slow
replicaIon
fork
progression.
PTHR13451 - MUS81

22. 22
"intra-S damage checkpoint signaling”
challenge #5
PTHR13451 - MUS81
v When
replicaIon
stress
is
encountered,
as
during
HU
exposure,
signals
are
transmibed
through
a
kinase
cascade.
First,
signals
are
transmibed
through
the
apical
kinase.
These
kinases
form
a
complex
with
adaptor
proteins
and
transmit
signals
through
transducers.
The
ulImate
target
is
the
effector
kinase.
The
process
is
well
known
in
budding
yeast,
fission
yeast,
and
human.

23. 23
SIGNALING or RESPONSE TO SIGNALING?
challenge #5
v "response
to
intra-­‐S
damage
checkpoint
signaling"
GO:0072429:
A
process
that
occurs
in
response
to
signals
generated
as
a
result
of
intra-­‐S
DNA
damage
checkpoint
signaling.
Source:
GOC:mtg_cell_cycle
v Conclusion:
given
the
literature,
propagaIng
the
annotaIon
regarding
the
“response”
to
the
signaling
cascade
(evidence
in
literature
for
human)
is
more
appropriate?
v QuesEon:
what
to
do
about
the
annotaEon?
See
note
below-­‐
v Btw,
this
is
what
I
feel
like
when
I
*think*
I'm
prepared
to
dispute
an
annotaIon.
hbps://youtu.be/xWpA-­‐2-­‐KdDo
à
IMP:
challenges
–
Karen
suggests
to
“move
on”.
It
may
not
look
like
a
“wrong”
annotaIon,
but
we
don’t
need
to
get
tangled
up.
If
terms
with
sufficient
support
explain
the
biology
appropriately,
then
use
those
to
beber
represent
the
biology,
don’t
worry
about
the
others.
PaulT:
“less
is
more”.
PTHR13451 - MUS81

24. EVIDENCE
summary
24PTHR13451 - MUS81
Molecular
funcIon:
v 20150706:
Eukaryota_PTN000335543
has
funcIon
crossover
juncIon
endodeoxyribonuclease
acIvity
(GO:0008821)
v 20150706:
Eukaryota_PTN000335543
has
funcIon
3'-­‐flap
endonuclease
acIvity
(GO:0048257)
Cellular
Component:
v 20150706:
Eukaryota_PTN000335543
located
in
nucleolus
(GO:0005730)
–
check
literature.
Unless
it
is
specifically
localized
in
nucleolus,
this
annotaIon
should
not
be
included.
Because
there
is
a
‘parent-­‐child’,
we’d
be
missing
the
‘nucleus’
annotaIon.
–
if
annotated
to
nucleolus,
it
implies
that
it
is
only
found
on
that
part
of
the
nucleus.
v 20150706:
Eukaryota_PTN000335543
located
in
nucleus
(GO:0005634)
v 20150706:
Eukaryota_PTN000335543
located
in
Holliday
juncIon
resolvase
complex
(GO:0048476)
Biological
process:
v 20150707:
Eukaryota_PTN000335543
parIcipates
in
response
to
intra-­‐S
DNA
damage
checkpoint
signaling
(GO:0072429)
v 20150707:
Eukaryota_PTN000335543
parIcipates
in
mitoIc
recombinaIon
(GO:0006312)
v 20150707:
Eukaryota_PTN000335543
parIcipates
in
DNA
catabolic
process,
endonucleolyIc
(GO:0000737):
this
may
already
be
covered
on
“endonuclease
acIvity”
in
MF.
Is
DNA
repair
really
a
“catabolic
process”?!
v 20150707:
Fungi_PTN001003088
parIcipates
in
resoluIon
of
meioIc
recombinaIon
intermediates
(GO:0000712)
v 20150707:
Fungi_PTN001003088
parIcipates
in
reciprocal
meioIc
recombinaIon
(GO:0007131)
Paul:
find
out
is
it
really
not
parIcipaIng
in
meitoic
recombinaIon
(in
DNA
repair)
in
the
ancestor
of
euks?
v 20150707:
Eukaryota_PTN000335543
parIcipates
in
replicaIon
fork
processing
(GO:0031297)
v 20150707:
Eukaryota_PTN000335543
parIcipates
in
postreplicaIon
repair
(GO:0006301)
v 20150706:
Eukaryota_PTN000335543
parIcipates
in
double-­‐strand
break
repair
via
break-­‐induced
replicaIon
(GO:
0000727)
v 20150706:
Eukaryota_PTN000335543
parIcipates
in
DNA
repair
(GO:0006281):
too
wide
–
this
is
already
captured
under
the
specifics
of
the
pathways.
Pruned
v 20150706:
Pruned
Eukaryota_PTN000981193

25. • Berkeley
BioinformaEcs
Open-­‐source
Projects
(BBOP),
Berkeley
Lab.
Gene
Ontology
team.
Suzanna
E.
Lewis
(PI).
• For
your
aXenEon,
thank
you!
Thank you. 25
PAINT
Suzanna
Lewis
Gene
Ontology
Chris
Mungall
Seth
Carbon
Heiko
Dietze
BBOP
GO:
hbp://GeneOntology.org
Thanks!