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PAINT Family PTHR13451-MUS81

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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

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PAINT Family PTHR13451-MUS81

  1. 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. 2. OUTLINE
 PAINT  PTHR13451  -­‐  MUS81   2OUTLINE •  DNA  REPAIR   “chicken  feet”  &  resolvases     •  MUS81   protein  &  endonuclease  complex     •  CHALLENGES   and  thought  process     •  EVIDENCE   summary  
  3. 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. 4 REPLICATION FORK STALLING AND RESTART
 repairing the damage PTHR13451 - MUS81
  5. 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. 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. 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. 8 PTHR13451
 MSA PTHR13451 - MUS81 EME1   MUS81  
  9. 9. 9 PTHR13451
 MSA PTHR13451 - MUS81 MUS81   ERCC4  –  pfam02732  
  10. 10. 10 PTHR13451
 MSA PTHR13451 - MUS81 EME1   ERCC4  –  pfam02732   EME1  
  11. 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. 12 MUS81 & EME1
 conclusion PTHR13451 - MUS81 ACTION:  “EME1”  porIon  of  the  tree  must  go  to  PTHR21077
  13. 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. 14 CELLULAR COMPONENT
 challenge #2 PTHR13451 - MUS81
  15. 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. 16 CONDENSED NUCLEAR CHROMOSOME
 challenge #3 PTHR13451 - MUS81
  17. 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. 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. 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. 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. 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. 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. 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. 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. 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!  

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