http://colil.dbcls.jp:18052/sparql?query=define%20sql%3Adescribe-mode%20%22CBD%22%20%20DESCRIBE%20%3Chttp%3A%2F%2Fpurl.jp%2Fbio%2F10%2Fcolil%2Fid%2F22761594%3E&output=application%2Fatom%2Bxml2024-03-29T21:27:39.130430ZOData Service and Descriptor DocumentnodeID://b4971800212024-03-29T21:27:39.130430Z4nodeID://b168483882024-03-29T21:27:39.130430ZThe molecular effects of MMS include the formation of N-7 methylguanine (which by spontaneous depurination can lead to an abasic site), N3- methyladenine, N3-cytosine and O6-methylguanine [>>48<<]. Although we cannot deduce from our in vivo analysis which of these damages underlies the cytotoxicity observed in nematodes, all of these base damages have residual coding capacity, and are less structurally perturbing than some of thenodeID://b168483192024-03-29T21:27:39.130430Zdefective Polη has clinical implications: Polη is the product of the gene mutated in Xeroderma Pigmentosum complementation group “Variant” (XPV), a syndrome that is associated with a high predisposition towards developing skin cancers [>>10<<]. In addition to a role in damage bypass some studies have suggested a role for Polη in homologous recombination, as the polymerase responsible for extension of the invading strand in the D-loop recombination intermediate [11], [12].nodeID://b168483242024-03-29T21:27:39.130430ZPolκ displays structural similarity to Polη but is considered to be the most evolutionarily conserved member of the Y-family showing homology to prokaryotic DinB [>>8<<], [9]. Its substrate specificity in vitro is limited, although Polκ is an efficient extender of mispaired primer termini and some guanyl adducted lesion sites [14], [15]. Furthermore, Polκ has been suggested as one of the gap-fillingnodeID://b168483572024-03-29T21:27:39.130430ZTransposition is desilenced in the germline of rde-3 mutants [32] and the ensuing DSBs predominantly rely on HR for their repair [>>33<<]. However, embryonic lethality was not increased in polh-1; rde-3 double mutants, in contrast to increased lethality in brc-1; rde-3 doubles (Table S2). As an independent and a direct method to address a possible in vivo role of C. elegansnodeID://b4971800182024-03-29T21:27:39.130430Z4nodeID://b4971800152024-03-29T21:27:39.130430Z5nodeID://b168483622024-03-29T21:27:39.130430ZIn addition we determined the apoptotic response in the germline after UV irradiation using a ced-1::GFP transgene that marks germ cells in the process of apoptosis [>>37<<]. In contrast to xpa-1 deficient animals [38], we found no reduction in the UV-dependent apoptotic response in polh-1 mutants as compared to wildtype animals (Figure S4). Together, these data indicate that NER is essential for normalnodeID://b168483312024-03-29T21:27:39.130430ZAll strains were cultured according to standard methods [>>54<<]. Wildtype N2 (Bristol) worms were used in all control experiments. The polh-1 (lf31) and polk-1 (lf29) mutants were isolated in our own laboratory.nodeID://b4971800322024-03-29T21:27:39.130430Z3nodeID://b4971800092024-03-29T21:27:39.130430Z7nodeID://b4971800232024-03-29T21:27:39.130430Z4nodeID://b4971800262024-03-29T21:27:39.130430Z4nodeID://b4971800402024-03-29T21:27:39.130430Z3nodeID://b4971800172024-03-29T21:27:39.130430Z4nodeID://b4971800312024-03-29T21:27:39.130430Z3nodeID://b4971800342024-03-29T21:27:39.130430Z3nodeID://b168483892024-03-29T21:27:39.130430ZOne of these genes, gei-17, was previously reported to regulate Polη; GEI-17 was shown to sumoylate POLH-1 near its PIP-box motif resulting in protection of the protein from degradation [>>13<<]. The profound effects of gei-17 RNAi on cellular tolerance to MMS suggest that this SUMO-ligase most likely acts on both POLH-1 and POLK-1 (Figure 5A and 5I); we note that C. elegans POLK-1 may also contain a PIP-box motif (Figure S1). InnodeID://b4971801012024-03-29T21:27:39.130430Z2nodeID://b168483582024-03-29T21:27:39.130430ZIt also implies that genes previously found to be involved in γ-irradiation protection are not necessarily involved in the repair of DNA breaks [>>35<<].nodeID://b4971800282024-03-29T21:27:39.130430Z3nodeID://b168483272024-03-29T21:27:39.130430ZIts substrate specificity in vitro is limited, although Polκ is an efficient extender of mispaired primer termini and some guanyl adducted lesion sites [14], [>>15<<]. Furthermore, Polκ has been suggested as one of the gap-filling polymerases in NER, explaining a moderate sensitivity of Polκ-deficient mammalian cells to UV [16], [17].nodeID://b168483322024-03-29T21:27:39.130430ZBCN2081, carrying a single copy integrated Pmyo::GFP transgene, was a gift from Ben Lehner (EMBL Centre for Genomic Regulation, Barcelona, Spain) [>>55<<]. All other alleles (xpa-1 (ok698); rde-3 (ne298); brc-1 (tm1145); dog-1 (gk10)) and the transgenic line MD701 (bcIs39[P(lim-7)ced-1::GFP+lin-15(+)]) were obtained from the C. elegans Genetics Center (St Paul, MN, USA). All mutant strainsnodeID://b4971800422024-03-29T21:27:39.130430Z3nodeID://b4971800452024-03-29T21:27:39.130430Z3nodeID://b168483702024-03-29T21:27:39.130430ZNER deficient xpa-1 larvae were profoundly more sensitive to MMS than either polk-1 or polh-1 deficient larvae (Figure S6B), while the opposite is true for embryonic stages: xpa-1 embryos are less sensitive to MMS than polh-1 embryos [>>6<<]. This again argues that TLS is more important than DNA repair at developmental stages that are characterized by fast replication cycles.nodeID://b168483652024-03-29T21:27:39.130430ZBefore migration and fusion with the maternal pronucleus, the paternal genome decondenses and is replicated in less than 12 minutes [>>39<<]. This time span is insufficient for NER to remove DNA damage. We hypothesize that the presence of unrepaired damage from the paternal DNA poses a problem on the first mitotic divisions in polh-1 early embryos. To address this hypothesis,nodeID://b4971801122024-03-29T21:27:39.130430Z2nodeID://b4971800362024-03-29T21:27:39.130430Z3nodeID://b4971800392024-03-29T21:27:39.130430Z3nodeID://b4971800502024-03-29T21:27:39.130430Z3nodeID://b4971800532024-03-29T21:27:39.130430Z3nodeID://b4971801032024-03-29T21:27:39.130430Z2nodeID://b4971801062024-03-29T21:27:39.130430Z2nodeID://b4971801202024-03-29T21:27:39.130430Z2nodeID://b4971800442024-03-29T21:27:39.130430Z3nodeID://b4971800472024-03-29T21:27:39.130430Z3nodeID://b4971801142024-03-29T21:27:39.130430Z2nodeID://b4971800612024-03-29T21:27:39.130430Z3nodeID://b4971801112024-03-29T21:27:39.130430Z2nodeID://b168483282024-03-29T21:27:39.130430ZFurthermore, Polκ has been suggested as one of the gap-filling polymerases in NER, explaining a moderate sensitivity of Polκ-deficient mammalian cells to UV [>>16<<], [17].nodeID://b4971801082024-03-29T21:27:39.130430Z2nodeID://b4971800552024-03-29T21:27:39.130430Z3nodeID://b4971800582024-03-29T21:27:39.130430Z3nodeID://b168483662024-03-29T21:27:39.130430ZHowever, akin to the outcome of published RNAi experiments [>>6<<], both polk-1 and polh-1 mutants are sensitive to chronic exposure to the alkylating agent MMS, albeit that the sensitivity in polh-1 mutants was much more pronounced (Figure 5A, Figure S6), indicating that both POLH-1 and POLK-1 play anodeID://b168483402024-03-29T21:27:39.130430ZIn addition, POLH-1 contains a C-terminal PIP box motif, which is essential for interaction with PCNA, and more recently, has also been shown to target the protein for degradation [13], [>>18<<]. The remaining part of the C-terminus is evolutionary less conserved. Human Polη/yeast Rad30 and human Polκ contain ubiquitin binding zinc finger (UBZ) domains, mediating their interaction with PCNA [19]. A UBZ domain was found in C.nodeID://b168483352024-03-29T21:27:39.130430ZThe ISceI expressing plasmid pRP3001 (hsp-16.41::ISceI ORF) [>>57<<], was modified to include the mCherry ORF leading to a functional ISceI/mCherry protein to visualize and monitor the expression of the ISceI endonuclease.nodeID://b4971800722024-03-29T21:27:39.130430Z2nodeID://b168483732024-03-29T21:27:39.130430Znpp-2 and npp-22 encode two components of the C. elegans nuclear pore complex (NPC)[>>42<<]. Null alleles of gei-17, npp-2 and npp-22 are embryonic lethal. Knockdowns of the four genes reduced tolerance to MMS to a similar extent as mutations in polh-1 and polk-1 (Figure 6K). In line with published data [6] we found that gei-17nodeID://b4971801222024-03-29T21:27:39.130430Z2nodeID://b4971801252024-03-29T21:27:39.130430Z2nodeID://b4971800492024-03-29T21:27:39.130430Z3nodeID://b4971800662024-03-29T21:27:39.130430Z2nodeID://b4971801162024-03-29T21:27:39.130430Z2nodeID://b4971801192024-03-29T21:27:39.130430Z2nodeID://b4971800632024-03-29T21:27:39.130430Z2nodeID://b4971801302024-03-29T21:27:39.130430Z2nodeID://b4971801332024-03-29T21:27:39.130430Z2nodeID://b4971800802024-03-29T21:27:39.130430Z2nodeID://b4971800572024-03-29T21:27:39.130430Z3nodeID://b4971801242024-03-29T21:27:39.130430Z2nodeID://b4971801272024-03-29T21:27:39.130430Z2nodeID://b4971800712024-03-29T21:27:39.130430Z2nodeID://b4971800742024-03-29T21:27:39.130430Z2nodeID://b4971801412024-03-29T21:27:39.130430Z2nodeID://b4971800682024-03-29T21:27:39.130430Z2nodeID://b168483362024-03-29T21:27:39.130430ZFor the analysis of apoptosis, transgenic MD701 animals, expressing a ced1::GFP fusion behind a lim-7 promotor, were used to visualize sheath cells surrounding apoptotic germ cells [>>37<<]. All microscopy was performed with a Leica DM6000 microscope.nodeID://b4971801352024-03-29T21:27:39.130430Z2nodeID://b168483742024-03-29T21:27:39.130430ZIn line with published data [>>6<<] we found that gei-17 knockdown led to abundant RAD51 foci in embryos treated on MMS, indicative of replication stress.nodeID://b4971801382024-03-29T21:27:39.130430Z2nodeID://b4971800822024-03-29T21:27:39.130430Z2nodeID://b4971800852024-03-29T21:27:39.130430Z2nodeID://b168483692024-03-29T21:27:39.130430ZFirst, polh-1; polk-1 double mutant embryos displayed foci of the DSB repair marker RAD51 (Figure 5C–5J), indicative of DSBs resulting from trying to replicate damaged genomes [>>40<<]. Second, DAPI staining revealed chromatin bridges and a disrupted nuclear morphology in the early embryo (Figure 5J), suggesting division of disentangled or incompletely replicated genomes. These phenotypes were less profound, butnodeID://b168483102024-03-29T21:27:39.130430ZTo remove these DNA lesions cells are equipped with various specialized repair mechanisms [>>1<<], including nucleotide excision repair (NER) that deals with helix-distorting obstructions [2].nodeID://b168483432024-03-29T21:27:39.130430ZFurthermore, C. elegans and yeast Polη and Polκ lack previously identified mammalian motifs that mediate an interaction with the deoxycytidyl transferase Rev1 [20], [>>21<<].nodeID://b4971801522024-03-29T21:27:39.130430Z2nodeID://b168483812024-03-29T21:27:39.130430Zindicates that TLS-proficient zygotes can replicate a damaged genome containing 10–50.000 UV lesions in less than 12 minutes - the time it takes for the paternal nucleus to double its genome content - without delaying cell division [>>39<<].nodeID://b4971801292024-03-29T21:27:39.130430Z2nodeID://b4971800762024-03-29T21:27:39.130430Z2nodeID://b4971800792024-03-29T21:27:39.130430Z2nodeID://b4971800932024-03-29T21:27:39.130430Z2nodeID://b4971801432024-03-29T21:27:39.130430Z2nodeID://b4971801462024-03-29T21:27:39.130430Z2nodeID://b4971800902024-03-29T21:27:39.130430Z2nodeID://b4971801602024-03-29T21:27:39.130430Z2nodeID://b4971800872024-03-29T21:27:39.130430Z2nodeID://b4971801372024-03-29T21:27:39.130430Z2nodeID://b4971800842024-03-29T21:27:39.130430Z2nodeID://b4971801512024-03-29T21:27:39.130430Z2nodeID://b4971801542024-03-29T21:27:39.130430Z2nodeID://b4971800982024-03-29T21:27:39.130430Z2nodeID://b4971801482024-03-29T21:27:39.130430Z2nodeID://b4971800952024-03-29T21:27:39.130430Z2nodeID://b168483442024-03-29T21:27:39.130430ZUsing a targeted mutagenesis approach [>>22<<] we isolated mutants for polh-1 and polk-1 (Figure 1B).nodeID://b168483392024-03-29T21:27:39.130430ZIn addition, POLH-1 contains a C-terminal PIP box motif, which is essential for interaction with PCNA, and more recently, has also been shown to target the protein for degradation [>>13<<], [18]. The remaining part of the C-terminus is evolutionary less conserved. Human Polη/yeast Rad30 and human Polκ contain ubiquitin binding zinc finger (UBZ) domains, mediating their interaction with PCNA [19]. A UBZ domain was found in C.nodeID://b4971801652024-03-29T21:27:39.130430Z2nodeID://b4971801622024-03-29T21:27:39.130430Z2nodeID://b168483772024-03-29T21:27:39.130430ZIndeed, we and others found that both for germ cell maturation and post-embryonic somatic development, NER is indispensable in response to specific DNA damages [>>26<<], [27], [38].nodeID://b168483822024-03-29T21:27:39.130430ZThis notion has been supported by studies in vertebrates, in which Polη was suggested to act in HR repair of DSBs by extending the D-loop intermediate structure [>>11<<], [12]. However, we observed a wild type response to either transposon-mediated or ISceI-induced DSBs, thus arguing against a role for POLH-1 in DSB repair, in either germline or somatic tissue of C. elegans. We ascribe the sensitivity ofnodeID://b168483132024-03-29T21:27:39.130430ZIn the nematode Caenorhabditis elegans a delay in replication is detrimental for the developmental program; timing of cell division is fixed and strictly regulated by the homologues of the checkpoint genes CHK1 and ATR [>>4<<]. Indeed, replication stress caused by depletion of nucleotide pools causes fatal errors in the correct timing of the first asynchronous divisions [5]. However, early embryos of C. elegans appear to be remarkably resistant to DNA damagingnodeID://b4971800892024-03-29T21:27:39.130430Z2nodeID://b168483512024-03-29T21:27:39.130430ZThis sensitivity was even more pronounced than for dog-1 mutant animals which are defective in the homolog of the Fanconi Anemia gene FANCJ, involved in crosslink repair [>>28<<].nodeID://b4971801592024-03-29T21:27:39.130430Z2nodeID://b4971801562024-03-29T21:27:39.130430Z2nodeID://b4971801732024-03-29T21:27:39.130430Z2nodeID://b4971801702024-03-29T21:27:39.130430Z2nodeID://b4971800972024-03-29T21:27:39.130430Z2nodeID://b4971801642024-03-29T21:27:39.130430Z2nodeID://b4971801672024-03-29T21:27:39.130430Z2nodeID://b4971801812024-03-29T21:27:39.130430Z2nodeID://b4971801752024-03-29T21:27:39.130430Z2nodeID://b4971801782024-03-29T21:27:39.130430Z2nodeID://b168483782024-03-29T21:27:39.130430ZIndeed, we and others found that both for germ cell maturation and post-embryonic somatic development, NER is indispensable in response to specific DNA damages [26], [>>27<<], [38]. However, and in line with previously published data [6], we found that immediately after fertilization of the oocyte, during stages of rapid cell divisions in the early embryo, survival is determined by TLS factors and not by NER.nodeID://b168483092024-03-29T21:27:39.130430ZintroductionnodeID://b168483142024-03-29T21:27:39.130430ZIndeed, replication stress caused by depletion of nucleotide pools causes fatal errors in the correct timing of the first asynchronous divisions [>>5<<]. However, early embryos of C. elegans appear to be remarkably resistant to DNA damaging agents, suggesting an efficient way to prevent the induction of replication stress by DNA damage [6].nodeID://b168483472024-03-29T21:27:39.130430ZIn contrast to Polη-defective yeast and mammalian cells, that display only a mild hypersensitivity to UV [>>25<<], [26], POLH-1 deficiency leads to extreme sensitivity to UV irradiation.nodeID://b168483522024-03-29T21:27:39.130430ZVertebrate Polη has been implicated as the polymerase responsible for extension of HR intermediates [>>12<<], [29]. Since HR is the predominant repair pathway in C. elegans for γ-irradiation-induced breaks in germ cells [30], [31], we exposed L4 animals to γ-irradiation and scored survival of the progeny (Figure 2C and Figure S2D).nodeID://b168483902024-03-29T21:27:39.130430ZSUMO proteases and ligases may anchor to the NPCs in order to sumoylate or desumoylate their targets [>>49<<]. Here we show that, similar to gei-17 and ulp-1, RNAi against nuclear pore components npp-2 and npp-22 is compatible with viability but results in sensitivity to UV lesions and MMS. However, sensitivity was not further increased in thenodeID://b4971801922024-03-29T21:27:39.130430Z2nodeID://b168483852024-03-29T21:27:39.130430ZIn human cells Rev1 is recruited to UV damages via an interaction with Polη [>>45<<]. Interestingly, Polκ has been shown to serve as a ‘backup’ polymerase in XPV cells in the bypass of both UV-induced CPDs as well as cisplatin adducts [46], [47]. We here show that in nematodes this genetic interaction is restricted tonodeID://b168483212024-03-29T21:27:39.130430ZIn addition to a role in damage bypass some studies have suggested a role for Polη in homologous recombination, as the polymerase responsible for extension of the invading strand in the D-loop recombination intermediate [11], [>>12<<]. Recently, it was reported that Polη plays a prominent role in early stages of nematode embryogenesis in C. elegans [6], [13]. Polκ displays structural similarity to Polη but is considered to be the most evolutionarily conserved member ofnodeID://b4971801692024-03-29T21:27:39.130430Z2nodeID://b4971801862024-03-29T21:27:39.130430Z2nodeID://b4971801832024-03-29T21:27:39.130430Z2nodeID://b4971801772024-03-29T21:27:39.130430Z2nodeID://b4971801912024-03-29T21:27:39.130430Z2nodeID://b4971801942024-03-29T21:27:39.130430Z2nodeID://b168483792024-03-29T21:27:39.130430ZIndeed, we and others found that both for germ cell maturation and post-embryonic somatic development, NER is indispensable in response to specific DNA damages [26], [27], [>>38<<]. However, and in line with previously published data [6], we found that immediately after fertilization of the oocyte, during stages of rapid cell divisions in the early embryo, survival is determined by TLS factors and not by NER. ThenodeID://b4971801882024-03-29T21:27:39.130430Z2nodeID://b168483482024-03-29T21:27:39.130430ZIn contrast to Polη-defective yeast and mammalian cells, that display only a mild hypersensitivity to UV [25], [>>26<<], POLH-1 deficiency leads to extreme sensitivity to UV irradiation.nodeID://b168483862024-03-29T21:27:39.130430ZInterestingly, Polκ has been shown to serve as a ‘backup’ polymerase in XPV cells in the bypass of both UV-induced CPDs as well as cisplatin adducts [>>46<<], [47]. We here show that in nematodes this genetic interaction is restricted to damage induced by the Sn1 methylating agent MMS. The molecular effects of MMS include the formation of N-7 methylguanine (which by spontaneous depurinationnodeID://b168483172024-03-29T21:27:39.130430ZIt allows for accommodation of a DNA lesion, but is also the basis for reduced fidelity [>>8<<]. The functional specificities of TLS polymerases are due to minor differences in the structural features of the active site.nodeID://b168483222024-03-29T21:27:39.130430ZRecently, it was reported that Polη plays a prominent role in early stages of nematode embryogenesis in C. elegans [>>6<<], [13]. Polκ displays structural similarity to Polη but is considered to be the most evolutionarily conserved member of the Y-family showing homology to prokaryotic DinB [8], [9]. Its substrate specificity in vitro is limited, althoughnodeID://b168483602024-03-29T21:27:39.130430ZAlthough xpa-1 mutants completely arrest in L1 after a low dose of UV (Figure 4A, [>>27<<]), in polh-1 mutants L1 development is only slightly delayed (Figure S3A).nodeID://b168483552024-03-29T21:27:39.130430ZSince HR is the predominant repair pathway in C. elegans for γ-irradiation-induced breaks in germ cells [30], [>>31<<], we exposed L4 animals to γ-irradiation and scored survival of the progeny (Figure 2C and Figure S2D).nodeID://b168483932024-03-29T21:27:39.130430ZDNA damage response in yeast was also suggested by Nagai et al., who showed relocation of damaged DNA to nuclear pores [51] and recently by Bermejo et al. who showed involvement of inner basket proteins in replication fork stability [>>52<<]. npp-22 encodes the C. elegans homolog of yeast and mammalian NDC1, which is crucial for nuclear pore assembly [53].nodeID://b4971801962024-03-29T21:27:39.130430Z2nodeID://b4971801992024-03-29T21:27:39.130430Z2nodeID://b4971800122024-03-29T21:27:39.130430Z5nodeID://b4971800202024-03-29T21:27:39.130430Z4nodeID://b4971800112024-03-29T21:27:39.130430Z6nodeID://b4971800142024-03-29T21:27:39.130430Z5nodeID://b168483182024-03-29T21:27:39.130430Zincluding UV-induced cyclobutane pyrimidine dimers (CPDs), 7,8-dihydro-8-oxoguanine, O 6-methylguanine, thymine glycol, cisplatin-induced intrastrand crosslinks, acetylaminofluorene-adducted guanine and benzo[a]pyrene-N 2-guanine [>>9<<]. In humans, defective Polη has clinical implications:nodeID://b168483562024-03-29T21:27:39.130430ZTransposition is desilenced in the germline of rde-3 mutants [>>32<<] and the ensuing DSBs predominantly rely on HR for their repair [33].nodeID://b4971800082024-03-29T21:27:39.130430Z7nodeID://b168483942024-03-29T21:27:39.130430Znpp-22 encodes the C. elegans homolog of yeast and mammalian NDC1, which is crucial for nuclear pore assembly [>>53<<]. Future work on gei-17, ulp-1 and the nuclear pore components npp-2 and npp-22 is needed to substantiate the role of sumoylation and desumoylation processes and a possible link to the NPC (subunits) in regulating TLS.nodeID://b168483302024-03-29T21:27:39.130430Zmaterials and methodsnodeID://b168483252024-03-29T21:27:39.130430ZPolκ displays structural similarity to Polη but is considered to be the most evolutionarily conserved member of the Y-family showing homology to prokaryotic DinB [8], [>>9<<]. Its substrate specificity in vitro is limited, although Polκ is an efficient extender of mispaired primer termini and some guanyl adducted lesion sites [14], [15]. Furthermore, Polκ has been suggested as one of the gap-fillingnodeID://b4971800222024-03-29T21:27:39.130430Z4nodeID://b4971800252024-03-29T21:27:39.130430Z4nodeID://b168483632024-03-29T21:27:39.130430ZIn contrast to xpa-1 deficient animals [>>38<<], we found no reduction in the UV-dependent apoptotic response in polh-1 mutants as compared to wildtype animals (Figure S4).nodeID://b4971800162024-03-29T21:27:39.130430Z5nodeID://b4971800192024-03-29T21:27:39.130430Z4nodeID://b4971800332024-03-29T21:27:39.130430Z3nodeID://b4971800302024-03-29T21:27:39.130430Z3nodeID://b4971801002024-03-29T21:27:39.130430Z2nodeID://b4971800242024-03-29T21:27:39.130430Z4nodeID://b4971800272024-03-29T21:27:39.130430Z3nodeID://b4971800412024-03-29T21:27:39.130430Z3nodeID://b168483262024-03-29T21:27:39.130430ZIts substrate specificity in vitro is limited, although Polκ is an efficient extender of mispaired primer termini and some guanyl adducted lesion sites [>>14<<], [15]. Furthermore, Polκ has been suggested as one of the gap-filling polymerases in NER, explaining a moderate sensitivity of Polκ-deficient mammalian cells to UV [16], [17].nodeID://b168483642024-03-29T21:27:39.130430ZHowever, limited time for DNA repair is available immediately upon fertilization, when a C. elegans embryo goes through a 3-hour period of rapid divisions, according to a fixed and time-constrained lineage program [>>31<<]. Thus, in this developmental stage incomplete removal of DNA damage could account for the severe embryonic lethality of UV-exposed polh-1 mutants.nodeID://b168483592024-03-29T21:27:39.130430Zhypothesized that the dependence on POLH-1 for damage tolerance might be specific for early embryonic development, when TLS by POLH-1 is the predominant mechanism to avoid checkpoint activation by replication fork blocks on damaged DNA [>>6<<]. In differentiated cells, NER or other repair pathways may dominate the damage response.nodeID://b4971800352024-03-29T21:27:39.130430Z3nodeID://b4971800382024-03-29T21:27:39.130430Z3nodeID://b168483332024-03-29T21:27:39.130430ZThe sensitivity of L1 larval stage animals to UV was measured as described previously [>>27<<]. L1s were synchronized by bleaching, and exposed to UV-C on empty NGM plates. Per plate, at least 100 L1 animals were counted.nodeID://b4971801052024-03-29T21:27:39.130430Z2nodeID://b4971800522024-03-29T21:27:39.130430Z3nodeID://b168483712024-03-29T21:27:39.130430Zgei-17 encodes a SUMO-protease that was previously shown to interact with POLH-1 after DNA damage [>>13<<]. ulp-1 encodes a ubiquitin-like protease (ULP) that deconjugates SUMO moieties from their target proteins [41]. npp-2 and npp-22 encode two components of the C. elegans nuclear pore complex (NPC)[42]. Null alleles of gei-17, npp-2 andnodeID://b4971801022024-03-29T21:27:39.130430Z2nodeID://b4971800292024-03-29T21:27:39.130430Z3nodeID://b4971800462024-03-29T21:27:39.130430Z3nodeID://b4971800432024-03-29T21:27:39.130430Z3nodeID://b4971801102024-03-29T21:27:39.130430Z2nodeID://b4971801132024-03-29T21:27:39.130430Z2nodeID://b4971800602024-03-29T21:27:39.130430Z3nodeID://b4971800372024-03-29T21:27:39.130430Z3nodeID://b4971801072024-03-29T21:27:39.130430Z2nodeID://b4971800542024-03-29T21:27:39.130430Z3nodeID://b4971800512024-03-29T21:27:39.130430Z3nodeID://b4971801042024-03-29T21:27:39.130430Z2nodeID://b4971801212024-03-29T21:27:39.130430Z2nodeID://b4971800482024-03-29T21:27:39.130430Z3nodeID://b168483292024-03-29T21:27:39.130430ZFurthermore, Polκ has been suggested as one of the gap-filling polymerases in NER, explaining a moderate sensitivity of Polκ-deficient mammalian cells to UV [16], [>>17<<].nodeID://b168483342024-03-29T21:27:39.130430ZA HR reporter plasmid was constructed consisting of a GFP/LacZ fusion under the control of the intestinal specific elt-2 promotor [>>56<<]. An ISceI recognition sequence was inserted that disrupted the GFP ORF. To provide a template for homologous recombination, part of the GFP coding region was PCR amplified and inserted downstream of the disrupted GFP/LacZ locus.nodeID://b4971800622024-03-29T21:27:39.130430Z2nodeID://b168483672024-03-29T21:27:39.130430ZPOLH-1 has previously been shown to be involved in avoiding DNA damage-induced checkpoint activation [>>6<<]. In C. elegans embryogenesis, checkpoints - mediated by the C. elegans homologs of the checkpoint genes ATR and CHK-1 - are used to time the first asynchronous cell divisions that are essential for embryonic patterning and thus embryonicnodeID://b4971800652024-03-29T21:27:39.130430Z2nodeID://b4971801152024-03-29T21:27:39.130430Z2nodeID://b4971801182024-03-29T21:27:39.130430Z2nodeID://b168483722024-03-29T21:27:39.130430Zulp-1 encodes a ubiquitin-like protease (ULP) that deconjugates SUMO moieties from their target proteins [>>41<<]. npp-2 and npp-22 encode two components of the C. elegans nuclear pore complex (NPC)[42]. Null alleles of gei-17, npp-2 and npp-22 are embryonic lethal. Knockdowns of the four genes reduced tolerance to MMS to a similar extent asnodeID://b168483412024-03-29T21:27:39.130430ZHuman Polη/yeast Rad30 and human Polκ contain ubiquitin binding zinc finger (UBZ) domains, mediating their interaction with PCNA [>>19<<]. A UBZ domain was found in C. elegans POLK-1 but not in C. elegans POLH-1 (Figure S1). Furthermore, C. elegans and yeast Polη and Polκ lack previously identified mammalian motifs that mediate an interaction with the deoxycytidylnodeID://b4971801322024-03-29T21:27:39.130430Z2nodeID://b4971800562024-03-29T21:27:39.130430Z3nodeID://b4971800592024-03-29T21:27:39.130430Z3nodeID://b4971801092024-03-29T21:27:39.130430Z2nodeID://b4971800732024-03-29T21:27:39.130430Z2nodeID://b4971801232024-03-29T21:27:39.130430Z2nodeID://b4971801262024-03-29T21:27:39.130430Z2nodeID://b4971800702024-03-29T21:27:39.130430Z2nodeID://b4971801402024-03-29T21:27:39.130430Z2nodeID://b4971800672024-03-29T21:27:39.130430Z2nodeID://b4971801172024-03-29T21:27:39.130430Z2nodeID://b4971800642024-03-29T21:27:39.130430Z2nodeID://b4971801342024-03-29T21:27:39.130430Z2nodeID://b4971800812024-03-29T21:27:39.130430Z2nodeID://b4971801312024-03-29T21:27:39.130430Z2nodeID://b4971801282024-03-29T21:27:39.130430Z2nodeID://b4971800752024-03-29T21:27:39.130430Z2nodeID://b4971800782024-03-29T21:27:39.130430Z2nodeID://b168483682024-03-29T21:27:39.130430Zembryogenesis, checkpoints - mediated by the C. elegans homologs of the checkpoint genes ATR and CHK-1 - are used to time the first asynchronous cell divisions that are essential for embryonic patterning and thus embryonic viability [>>5<<]. Checkpoint activation due to DNA damage interferes with the developmental role of the checkpoint, causing patterning defects and embryonic lethality.nodeID://b168483372024-03-29T21:27:39.130430ZThe procedure is an adaptation from a genome-wide RNAi screen for radiation sensitivity by Van Haaften et al, described in detail in their supplementary data [>>35<<]. Briefly, L1 worms were grown to L4s in liquid on RNAi food. At the L4 stage MMS was added to a concentration of 0.01%. After three days survival of the progeny was scored by visual inspection. For knockdown of polk-1, gei-17, ulp-1,nodeID://b168483422024-03-29T21:27:39.130430ZFurthermore, C. elegans and yeast Polη and Polκ lack previously identified mammalian motifs that mediate an interaction with the deoxycytidyl transferase Rev1 [>>20<<], [21].nodeID://b4971801452024-03-29T21:27:39.130430Z2nodeID://b168483802024-03-29T21:27:39.130430ZHowever, and in line with previously published data [>>6<<], we found that immediately after fertilization of the oocyte, during stages of rapid cell divisions in the early embryo, survival is determined by TLS factors and not by NER.nodeID://b168483752024-03-29T21:27:39.130430ZThe SUMO protease gene gei-17 has previously been shown to promote damage tolerance by sumoylating POLH-1 [>>13<<]. Our results suggest that GEI-17 is implicated in TLS mediated by both POLH-1 and POLK-1.nodeID://b4971800922024-03-29T21:27:39.130430Z2nodeID://b4971801422024-03-29T21:27:39.130430Z2nodeID://b168483112024-03-29T21:27:39.130430ZTo remove these DNA lesions cells are equipped with various specialized repair mechanisms [1], including nucleotide excision repair (NER) that deals with helix-distorting obstructions [>>2<<]. However, during embryogenesis, which entails phases of rapid cell division, only a limited time window is available for repair processes [3]. Consequently, unrepaired DNA damage may delay replication and cell cycle progression. In thenodeID://b4971800692024-03-29T21:27:39.130430Z2nodeID://b4971800832024-03-29T21:27:39.130430Z2nodeID://b4971801362024-03-29T21:27:39.130430Z2nodeID://b4971800862024-03-29T21:27:39.130430Z2nodeID://b4971801392024-03-29T21:27:39.130430Z2nodeID://b4971801532024-03-29T21:27:39.130430Z2nodeID://b4971801502024-03-29T21:27:39.130430Z2nodeID://b4971800772024-03-29T21:27:39.130430Z2nodeID://b4971800942024-03-29T21:27:39.130430Z2nodeID://b4971801442024-03-29T21:27:39.130430Z2nodeID://b4971801472024-03-29T21:27:39.130430Z2nodeID://b4971800912024-03-29T21:27:39.130430Z2nodeID://b4971801612024-03-29T21:27:39.130430Z2nodeID://b4971800882024-03-29T21:27:39.130430Z2nodeID://b168483382024-03-29T21:27:39.130430ZresultsnodeID://b4971801582024-03-29T21:27:39.130430Z2nodeID://b168483762024-03-29T21:27:39.130430ZdiscussionnodeID://b4971801552024-03-29T21:27:39.130430Z2nodeID://b168483122024-03-29T21:27:39.130430ZHowever, during embryogenesis, which entails phases of rapid cell division, only a limited time window is available for repair processes [>>3<<]. Consequently, unrepaired DNA damage may delay replication and cell cycle progression. In the nematode Caenorhabditis elegans a delay in replication is detrimental for the developmental program; timing of cell division is fixed andnodeID://b168483502024-03-29T21:27:39.130430ZBoth polh-1(lf31) and polh-1(ok3317) mutants are more sensitive to UV than animals carrying mutations in xpa-1, the worm homolog of NER gene XPA, which is essential for repair of UV damage [26], [>>27<<]. NER contributes to UV survival also in polh-1 compromised conditions as animals defective in both xpa-1 and polh-1 are more sensitive than either of the single mutants. (Figure S2B). In line with mammalian data, we observed that thenodeID://b168483452024-03-29T21:27:39.130430ZBecause UV-induced CPDs are excellent substrates for Polη-mediated TLS in yeast and mammals [>>23<<], [24], we tested the sensitivity of polh-1 mutant animals to UV light by irradiating young adults and scoring progeny survival (Figure 2A and Figure S2A).nodeID://b168483832024-03-29T21:27:39.130430ZThis notion has been supported by studies in vertebrates, in which Polη was suggested to act in HR repair of DSBs by extending the D-loop intermediate structure [11], [>>12<<]. However, we observed a wild type response to either transposon-mediated or ISceI-induced DSBs, thus arguing against a role for POLH-1 in DSB repair, in either germline or somatic tissue of C. elegans. We ascribe the sensitivity of polh-1nodeID://b4971801722024-03-29T21:27:39.130430Z2nodeID://b4971800992024-03-29T21:27:39.130430Z2nodeID://b4971801492024-03-29T21:27:39.130430Z2nodeID://b4971800962024-03-29T21:27:39.130430Z2nodeID://b4971801662024-03-29T21:27:39.130430Z2nodeID://b4971801632024-03-29T21:27:39.130430Z2nodeID://b4971801802024-03-29T21:27:39.130430Z2nodeID://b4971801572024-03-29T21:27:39.130430Z2nodeID://b4971801742024-03-29T21:27:39.130430Z2nodeID://b4971801712024-03-29T21:27:39.130430Z2nodeID://b4971801682024-03-29T21:27:39.130430Z2nodeID://b168483462024-03-29T21:27:39.130430ZBecause UV-induced CPDs are excellent substrates for Polη-mediated TLS in yeast and mammals [23], [>>24<<], we tested the sensitivity of polh-1 mutant animals to UV light by irradiating young adults and scoring progeny survival (Figure 2A and Figure S2A).nodeID://b4971801822024-03-29T21:27:39.130430Z2nodeID://b4971801852024-03-29T21:27:39.130430Z2nodeID://b168483842024-03-29T21:27:39.130430ZThe induction of free radicals by ionizing radiation causes a plethora of lesions in the DNA, such as 8-oxo-dG sites, which may require Polη-mediated bypass to prevent checkpoint activation [>>43<<].nodeID://b168483202024-03-29T21:27:39.130430ZIn addition to a role in damage bypass some studies have suggested a role for Polη in homologous recombination, as the polymerase responsible for extension of the invading strand in the D-loop recombination intermediate [>>11<<], [12]. Recently, it was reported that Polη plays a prominent role in early stages of nematode embryogenesis in C. elegans [6], [13]. Polκ displays structural similarity to Polη but is considered to be the most evolutionarily conservednodeID://b168483152024-03-29T21:27:39.130430ZHowever, early embryos of C. elegans appear to be remarkably resistant to DNA damaging agents, suggesting an efficient way to prevent the induction of replication stress by DNA damage [>>6<<].nodeID://b168483532024-03-29T21:27:39.130430ZVertebrate Polη has been implicated as the polymerase responsible for extension of HR intermediates [12], [>>29<<]. Since HR is the predominant repair pathway in C. elegans for γ-irradiation-induced breaks in germ cells [30], [31], we exposed L4 animals to γ-irradiation and scored survival of the progeny (Figure 2C and Figure S2D).nodeID://b168483912024-03-29T21:27:39.130430ZIn yeast, mutants in the Nup84 and Nup60/Mlp1-2 complexes have similar phenotypes in the response to DNA damage as Ulp1 mutants [>>50<<]. A direct link of NPCs to the DNA damage response in yeast was also suggested by Nagai et al., who showed relocation of damaged DNA to nuclear pores [51] and recently by Bermejo et al. who showed involvement of inner basket proteins innodeID://b4971801762024-03-29T21:27:39.130430Z2nodeID://b4971801792024-03-29T21:27:39.130430Z2nodeID://b4971801902024-03-29T21:27:39.130430Z2nodeID://b4971801932024-03-29T21:27:39.130430Z2nodeID://b4971801842024-03-29T21:27:39.130430Z2nodeID://b4971801872024-03-29T21:27:39.130430Z2nodeID://b4971801982024-03-29T21:27:39.130430Z2nodeID://b4971801952024-03-29T21:27:39.130430Z2nodeID://b168483162024-03-29T21:27:39.130430ZTo be able to deal with replication obstructions, organisms evolved ways that allow bypass of the damaged template, thus ensuring continuity of the replication process [>>7<<]. Specialized TLS polymerases are capable of direct bypass of DNA lesions in an error free or error prone fashion, depending on their affinities for the specific lesion site.nodeID://b168483542024-03-29T21:27:39.130430ZSince HR is the predominant repair pathway in C. elegans for γ-irradiation-induced breaks in germ cells [>>30<<], [31], we exposed L4 animals to γ-irradiation and scored survival of the progeny (Figure 2C and Figure S2D).nodeID://b168483492024-03-29T21:27:39.130430ZBoth polh-1(lf31) and polh-1(ok3317) mutants are more sensitive to UV than animals carrying mutations in xpa-1, the worm homolog of NER gene XPA, which is essential for repair of UV damage [>>26<<], [27]. NER contributes to UV survival also in polh-1 compromised conditions as animals defective in both xpa-1 and polh-1 are more sensitive than either of the single mutants. (Figure S2B). In line with mammalian data, we observed thatnodeID://b168483872024-03-29T21:27:39.130430ZInterestingly, Polκ has been shown to serve as a ‘backup’ polymerase in XPV cells in the bypass of both UV-induced CPDs as well as cisplatin adducts [46], [>>47<<]. We here show that in nematodes this genetic interaction is restricted to damage induced by the Sn1 methylating agent MMS. The molecular effects of MMS include the formation of N-7 methylguanine (which by spontaneous depurination can leadnodeID://b168483922024-03-29T21:27:39.130430ZA direct link of NPCs to the DNA damage response in yeast was also suggested by Nagai et al., who showed relocation of damaged DNA to nuclear pores [>>51<<] and recently by Bermejo et al. who showed involvement of inner basket proteins in replication fork stability [52].http://purl.jp/bio/10/colil/id/227615942024-03-29T21:27:39.130430Z10.1371%2Fjournal.pgen.1002800PMC0nodeID://b168483232024-03-29T21:27:39.130430ZRecently, it was reported that Polη plays a prominent role in early stages of nematode embryogenesis in C. elegans [6], [>>13<<]. Polκ displays structural similarity to Polη but is considered to be the most evolutionarily conserved member of the Y-family showing homology to prokaryotic DinB [8], [9]. Its substrate specificity in vitro is limited, although Polκ isnodeID://b4971801892024-03-29T21:27:39.130430Z2nodeID://b168483612024-03-29T21:27:39.130430Zpolh-1 mutants was comparable to wildtype following UV exposure (Figure 4B–4E), in contrast to xpa-1 mutants that (i) display an UV-induced expansion of the pachytene region and (ii) fail to generate normal-sized oocytes (Figure 4D), [>>27<<]. In addition we determined the apoptotic response in the germline after UV irradiation using a ced-1:nodeID://b4971801972024-03-29T21:27:39.130430Z2nodeID://b4971800132024-03-29T21:27:39.130430Z5nodeID://b4971800102024-03-29T21:27:39.130430Z6