FAM111A is a nuclear serine endopeptidase that binds single-stranded DNA and associates with replication-associated chromatin through a PCNA-interacting motif. Its protease, DNA-binding and PCNA-interaction regions help replication forks traverse protein obstacles and support removal of covalent TOP1–DNA complexes. FAM111A can cleave itself, including between separate protein molecules. It also restricts selected DNA viruses through context-dependent interactions and redistribution to the cytoplasm; during vaccinia-virus infection, cytoplasmic FAM111A promotes loss of viral I3 through cellular degradation machinery. Disease-associated substitutions can increase proteolysis and disrupt nuclear functions in experimental systems.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000785 chromatin | IBA GO_REF:0000033 | ACCEPT | Summary: FAM111A associates with nascent chromatin and replication sites. Reason: The actual chromatin IBD at PTN002683271 uses human Q96PZ2 descendant evidence. PMID:24561620 reports nascent-chromatin capture and immunoblot confirmation in human cells, together with Triton-resistant GFP-FAM111A colocalization with PCNA. PMID:33369867 independently visualizes endogenous FAM111A at PCNA-positive replication sites. These support chromatin association without equating every nuclear focus with replicating chromatin. The exact 611-residue target MSA matches current human FAM111A, and a subsequently recovered PANTHER version 19 tree places PTN002494620 beneath the asserted PTN002683271; target self-WITH is legitimate experimental grounding. Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN002683271 · PTN002683271 SUPPORTS TRANSFER The matching chromatin IBD cites Q96PZ2; human nascent-chromatin capture and endogenous replication-site imaging support the target. The version 19 PTHR14389 tree verifies descent of the sequence-matched human leaf PTN002494620 from PTN002683271. The matching IBD and bounded target-path NOT check support transfer; public source locators and scope are given in the source guide. |
| GO:0000785 chromatin | IDA PMID:24561620 Nascent chromatin capture proteomics determines chromatin dy... | ACCEPT | Summary: FAM111A associates with newly replicated human chromatin. Reason: PMID:24561620 identifies FAM111A by nascent-chromatin capture in human HeLa S3 cells and confirms its enrichment by western blot. Tagged FAM111A in U-2-OS cells is nuclear, resistant to Triton pre-extraction and colocalizes with PCNA (Results and Figures 6–7). These biochemical and imaging observations support chromatin residence; they do not make every molecule constitutively chromatin bound or establish sequence equivalence of the historical expression plasmid to every current transcript. Supporting Evidence: |
| GO:0003697 single-stranded DNA binding | IDA PMID:32165630 FAM111A protects replication forks from protein obstacles vi... | ACCEPT | Summary: Purified human FAM111A preferentially binds single-stranded DNA. Reason: PMID:32165630 Figure 2 and the EMSA/fluorescence-polarization Methods directly test recombinant human FAM111A. Full-length Sf21-produced protein binds ssDNA and Y-fork oligonucleotides, more weakly dsDNA, with antibody supershift and BSA controls; bacterial MBP fragments map a sufficient region to residues 176–282, and F231A impairs binding. The reported full-length affinity is about 60 nM under the tested conditions. This is preferential, not exclusive, ssDNA recognition; tagged full-length and engineered fragments are kept distinct. Supporting Evidence: |
| GO:0005515 protein binding | IPI PMID:23093934 Identification of FAM111A as an SV40 host range restriction ... | KEEP AS NON CORE | Summary: FAM111A physically associates with SV40 large T antigen. Reason: PMID:23093934 Figures 3–5 combines affinity proteomics, reciprocal co-immunoprecipitation in human U-2-OS cells and pairwise yeast two-hybrid mapping of human FAM111A residues 336–611 with the LT C-terminal region. Current UniProt P03070 identifies SV40 large T antigen. Targeted ontology searches did not establish an informative FAM111A molecular role from this contact: the paper does not show FAM111A cleavage of LT or direct inhibition of its helicase chemistry. Retain this supported generic interaction as non-core under the campaign exception rather than inventing such activities. Human binding assays and monkey host-range infection assays are separate. Supporting Evidence: |
| GO:0005515 protein binding | IPI PMID:24561620 Nascent chromatin capture proteomics determines chromatin dy... | KEEP AS NON CORE | Summary: The FAM111A PIP box mediates PCNA binding. Reason: PMID:24561620 Figure 6g and GST-pull-down Methods show in-vitro-translated human FAM111A binding recombinant GST-PCNA, reduced by the Y24A/F25A PIP mutation; cell imaging independently places FAM111A with PCNA at replication sites. Current UniProt P12004 is human DNA sliding clamp PCNA. A search for PCNA/clamp-binding specificity did not establish that FAM111A performs clamp loading or unloading. The 2014 PCNA-loading phenotype also differs from largely unchanged chromatin PCNA in the 2020 HAP1 knockout context. Keep the demonstrated interaction as non-core under the supported-binding exception; do not assign PCNA or clamp-loader activity to FAM111A. |
| GO:0005634 nucleus | EXP PMID:37607234 Human FAM111A inhibits vaccinia virus replication by degradi... | ACCEPT | Summary: FAM111A occupies the nucleus before infection-dependent redistribution. Reason: PMID:37607234 Figure 3 and Immunofluorescence Methods show nuclear FAM111A in mock-infected human A549 cells, using transfected WT and catalytic mutants, and a separate infection time course measures endogenous nuclear/cytoplasmic fractions with histone H3 and beta-actin controls. MVA infection shifts WT FAM111A toward the cytoplasm; inactive mutants remain nuclear. Nuclear residence is therefore supported without implying exclusivity across infection states. Independent human fractionation and replication-site imaging in PMID:23093934 and PMID:24561620 corroborate the location. |
| GO:0005634 nucleus | IBA GO_REF:0000033 | ACCEPT | Summary: Nuclear localization is supported directly in human cells. Reason: The recovered nucleus IBD at PTN002683271 cites human FAM111A/Q96PZ2 and FAM111B/Q6SJ93. Human FAM111A is detected in nuclear fractions in PMID:23093934, at nuclear replication sites in PMID:24561620 and PMID:33369867, and in uninfected A549 nuclei in PMID:37607234. The broad nuclear term remains informative because the observed pools are not confined to chromatin or one subnuclear compartment. The target experiments support this annotation independently of the subsequently verified PANTHER version 19 target path; the donor count and target self-WITH are not treated as defects. Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN002683271 · PTN002683271 SUPPORTS TRANSFER The matching nucleus IBD lists Q6SJ93 and Q96PZ2. Current FAM111B donor GOA contains experimental nucleus assertions, while independent target localization is directly read; those donor localization papers are not used as FAM111A target evidence. The version 19 PTHR14389 tree verifies descent of the sequence-matched human leaf PTN002494620 from PTN002683271. The matching IBD and bounded target-path NOT check support transfer; public source locators and scope are given in the source guide. |
| GO:0005634 nucleus | IDA PMID:23093934 Identification of FAM111A as an SV40 host range restriction ... | ACCEPT | Summary: Endogenous human FAM111A is present in the nuclear fraction. Reason: PMID:23093934 Figure 6a and Western blots Methods identify endogenous FAM111A in nuclear and cytoplasmic fractions of human U-2-OS cells, with lamin and tubulin as compartment markers. The positive nuclear observation is consistent with later human replication-site imaging and infection studies. Retain nucleus at the gene level without treating the concurrent cytoplasmic pool as a contradiction or assigning all subnuclear structures. |
| GO:0005634 nucleus | IDA PMID:24561620 Nascent chromatin capture proteomics determines chromatin dy... | ACCEPT | Summary: Human FAM111A localizes to nuclear replication-associated chromatin. Reason: PMID:24561620 Figures 6–7 show tagged FAM111A in human U-2-OS nuclei, with Triton-resistant foci and PCNA colocalization; the PIP mutant redistributes within the nucleus. Nascent-chromatin capture and human TIG-3/U-2-OS perturbation assays independently place its role in the nuclear DNA-replication context. Broad nucleus is valid alongside the more specific chromatin observation, without claiming exclusively nuclear residence in other cellular states. |
| GO:0005634 nucleus | IEA GO_REF:0000044 | ACCEPT | Summary: The curated nuclear-location mapping is experimentally corroborated. Reason: The actual UniProt SL-0191 record maps nucleus to GO:0005634, and the human FAM111A SUBCELLULAR LOCATION entry cites the same target studies. Original human fractionation, nuclear imaging and PCNA-associated foci support the mapping. Retain broad nucleus alongside chromatin because nucleolar and other nuclear pools were also observed; neither infection-dependent export nor cytoplasmic fractions erase the positive nuclear evidence. |
| GO:0005694 chromosome | EXP PMID:24561620 Nascent chromatin capture proteomics determines chromatin dy... | MODIFY | Summary: The demonstrated chromosome-associated pool is nascent chromatin. Reason: PMID:24561620 Figures 6–7 and Nascent Chromatin Capture Methods directly resolve FAM111A association with newly replicated chromatin and replication foci. GO:0000785 chromatin is a current part_of descendant of chromosome GO:0005694 and matches this experimental resolution. Refine the broad location to chromatin without claiming a constitutive chromosome scaffold, a mitotic chromosome-specific assay or direct binding to every chromatin component. Proposed replacements: chromatin Supporting Evidence: |
| GO:0005694 chromosome | IEA GO_REF:0000044 | MODIFY | Summary: The mapped chromosome association is more precisely supported as chromatin association. Reason: UniProt SL-0468 genuinely maps to chromosome; the mapping is not a source error. The underlying human study, PMID:24561620, specifically captures newly replicated chromatin, confirms FAM111A in the captured material by immunoblot, and shows extraction-resistant PCNA-associated foci. Chromatin is the DNA-protein complex forming chromosomes and is part_of chromosome in the current ontology. Refine to that assayed component rather than imply a separate mitotic-chromosome or kinetochore localization. The native capture and tagged imaging are distinct evidence streams. Proposed replacements: chromatin |
| GO:0005737 cytoplasm | EXP PMID:37607234 Human FAM111A inhibits vaccinia virus replication by degradi... | KEEP AS NON CORE | Summary: FAM111A gains a cytoplasmic pool during the tested poxvirus infection. Reason: PMID:37607234 Figure 3 uses human A549 imaging and endogenous fractionation to show MVA-dependent nuclear-to-cytoplasmic redistribution, with a catalytic-mutant comparison. This is a positive, context-dependent localization, retained as non-core relative to replication-associated nuclear work. Figures 6–7 further distinguish protease-dependent export from subsequent I3 degradation: an inactive FAM111A construct forced into the cytoplasm can promote I3 loss, with autophagy-pathway evidence. Cytoplasmic residence therefore does not by itself establish direct proteolytic cleavage of I3, nor universal export in all cells or infections. Supporting Evidence: |
| GO:0005737 cytoplasm | IDA PMID:23093934 Identification of FAM111A as an SV40 host range restriction ... | KEEP AS NON CORE | Summary: Human U-2-OS fractionation detects a cytoplasmic FAM111A pool. Reason: PMID:23093934 Figure 6a and Western blots Methods directly report FAM111A in both cytoplasmic and nuclear human U-2-OS fractions, with tubulin and lamin controls. Retain the positive cytoplasmic observation as non-core; there is no basis to discard it as contamination or to infer a specific organelle from this fraction. The infection-dependent cytoplasmic pool in PMID:37607234 is independent corroboration in a different state, not a substitute for this original assay. |
| GO:0005737 cytoplasm | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: A positive cytoplasmic pool is retained as contextual localization. Reason: The official SL-0086 mapping supports the imported cytoplasm term. PMID:23093934 detects endogenous FAM111A in both nuclear and cytoplasmic U-2 OS fractions with tubulin/lamin controls. PMID:37607234 additionally reports nuclear-to-cytoplasmic redistribution during MVA infection, using transfected human A549 imaging and separate endogenous fractionation. Thus cytoplasm is not dismissed as an artifact or restricted solely to infection. It is retained as a contextual pool alongside the predominant nuclear replication/repair role. In the poxvirus work, cytoplasmic FAM111A can promote I3 loss through cellular autophagy even when its protease is inactive; this is not direct FAM111A cleavage of I3. |
| GO:0006260 DNA replication | IMP PMID:24561620 Nascent chromatin capture proteomics determines chromatin dy... | ACCEPT | Summary: FAM111A contributes to nuclear DNA replication in the tested human contexts. Reason: PMID:24561620 Figure 7 shows reduced EdU incorporation, altered PCNA loading and delayed S-phase entry after FAM111A depletion in human TIG-3 and U-2-OS cells, with two siRNAs and PIP-dependent localization/binding evidence. The gene-level participation is strengthened by PMID:32165630 DNA-combing rescue experiments, which require intact FAM111A catalytic, DNA-binding and PCNA-interaction regions to traverse protein obstacles. This supports a replication-associated contribution rather than DNA polymerase activity or universal requirement for basal fork speed: in the later HAP1 knockout study, untreated fork speed and chromatin PCNA were largely unchanged. Retain the broader source-specific annotation because the 2014 bulk DNA-synthesis and S-phase-entry experiments were not restricted to restarting drug-stalled forks. The later obstacle-specific fork-processing experiments therefore do not exhaust this independently supported replication context. GO places fork processing within DNA replication through part_of ancestry; this does not make the two experimental claims identical, and the broad term is not duplicated as an additional core process. |
| GO:0006508 proteolysis | IDA PMID:32165630 FAM111A protects replication forks from protein obstacles vi... | MODIFY | Summary: Refine the directly measured FAM111A cleavage process to protein autoprocessing. Reason: PMID:32165630 Figure 3 shows FAM111A cleavage in human 293T cells, loss of cleavage with S541A, increased cleavage with tested disease mutants, and intermolecular cleavage of inactive tagged FAM111A by an active counterpart. Edman sequencing of an R569H-derived fragment produced in Sf21 maps an internal cut after Phe334; cleavage-site substitutions reduce processing. Thus FAM111A does the cleaving rather than merely serving as a substrate. The directly assayed process is cleavage of FAM111A molecules by FAM111A, so GO:0016540 protein autoprocessing is a supported, more specific replacement for broad proteolysis. Current GO places autoprocessing under proteolysis; the existing specific annotation is retained, and no new process assertion is created. This refinement describes the demonstrated substrate and does not deny possible other proteolytic roles. PMID:32165630 did not obtain measurable activity from its purified preparation or demonstrate direct purified TOP1/PARP1 cleavage. Independent PMID:32776417 Figure 1G and Figure EV1C/D/F reports positive autocleavage of yeast-purified full-length human FLAG-FAM111A, with catalytic D439N and AEBSF controls. This separate positive preparation supports intrinsic activity without changing the earlier paper’s result or establishing purified TOP1/PARP1 cleavage. Proposed replacements: protein autoprocessing |
| GO:0006974 DNA damage response | IDA PMID:32165630 FAM111A protects replication forks from protein obstacles vi... | MODIFY | Summary: The supported damage response comprises protein-obstacle fork processing and DNA–protein cross-link repair. Reason: PMID:32165630 Figures 4–6 connect FAM111A to TOP1 covalent-complex removal and replication through trapped protein obstacles. Human knockout/rescue experiments require the catalytic serine, PIP box and DNA-binding region; PARP1 knockout distinguishes trapping from loss of PARP catalytic signaling. Increased phospho-Chk1 and gamma-H2AX are downstream readouts, not evidence that FAM111A itself is a checkpoint kinase or general damage sensor. Refine the broad response to the two already independently supported processes. TOP1 covalent complexes and usually noncovalent PARP traps remain distinct; no purified lesion-cleavage reconstitution is claimed. Proposed replacements: replication fork processing protein-DNA covalent cross-linking repair Supporting Evidence: |
| GO:0008236 serine-type peptidase activity | IBA GO_REF:0000033 | MODIFY | Summary: Internal cleavage supports serine-type endopeptidase specificity. Reason: The actual peptidase IBD is grounded in human FAM111B/Q6SJ93, whose current IDA cites PMID:32776417. That complete original also directly assays full-length human FAM111A: yeast-purified FLAG protein forms autocleavage products at 37 degrees C, with catalytic-mutant and AEBSF controls. Independently, PMID:32165630 maps an internal cut after Phe334 in Sf21-produced R569H material and shows catalytic-serine-dependent cleavage and intermolecular processing in human cells. Together these support the internal-bond specificity of GO:0004252, a child of GO:0008236. The earlier paper failed to reproduce purified activity under its own conditions; the later positive preparation does not establish purified TOP1/PARP1 cleavage or universal activity of all constructs. The real IBD is now linked to the exact human target by the subsequently recovered PANTHER version 19 tree and matching 611-residue MSA. Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN002683271 · PTN002683271 SUPPORTS TRANSFER The matching broad peptidase IBD cites Q6SJ93. Actual PMID:32776417 validates donor FAM111B and target FAM111A autocleavage with catalytic controls; target internal-bond specificity additionally comes from PMID:32165630. No failure of the ancestral broad activity is inferred. The version 19 PTHR14389 tree verifies descent of the sequence-matched human leaf PTN002494620 from PTN002683271. The matching IBD and bounded target-path NOT check support transfer; public source locators and scope are given in the source guide. Proposed replacements: serine-type endopeptidase activity |
| GO:0016540 protein autoprocessing | IDA PMID:32165630 FAM111A protects replication forks from protein obstacles vi... | ACCEPT | Summary: FAM111A can process FAM111A molecules through protease-dependent cleavage. Reason: PMID:32165630 Figure 3 and Peptide sequencing Methods provide active-site and cleavage-site controls, an internal Phe334 cut mapped in Sf21-produced R569H material, and trans-cleavage of an inactive tagged molecule by its active counterpart in human 293T cells. These observations support protein autoprocessing, which need not be restricted to an intramolecular event. They do not establish that autocleavage is obligatory physiological maturation, that the fragments are alternative splice products, or that every disease allele behaves identically. PMID:32165630’s negative purified in-vitro activity result remains specific to that preparation. Independent PMID:32776417 Figure 1G and Figure EV1C/D/F reports positive autocleavage of yeast-purified full-length human FLAG-FAM111A, with catalytic D439N and AEBSF controls. This separate positive preparation supports intrinsic activity without changing the earlier paper’s result or establishing purified TOP1/PARP1 cleavage. |
| GO:0031297 replication fork processing | IBA GO_REF:0000033 | ACCEPT | Summary: FAM111A supports fork progression through protein obstacles. Reason: The matching PTN002683271 IBD uses target Q96PZ2 experimental evidence. In PMID:32165630, sequential CldU/IdU DNA-combing assays in human HAP1 cells show that FAM111A loss impairs fork progression under camptothecin or niraparib, rescued by WT but not catalytic-serine, PIP-box or DNA-binding mutants. This supports processing of protein-obstructed forks rather than merely a generic survival effect. Untreated fork speed was largely unchanged, and the hydroxyurea experiments did not reveal a general fork-stability defect. Trapped PARP complexes need not be covalent lesions. Accept this bounded process; the subsequently recovered PANTHER version 19 tree now verifies descent of the exact human target from the asserted node. Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN002683271 · PTN002683271 SUPPORTS TRANSFER The actual fork-processing IBD cites the target itself, with independently read human DNA-combing and rescue experiments. Self-WITH is valid descendant evidence, not circularity. The version 19 PTHR14389 tree verifies descent of the sequence-matched human leaf PTN002494620 from PTN002683271. The matching IBD and bounded target-path NOT check support transfer; public source locators and scope are given in the source guide. |
| GO:0031297 replication fork processing | IDA PMID:32165630 FAM111A protects replication forks from protein obstacles vi... | ACCEPT | Summary: FAM111A helps forks traverse DNA-bound protein obstacles. Reason: PMID:32165630 Figure 5 and DNA combing Methods directly measure sequentially labeled replication tracks in human HAP1 knockout and rescue cells during niraparib or camptothecin exposure. WT FAM111A restores fork progression, whereas catalytic S541A, PIP-box YFAA or DNA-binding F231A mutants do not. This supports replication fork processing at protein obstacles, not an assertion that FAM111A catalyzes DNA synthesis or all forms of fork restart. Untreated fork speed is largely unaffected, and the hydroxyurea experiment does not show increased fork collapse after FAM111A loss. Supporting Evidence: |
| GO:0045071 negative regulation of viral genome replication | IMP PMID:23093934 Identification of FAM111A as an SV40 host range restriction ... | KEEP AS NON CORE | Summary: FAM111A restricts viral replication in defined host and viral contexts. Reason: PMID:23093934 Figures 7–8 show that FAM111A depletion sustains SV40 host-range-mutant viral expression in human U-2-OS cells and restores mutant-virus or adenovirus growth in African green monkey CV-1P cells; the plaque and burst assays are not human-cell assays. Independently, PMID:37607234 Figure 1 measures viral DNA and titers in human A549 depletion/knockout and overexpression experiments, especially for SPI-1-deficient viruses. These positive host-restriction observations support retaining the process as a contextual non-core role. They do not establish broad antiviral efficacy or that the SV40 LT protein is a direct proteolytic substrate. |
| GO:0106300 protein-DNA covalent cross-linking repair | IBA GO_REF:0000033 | ACCEPT | Summary: FAM111A participates in repair of covalent TOP1-DNA complexes. Reason: The actual PTN002683271 IBD cites Q96PZ2. PMID:32165630 measures increased TOP1 covalent-complex foci after FAM111A loss in human cells and rescue by WT but not catalytic-serine or PIP-box mutants. Controlled FAM111A proteolysis and the independent purified autocleavage in PMID:32776417 support a catalytic repair contribution, rather than identifying FAM111A merely as the damaged substrate. A defined purified TOP1-DNA cleavage reaction was not reconstituted, so its exact lesion-processing mechanism remains bounded. PARP trapping is assessed separately and is not automatically called a covalent cross-link. The original target evidence supports the annotation, and the subsequently recovered PANTHER version 19 tree independently verifies target descent from the asserted node. Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN002683271 · PTN002683271 SUPPORTS TRANSFER The matching DPC-repair IBD cites Q96PZ2; TOP1cc accumulation and domain-dependent rescue were independently read. The mechanism is not upgraded to a purified lesion-cleavage assay. The version 19 PTHR14389 tree verifies descent of the sequence-matched human leaf PTN002494620 from PTN002683271. The matching IBD and bounded target-path NOT check support transfer; public source locators and scope are given in the source guide. |
| GO:0106300 protein-DNA covalent cross-linking repair | IDA PMID:32165630 FAM111A protects replication forks from protein obstacles vi... | ACCEPT | Summary: FAM111A contributes to removal of protein–DNA covalent cross-links. Reason: PMID:32165630 Figure 4 and associated Methods show increased TOP1 covalent-complex foci after FAM111A loss in human cells, reduced by WT but not catalytic-serine or PIP-box mutant rescue; Figure 5 links the activity to progression through camptothecin-induced obstacles. This is role-specific evidence for the existing cross-link-repair assertion, supported by controlled cellular proteolysis, rather than necessity evidence alone. The paper did not reconstitute purified FAM111A cleavage of a defined TOP1–DNA substrate, and trapped PARP1 complexes are not automatically classified as covalent lesions. Supporting Evidence: |
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Download this section (compressed HTML)Q: Which endogenous protein obstacles are directly cleaved by FAM111A, and which TOP1cc-repair or PARP-trap phenotypes depend on cooperation with other degradation systems?
Q: How do DNA engagement, PCNA binding and cellular context regulate FAM111A autocleavage and protease activity without causing inappropriate nuclear damage?
Q: Which cytoplasmic factors connect FAM111A to I3 degradation during infection, and how does that route differ from its nuclear replication-associated role?
Experiment: Reconstitute cleavage with purified full-length human FAM111A and defined covalent TOP1–DNA substrates, alongside noncovalent protein–DNA obstacles and FAM111A autocleavage controls. Compare WT with catalytic and DNA-binding mutants, with and without PCNA-loaded DNA and candidate degradation partners; measure peptide products and release of the DNA-linked adduct separately.
Hypothesis: If FAM111A directly attacks a lesion-bound protein, catalytic activity should generate mapped cleavage products in the defined system; a requirement for added partners would distinguish cooperative processing from autonomous turnover.
Experiment: Compare endogenous-level WT, catalytic, PIP-box and F231 mutant replacement in matched human cell backgrounds under untreated, TOP1-trapping and PARP-trapping conditions. Measure fork progression, chromatin PCNA, FAM111A cleavage products and nuclear integrity while controlling protein abundance and cell-cycle state.
Hypothesis: Cell context and protein-obstacle load determine when DNA and PCNA engagement are required, separating the 2014 basal replication phenotype from the 2020 obstacle-dependent phenotype and distinguishing controlled activity from toxicity.
Experiment: In infected human cells, compare endogenous-level WT FAM111A with protease-inactive and nuclear-export-targeted constructs, then perturb candidate autophagy and proteasome components while measuring FAM111A localization, I3 turnover and viral DNA accumulation separately.
Hypothesis: Cytoplasmic delivery and engagement of cellular degradation machinery can be separable from FAM111A peptide-bond cleavage, so I3 loss need not identify I3 as a direct protease substrate.
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