Analysis of DNA hurt reaction in lem-3（op444） mutantsBased on our information earlier mentioned, we upcoming requested which fix pathwayLEM-3 could take part in. We initially investigated double strandbreak repair, as this method occurs extensively in the germ line as part of meiotic recombination. Failure to resolve the double strand breaksinduced by the SPO-eleven endonuclease final results in improved germ cellapoptosis because of to activation of CEP-one/p53 and enhanced meiotic nondisjunction.
embryoniclethality and a high incidence of males. Nonetheless, none of thesephenotypes is clear in lem-3（op444） mutants in the absence ofexogenous DNA damage （Figure 1A, 4D, and data not shown）。 Therefore,we conclude that lem-3 mutants are proficient in the two meiosis and theresolution of dsDNA breaks induced during meiotic recombination.
Rad54 has been implicated in several actions in the course of homologousrecombination and restore [28]. Down-regulation of rad-fifty four byRNAi results in improved apoptosis and radiation-inducedembryonic lethality [29]. We have by now proven that RAD-54forms foci following IR, probable marking web sites of lively mend [thirty].
To get even further insights into the capacity of lem-3（op444） mutants torepair exogenously produced dsDNA breaks, we analyzed thesubcellular localisation of YFP::RAD-fifty four. In the absence ofexogenous damage, YFP::RAD-54（opIs257） localized to distinctfoci in the meiotic zone of the germ line, but showed a diffusenuclear staining pattern in the mitotic zone （Figure 5A）。 This is inline with the acknowledged position of RAD-fifty four for the duration of homologousrecombination. Upon irradiation, foci also appeared in the mitotic zone （Figure 5B）。 Quantitative examination exposed that the numberof RAD-fifty four foci for each nucleus ended up similar in wild variety and lem-3（op444） （Figure 5D）。 Soon after seventeen hours, most cells in the mitotic zonehad efficiently eradicated RAD-fifty four in each wild form and lem-3（op444）。 Only a couple of, enlarged nuclei nonetheless confirmed RAD-54 foci（Figure 5C）。 These very likely symbolize cells with persistent DNAdamage that unsuccessful to re-enter the cell cycle. We conclude fromthese info that the mend of exogenously created dsDNA breaksis normal lem-3（op444）。lem-three and xpa-1 act in different pathwaysSince lem-3 mutants are also hypersensitive to UV-C light, weinvestigated a possible involvement of lem-3 in the NER pathway.
We created and analyzed a double mutant with xpa-1（ok698）， a component typical to transcription-coupled repair and international genomic mend,the two branches of the NER pathway. Curiously, we found thatthe double mutant showed a major improve in embryoniclethality in contrast to the two single mutants （Figure 3A）。 Consequently, weconclude that lem-3 and xpa-1 likely do not act in a linear pathway.
LEM-3 is necessary for suitable chromosome segregationfollowing DNA problems
As we could not locate any obvious problems in the germ line of lem-3（op444） mutants, we investigated in additional detail the cause of thelethality noticed adhering to DNA hurt. To get a very first insightinto the nature of the defect, we stained chromatin in early stageembryos derived from irradiated hermaphrodite worms. Whereaschromosome segregation was generally usual in irradiated wildtype embryos （91%, n= 67）， 77% （n= 31） of irradiated lem-3（op444） embryos confirmed chromosome mis-segregation starting atthe second cell division, as evidenced by the technology of extranuclear chromatin material and chromatin bridges followingmitosis （Figure 6）。 We verified this consequence by using a GFP::H2Btransgene to adhere to chromosome segregation in early embryos bytime lapse microscopy （Movie S1 and Film S2）。 To determinewhether LEM-3 is also necessary for proper chromosomesegregation in the course of larval progress, we irradiated freshlyhatched L1 larvae and scored the animals as grownups for locomotiondefects or protruding vulvae – phenotypes arising from defectiveproliferation/division of the Pn.a neuroblast or Pn.p vulvalprecursor cells, respectively. These phenotypes can also beobserved next irradiation of mutants of the non-homologousend joining pathway genes cku-70, cku-eighty and lig-4 [31]. Only 2%of irradiated lem-3 mutants developed to grownups without having anydevelopmental flaws （96% shown an uncoordinated phenotype（Unc）， sixty one% had a protruding vulva （P-vul））。 In contrast, 86%of irradiated wild sort L1 larvae produced to phenotypicallynormal adults （Figure 1D）。 These effects propose that lem-three mutantembryos die after irradiation simply because of reduction of genomic integritydue to chromosome segregation problems. Moreover, we concludethat lem-3 is also required during post-embryonic cell divisionsafter genotoxic strain to ensure normal mobile proliferation.LEM-3 genetically interacts with BAF-1Three LEM-domain proteins, EMR-1 （Ce-emerin）， LEM-2（Ce-MAN1）， and LEM-3 have been identified in C. elegans. EMR-1and LEM-2 have a transmembrane domain and an N-terminallylocated LEM domain [fourteen], while LEM-3 lacks a transmembranedomain, and has a LEM-domain situated in the middle ofthe protein [14]. EMR-1 and LEM-two bind LMN-1, the C. elegans lamin homologue, and BAF-1 （barrier-to-autointegration factor）[fifteen]. BAF is an evolutionarily conserved and necessary proteinwhich was proven to interact with dsDNA, chromatin, nuclearlamina proteins, histones and transcription variables [32]. Downregulationof possibly lmn-1 or baf-one or co-depletion of emr-1 and lem-two prospects to extreme defects in mitosis, including abnormalchromosome segregation and anaphase bridges, which ultimatelyleads to embryonic lethality [13,15,18,33]. BAF-one is crucial fornuclear envelope development [34] and essential to assemble LMN-one, EMR-one and LEM-2 on the nuclear envelope [17].
As downregulation of possibly baf-one or co-depletion of emr-one andlem-2 potential customers to embryonic lethality [thirteen,fifteen,18] with phenotypessimilar to people noticed in lem-3 mutants next IR, weanalyzed regardless of whether baf-1, lem-2 or emr-1 mutants may well be sensitiveto DNA harm. We found that emr-1 mutants were being weaklysensitive, whilst lem-2 mutants shown reasonable sensitivity to IR（Figure 7B）。 The temperature sensitive baf-1（t1639ts） mutant atpermissive temperature was also hypersensitive to irradiationtreatment （Figure 7A）。 To check no matter if baf-1 and lem-three interactgenetically, we created double mutants with the two lem-3 alleles andanalyzed their sensitivity to irradiation. Apparently, the completeloss of LEM-three perform （lem-3（mn155）） drastically greater thelethality of the baf-1（t1639） mutation, even in the absence ofexogenous DNA injury. By distinction, the double mutant with thepoint mutation （lem-3（op444）） showed only a insignificant boost inlethality, probable due to residual action of the LEM-3 （L659F）protein. This end result suggests that when BAF-1 purpose is restricting,LEM-3 perform gets critical even in the absence of DNAdamage. Taken together, the phenotypes explained in irradiatedlem-three mutants and animals depleted of either lmn-1 or baf-1, or codepletedof emr-1 and lem-two suggest a possible typical result in of theobserved molecular defects.
