RFC3

RFC3 encodes a subunit of the heteropentameric Replication Factor C (RF-C) clamp loader, an essential complex required for processive DNA replication by DNA polymerases delta and epsilon (PMID:8302859). The purified protein displays ATPase activity strongly stimulated by single-stranded DNA, and its conserved Walker A lysine (K59) is essential for ATP hydrolysis, DNA binding, and loading of the PCNA clamp onto DNA (PMID:8302859, PMID:11432854). This replication role is genetically essential and conserved across species: the S. pombe ortholog is required for DNA replication and is functionally complemented by both the S. cerevisiae and human RFC3 (hRFC36) proteins (PMID:10794172). Consistent with its replication function, RFC3 is required for cell proliferation and S-phase progression, with knockdown arresting cells in S phase and altering expression of cell cycle regulators p21, p53, p57, and cyclin A across multiple cancer contexts (PMID:22328562, PMID:26397132, PMID:26464638). RFC3 is transcriptionally activated by the YAP1/TEAD axis, which binds its promoter to drive tumor growth and metastasis (PMID:38383698), and it physically interacts with KIF14 to promote cancer cell proliferation, migration, and invasion (PMID:38590579). Independently of replication, RFC3 acts as a host antiviral restriction factor downstream of an IRF2–FAM111A axis, limiting replication of orthopoxvirus and Zika virus (PMID:28320935, PMID:34930359).

1. 1994 High

   Established RFC3 as the 40-kDa subunit of the essential RF-C clamp loader and showed it is an intrinsic ATPase, answering what biochemical activity it contributes to replication.

   Evidence Molecular cloning, gene disruption, purification to homogeneity, and in vitro ATPase assays with defined nucleic acid substrates in yeast

   PMID:8302859

   Open questions at the time

   • Did not resolve how the subunit assembles within the pentameric complex
   • Did not define structural basis of ssDNA-stimulated ATPase
2. 2000 High

   Demonstrated cross-species functional conservation, showing the replication role of RFC3 is preserved from yeast to human.

   Evidence S. pombe gene disruption and heterologous complementation with S. cerevisiae Rfc3 and human hRFC36

   PMID:10794172

   Open questions at the time

   • Did not characterize human RFC3 biochemistry directly
   • Did not address non-replication functions
3. 2001 High

   Pinpointed the conserved Walker A lysine (K59) as the active-site residue coupling ATP binding to DNA recognition and PCNA loading, defining the mechanism of RFC3's contribution to clamp loading.

   Evidence Site-directed mutagenesis (K59E, K59R) combined with reconstituted ATPase, DNA binding, clamp loading, and PCNA interaction assays

   PMID:11432854

   Open questions at the time

   • Did not resolve subunit-specific roles within the assembled clamp loader
   • Performed in yeast system
4. 2015 Medium

   Linked RFC3 to S-phase progression and proliferation in cancer cells, showing its loss arrests the cell cycle and perturbs cell cycle regulators.

   Evidence Lentiviral shRNA knockdown with flow cytometry and western blot of p21/p53/p57/cyclin A in hepatocellular and ovarian carcinoma cells

   PMID:22328562 PMID:26397132 PMID:26464638

   Open questions at the time

   • Cell cycle protein changes are correlative, not mechanistically dissected
   • Did not establish whether effects are downstream of replication clamp loading or a separate role
5. 2017 Medium

   Revealed an unexpected antiviral role, placing the RFC complex including RFC3 as a host restriction factor against poxvirus downstream of IRF2–FAM111A.

   Evidence Genome-wide siRNA screen with secondary validation, viral replication/spread assays, and expression profiling

   PMID:28320935

   Open questions at the time

   • Molecular mechanism of restriction not defined
   • Relationship between clamp loading and antiviral activity unresolved
6. 2021 Medium

   Extended the IRF2–FAM111A–RFC3 restriction axis to Zika virus, generalizing RFC3's antiviral function.

   Evidence siRNA epistasis of IRF2/FAM111A/RFC3 with viral replication readouts across A549, 2FTGH, and U5A cells

   PMID:34930359

   Open questions at the time

   • How RFC3 biochemically restricts viral replication is unknown
   • Direct interaction with FAM111A not demonstrated
7. 2024 Medium

   Identified KIF14 as a physical partner and downstream effector, and YAP1/TEAD as a transcriptional activator, situating RFC3 in oncogenic signaling networks.

   Evidence Co-immunoprecipitation and KIF14 overexpression rescue in colorectal cancer; dual luciferase promoter reporter and shRNA knockdown with xenograft assays in gastric cancer

   PMID:38383698 PMID:38590579

   Open questions at the time

   • KIF14 interaction rests on a single Co-IP without reciprocal validation
   • Mechanistic link between KIF14 binding and RFC3 replication function unclear
8. 2025 Low

   Connected RFC3 to chemotherapy resistance via ferroptosis, suggesting modulation of a Wnt/β-catenin/GPX4 axis.

   Evidence siRNA knockdown with oxaliplatin sensitivity, ferroptosis readouts, and immunoblot of pathway proteins in colorectal cancer cells

   PMID:39749364

   Open questions at the time

   • Mechanism inferred from immunoblot expression changes only, without direct pathway reconstitution
   • Causality between RFC3 and GPX4 axis not established

• How RFC3's core clamp-loading biochemistry mechanistically connects to its antiviral restriction and oncogenic signaling roles remains unresolved.
• No structural model of human RFC3 within the assembled clamp loader in the corpus
• Molecular basis of viral restriction undefined
• Unknown whether non-replication functions require ATPase/clamp-loading activity