Gen (CG10670, Q9VRJ0) — manual literature review

No deep-research provider was available for this session; this summary was compiled by hand
from the cached publications in publications/ and PubMed searches
("Drosophila Gen endonuclease GEN1 mus324"; "Drosophila GEN Holliday junction resolvase").

Identity and family

• Gen (DmGEN, "XPG-like endonuclease"; FBgn0263831; ORF CG10670) encodes a 726-aa member of the
  XPG/RAD2 structure-specific nuclease family, GEN subfamily (class IV), the ortholog of human GEN1 and
  budding-yeast Yen1. It has XPG-N and XPG-I nuclease domains positioned close together, as in FEN1/EXO1,
  followed by an H3TH (HhH2) DNA-binding region and a chromodomain-like region (UniProt Q9VRJ0; InterPro
  IPR041012 GEN_chromo).
  PMID:15576351
• The original genetic name for the locus was mus324; the two mus324 alleles carry the same frameshift
  in Gen at codons 374–375.
  PMID:22022278

Biochemistry

• Ishikawa et al. 2004 first purified DmGEN and reported endonuclease and both 3'-5' and 5'-3' exonuclease
  activities PMID:15576351.
  UniProt considers the 3'-5' exonuclease probably artifactual (CAUTION in Q9VRJ0: contaminating nucleases).
• Kanai et al. 2007, using an E143A/E145A active-site mutant as a control, established 5'-flap endonuclease
  activity with a preference for blocked flaps, cleavage of the lagging strand of a model replication fork,
  weak nick-dependent 5'-3' exonuclease, and no bubble incision
  PMID:17614965.
  They reported (abstract-only in cache; via UniProt FUNCTION) no Holliday junction cleavage.
• Bellendir et al. 2017 (full text) showed with recombinant full-length and truncated DmGen that it cleaves
  5' flaps, replication-fork structures, splayed arms and fixed, mobile and nicked Holliday junctions, but not
  3' flaps, unbranched or nicked duplex PMID:28369583.
  HJ cleavage is symmetric and yields ligatable nicked duplexes; DmGen binds HJs with sub-nanomolar affinity
  (EMSA, K1/2 ~0.2 nM) and forms dimers on HJs; flap cleavage is much faster than HJ cleavage because
  productive dimer assembly on the HJ is rate-limiting. This supersedes the 2007 report of no HJ cleavage.

Localization

• Kanai 2007: anti-DmGEN immunostaining placed the protein in nuclei of proliferating early embryos
  PMID:17614965.
• Bellendir 2017: a different antibody and His-tagged overexpression in S2 cells showed predominantly or
  exclusively cytoplasmic localization in interphase, like human GEN1 (cytoplasmic until nuclear envelope
  breakdown) and yeast Yen1 PMID:28369583.
  The two reports conflict; the more recent, better-controlled work favours interphase cytoplasmic
  sequestration with presumed access to DNA in mitosis.

Genetics / in vivo roles

• Gen mutants are hypersensitive to MMS and nitrogen mustard (HN2) — more so than mus81 mutants
  PMID:22022278 — and to camptothecin, hydroxyurea and ionizing radiation PMID:28369583.
• mus81 Gen double mutants show elevated apoptosis in imaginal discs and stronger hypersensitivity than
  either single mutant: MUS81 is a backup to GEN in flies (reverse of yeast/human).
• Gen Blm (mus309) double mutants die as first-instar larvae; lethality is partially suppressed by loss of
  Rad51 (spn-A), implicating recombination intermediates PMID:22022278.
• Gen is also essential (together with MUS81 or SLX1) in Fancm mutants PMID:25205745.
• In an I-SceI DSB repair screen (before mus324 was known to be Gen), mus324 modestly reduced
  single-strand annealing under a mild heat-shock regime only PMID:17660539.

Synthesis

Gen encodes the fly GEN1/Yen1 structure-selective endonuclease, cleaving 5' flaps, replication-fork-like
structures and Holliday junctions (symmetrically). It is the predominant structure-selective nuclease for
damage tolerance in Drosophila, acting in DNA repair (replication-associated damage, crosslinks, DSBs) with
MUS81-MMS4 as backup; it becomes essential when BLM or FANCM is absent. Whether its key in vivo substrates
are HJs or simpler branched intermediates remains unresolved.