POLD2 (p50) is the essential B-subunit of human DNA polymerase delta (Polฮด), a B-family replicative polymerase that synthesizes DNA at both the lagging and leading strands and participates in multiple repair-associated DNA synthesis pathways (BER long-patch, NER, MMR, HRR, break-induced replication, TLS). Polฮด is a heterotetramer of the catalytic subunit POLD1 (p125) plus the accessory subunits POLD2 (p50), POLD3 (p66/p68), and POLD4 (p12); POLD2 is non-catalytic but has a central structural/organizational role, bridging POLD1 with POLD3 and stabilizing the complex (Conde et al. 2019; Alli et al. 2024). POLD2 and POLD3 are also shared with translesion polymerase Pol ฮถ4 (REV3-REV7-POLD2-POLD3), providing a structural platform for Pol ฮดโPol ฮถ catalytic-subunit switching at replication-blocking lesions (Lee et al. 2014/Makarova et al. 2012; Wu et al. 2023). POLD2 directly contacts the [4Fe-4S] cluster of the POLD1/REV3 C-terminal domain and is the principal interaction partner for Pol ฮท's FF483-484 motif, linking POLD2 to polymerase switching during damage tolerance (Baldeck et al. 2015). POLD2 is essential in human cells; a homozygous p.Asp293Asn variant destabilizes the Polฮด complex and causes an autosomal-recessive syndrome of replicative stress, neurodevelopmental abnormalities, and combined immunodeficiency (Conde et al. 2019). Its core localization is nuclear (nucleoplasm/replication factories) where it acts as part of Polฮด and Polฮถ4 complexes.
Definition: Although GO:0006273 (lagging strand elongation) exists and could be added to POLD2 annotations, no new term is strictly required; consider IBA propagation of GO:0006273 to POLD2 to capture its lagging-strand-specific contribution as part of Polฮด.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0043625 delta DNA polymerase complex | IBA GO_REF:0000033 | ACCEPT | Summary: Core, well-supported annotation. POLD2 is a canonical accessory subunit of the heterotetrameric Polฮด complex, conserved from yeast to human. ACCEPT as core. Supporting Evidence: PMID:31449058 The mammalian polymerase ฮด complex is a heterotetramer consisting of the catalytic subunit POLD1 and the accessory subunits POLD2, POLD3, and POLD4 file:human/POLD2/POLD2-deep-research-falcon.md Human Polฮด is widely described as a **heterotetramer** composed of **POLD1 (p125)** plus accessory subunits **POLD2 (p50)**, **POLD3 (p66/p68)**, and **POLD4 (p12)**. |
| GO:0006271 DNA strand elongation involved in DNA replication | IBA GO_REF:0000033 | ACCEPT | Summary: Accept as a core process annotation. POLD2 is part of the Polฮด holoenzyme that carries out template-directed DNA strand elongation at the replication fork. As a non-catalytic subunit it contributes to (rather than enables) elongation. Supporting Evidence: PMID:31449058 Polymerase ฮด is essential for eukaryotic genome duplication and synthesizes DNA at both the leading and lagging strands. |
| GO:0003677 DNA binding | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: IEA from InterPro. POLD2 in isolation has not been shown to bind DNA directly; the cocrystal structure of POLD2 with the POLD3 NTD shows "a primarily negatively charged molecular surface without obvious features for DNA binding" (Lee et al. 2014). DNA contacts in Polฮด are dominated by POLD1/POLD3. This is a generic IEA that is at best indirect; mark as over-annotated. Supporting Evidence: PMID:24449906 The cocrystal structure of human PolD2 and the NTD of PolD3 has been determined ( 35 ) and shows a primarily negatively charged molecular surface without obvious features for DNA binding. |
| GO:0005634 nucleus | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: Correct but very general. POLD2 acts in the nucleus as part of Polฮด/Polฮถ4. The more specific nucleoplasm annotations are also present and preferred. |
| GO:0006260 DNA replication | IEA GO_REF:0000120 | ACCEPT | Summary: Core process. POLD2 is part of the essential replicative polymerase Polฮด and is required for normal eukaryotic genome duplication. Supporting Evidence: PMID:31449058 Polymerase ฮด is essential for eukaryotic genome duplication and synthesizes DNA at both the leading and lagging strands. |
| GO:0006281 DNA repair | IEA GO_REF:0000043 | ACCEPT | Summary: Core process. Polฮด participates in BER long-patch synthesis, NER repair synthesis, MMR resynthesis, HRR synthesis, and translesion synthesis via Polฮถ4; POLD2 is required as a structural component of these complexes. Supporting Evidence: PMID:31449058 Beyond DNA replication, the polymerase ฮด complex has emerged as a central element in the safeguarding of genome integrity by controlling processes such as break-induced replication ( 7 ) and homologous recombination (HR) |
| GO:0006974 DNA damage response | IEA GO_REF:0000043 | KEEP AS NON CORE | Summary: Accept as non-core. Polฮด-mediated repair synthesis contributes to the cellular DNA damage response. POLD2 deficiency leads to replicative stress and 53BP1 foci, indicating its loss compromises the DDR. The more direct annotations are DNA repair and replication; "DNA damage response" is broader. Supporting Evidence: PMID:31449058 Patientsโ cells showed impaired cell-cycle progression and replication-associated DNA lesions that were reversible upon overexpression of polymerase ฮด. |
| GO:0043625 delta DNA polymerase complex | IEA GO_REF:0000120 | ACCEPT | Summary: Duplicate of core IBA/IDA-supported delta DNA polymerase complex annotation. |
| GO:0071897 DNA biosynthetic process | IEA GO_REF:0000120 | MARK AS OVER ANNOTATED | Summary: True but too general; more specific terms (DNA replication, DNA repair, lagging strand elongation) are already annotated and capture POLD2's actual contributions. |
| GO:0005515 protein binding | IPI PMID:12403614 Reconstitution and characterization of the human DNA polymer... | MARK AS OVER ANNOTATED | Summary: Non-informative MF. The supporting paper documents reconstitution of the 4-subunit Polฮด holoenzyme with POLD2 as a core subunit, captured by the delta DNA polymerase complex CC annotation. "Protein binding" adds no MF information. Supporting Evidence: PMID:12403614 Reconstitution and characterization of the human DNA polymerase delta four-subunit holoenzyme. |
| GO:0005515 protein binding | IPI PMID:15670210 Human Werner helicase interacting protein 1 (WRNIP1) functio... | MARK AS OVER ANNOTATED | Summary: Non-informative MF. Documents WRNIP1 binding to Polฮด; the functional content (modulation of Polฮด) is captured by the holoenzyme annotation. Supporting Evidence: PMID:15670210 the purified human WRNIP1 complex interacted physically with human DNA polymerase delta (pol delta), stimulating its DNA synthesis activity more than fivefold in the presence or absence of proliferating cell nuclear antigen |
| GO:0005515 protein binding | IPI PMID:16000169 An in vivo analysis of the localisation and interactions of ... | MARK AS OVER ANNOTATED | Summary: Non-informative MF. Documents the POLD2-POLD3 interaction within the Polฮด complex (captured by the CC annotation). Supporting Evidence: PMID:16000169 we have mapped the interaction domains for binding to the p50 polymerase delta subunit and with PCNA to the N-terminus and the C-terminus of p66 |
| GO:0005515 protein binding | IPI PMID:16510448 Functional roles of p12, the fourth subunit of human DNA pol... | MARK AS OVER ANNOTATED | Summary: Non-informative MF. Documents POLD2-POLD4 (p12) interaction; covered by Polฮด complex CC. Supporting Evidence: PMID:16510448 which is tightly associated with the p50 subunit |
| GO:0005515 protein binding | IPI PMID:26030842 A fluorescent bimolecular complementation screen reveals MAF... | MARK AS OVER ANNOTATED | Summary: Non-informative MF from a high-throughput PCNA-interactome screen; no specific function captured. |
| GO:0005515 protein binding | IPI PMID:26496610 A human interactome in three quantitative dimensions organiz... | MARK AS OVER ANNOTATED | Summary: Non-informative MF from a high-throughput interactome study. |
| GO:0005515 protein binding | IPI PMID:28514442 Architecture of the human interactome defines protein commun... | MARK AS OVER ANNOTATED | Summary: Non-informative MF from a global interactome/community-detection study. |
| GO:0005515 protein binding | IPI PMID:31449058 Polymerase ฮด deficiency causes syndromic immunodeficiency wi... | MARK AS OVER ANNOTATED | Summary: Non-informative MF. The Conde et al. study is mechanistically important (defines POLD2-POLD3 interface and Polฮด complex stability) and is captured by core CC/BP annotations; the generic protein binding term should be over-annotated. Supporting Evidence: PMID:31449058 The specific function of the POLD2 subunit has been poorly explored, despite its central structural role in interacting with both POLD1 and POLD3 |
| GO:0005515 protein binding | IPI PMID:33961781 Dual proteome-scale networks reveal cell-specific remodeling... | MARK AS OVER ANNOTATED | Summary: Non-informative MF from a high-throughput BioPlex interactome dataset. |
| GO:0005515 protein binding | IPI PMID:38554706 The SPATA5-SPATA5L1 ATPase complex directs replisome proteos... | MARK AS OVER ANNOTATED | Summary: Non-informative MF; SPATA5-SPATA5L1 study describes replisome proteostasis but the POLD2 interaction is not the central finding. |
| GO:0006261 DNA-templated DNA replication | IDA PMID:20334433 The p12 subunit of human polymerase delta modulates the rate... | ACCEPT | Summary: Accept. Direct biochemical evidence using reconstituted four-subunit human Polฮด holoenzyme containing POLD2 catalyzing template-dependent DNA synthesis. Supporting Evidence: PMID:20334433 This study examines the role of the p12 subunit in the function of the human DNA polymerase ฮด (Pol ฮด) holoenzyme by comparing the kinetics of DNA synthesis and degradation catalyzed by the four subunit complex |
| GO:0016035 zeta DNA polymerase complex | IPI PMID:24449906 Human Pol ฮถ purified with accessory subunits is active in tr... | ACCEPT | Summary: Accept. Lee et al. directly demonstrated that purified human Pol ฮถ4 is the four-subunit Rev3-Rev7-POLD2-POLD3 complex. POLD2 is therefore a bona fide subunit of the zeta DNA polymerase (Pol ฮถ4) complex as well as Polฮด. Supporting Evidence: PMID:24449906 The purified four-subunit Pol ฮถ4 (Rev3โRev7โPolD2โPolD3) is much more efficient and more processive at bypassing a 1,2-intrastrand d(GpG)-cisplatin cross-link than the two-subunit Pol ฮถ2 (Rev3โRev7). |
| GO:0042276 error-prone translesion synthesis | IDA PMID:24449906 Human Pol ฮถ purified with accessory subunits is active in tr... | KEEP AS NON CORE | Summary: Accept as non-core. Pol ฮถ4 (containing POLD2) catalyzes extension across cisplatin and other lesions; this is a translesion-synthesis activity that POLD2 contributes to as a Pol ฮถ4 subunit. Not POLD2's primary cellular function but a validated, mechanistically supported role. Supporting Evidence: PMID:24449906 We show that complete bypass of cisplatin lesions requires Pol ฮท to insert dCTP opposite the 3โฒ guanine and Pol ฮถ4 to extend the primers. |
| GO:0043625 delta DNA polymerase complex | IPI PMID:12403614 Reconstitution and characterization of the human DNA polymer... | ACCEPT | Summary: Core IDA-supported localization; same complex as IBA. Accept. Supporting Evidence: PMID:12403614 Reconstitution and characterization of the human DNA polymerase delta four-subunit holoenzyme. |
| GO:0005515 protein binding | IPI PMID:12522211 Identification of a novel protein, PDIP38, that interacts wi... | MARK AS OVER ANNOTATED | Summary: Non-informative MF. Documents PDIP38 interaction with POLD2 (p50); does not convey POLD2's molecular function. Supporting Evidence: PMID:12522211 Identification of a novel protein, PDIP38, that interacts with the p50 subunit of DNA polymerase delta and proliferating cell nuclear antigen. |
| GO:0005654 nucleoplasm | IDA GO_REF:0000052 | ACCEPT | Summary: Accept. Direct immunofluorescence places POLD2 in the nucleoplasm. |
| GO:0043625 delta DNA polymerase complex | IDA PMID:11595739 Mediation of proliferating cell nuclear antigen (PCNA)-depen... | ACCEPT | Summary: Core CC annotation; ACCEPT. Supporting Evidence: PMID:11595739 the third subunit of human DNA polymerase delta, p66, interacts with PCNA through a canonical PCNA-binding sequence located in its C terminus |
| GO:0071897 DNA biosynthetic process | IDA PMID:11595739 Mediation of proliferating cell nuclear antigen (PCNA)-depen... | MARK AS OVER ANNOTATED | Summary: Over-annotated relative to more specific DNA replication and lagging strand elongation annotations. Supporting Evidence: PMID:11595739 Direct participation of p66 in PCNA-dependent DNA replication in vivo is demonstrated by co-localization of p66 with PCNA and DNA polymerase delta within DNA replication foci |
| GO:0016035 zeta DNA polymerase complex | IDA PMID:22465957 DNA polymerase ฮด and ฮถ switch by sharing accessory subunits ... | ACCEPT | Summary: Accept. Makarova et al. demonstrated that POLD2 (p50, B-subunit) binds equally well to the catalytic subunits of either Polฮด (p125) or Polฮถ (REV3), and that Polฮด and Polฮถ switch by sharing accessory subunits including POLD2. Supporting Evidence: PMID:22465957 ( i ) the B-subunit of Pol ฮด binds equally well to the catalytic subunit of either Pol ฮด or Pol ฮถ |
| GO:0043625 delta DNA polymerase complex | IDA PMID:22465957 DNA polymerase ฮด and ฮถ switch by sharing accessory subunits ... | ACCEPT | Summary: Core CC; ACCEPT. Supporting Evidence: PMID:22465957 DNA polymerase ฮด and ฮถ switch by sharing accessory subunits of DNA polymerase ฮด. |
| GO:0005654 nucleoplasm | TAS Reactome:R-HSA-110363 | ACCEPT | Summary: Reactome-derived nucleoplasm localization, consistent with IDA evidence. |
| GO:0005654 nucleoplasm | TAS Reactome:R-HSA-110364 | ACCEPT | Summary: Reactome BER pathway localization; consistent with core nuclear function. |
| GO:0005654 nucleoplasm | TAS Reactome:R-HSA-110368 | ACCEPT | Summary: Reactome long-patch BER strand-displacement synthesis; supports core nuclear repair role. |
| GO:0005654 nucleoplasm | TAS Reactome:R-HSA-110371 | ACCEPT | Summary: Reactome BER ligation step; supports nuclear localization. |
| GO:0005654 nucleoplasm | TAS Reactome:R-HSA-174438 | ACCEPT | Summary: Reactome telomere C-strand flap formation; supports nuclear localization. |
| GO:0005654 nucleoplasm | TAS Reactome:R-HSA-174441 | ACCEPT | Summary: Reactome RNA primer removal/lagging strand; supports nuclear localization. |
| GO:0005654 nucleoplasm | TAS Reactome:R-HSA-174444 | ACCEPT | Summary: Reactome C-strand Okazaki fragment formation; supports nuclear localization. |
| GO:0005654 nucleoplasm | TAS Reactome:R-HSA-174445 | ACCEPT | Summary: Reactome RPA binding to C-strand flap; supports nuclear localization. |
| GO:0005654 nucleoplasm | TAS Reactome:R-HSA-174446 | ACCEPT | Summary: Reactome flap removal at C-strand; supports nuclear localization. |
| GO:0005654 nucleoplasm | TAS Reactome:R-HSA-174448 | ACCEPT | Summary: Reactome telomere processive complex formation; supports nuclear localization. |
| GO:0005654 nucleoplasm | TAS Reactome:R-HSA-174451 | ACCEPT | Summary: Reactome DNA2 recruitment at C-strand; supports nuclear localization. |
| GO:0005654 nucleoplasm | TAS Reactome:R-HSA-174456 | ACCEPT | Summary: Reactome Okazaki fragment joining (C-strand); supports nuclear localization. |
| GO:0005654 nucleoplasm | TAS Reactome:R-HSA-176702 | ACCEPT | Summary: Reactome telomere processive complex dissociation; supports nuclear localization. |
| GO:0005654 nucleoplasm | TAS Reactome:R-HSA-5358579 | ACCEPT | Summary: Reactome Polฮด gap-filling DNA synthesis; supports nuclear localization. |
| GO:0005654 nucleoplasm | TAS Reactome:R-HSA-5651805 | ACCEPT | Summary: Reactome BER LIG1 step; supports nuclear localization. |
| GO:0005654 nucleoplasm | TAS Reactome:R-HSA-5651809 | ACCEPT | Summary: Reactome BER complex dissociation; supports nuclear localization. |
| GO:0005654 nucleoplasm | TAS Reactome:R-HSA-5651992 | ACCEPT | Summary: Reactome PCNA replication complex on damaged dsDNA; supports nuclear localization. |
| GO:0005654 nucleoplasm | TAS Reactome:R-HSA-5652005 | ACCEPT | Summary: Reactome RAD18/CUL4-DDB1-DTL ubiquitin ligase binding to PCNA replication complex; supports nuclear localization. |
| GO:0005654 nucleoplasm | TAS Reactome:R-HSA-5652009 | ACCEPT | Summary: Reactome PCNA monoubiquitination step; supports nuclear localization. |
| GO:0005654 nucleoplasm | TAS Reactome:R-HSA-5653838 | ACCEPT | Summary: Reactome post-TLS Polฮด/ฮต re-binding deISGylated PCNA; supports nuclear localization. |
| GO:0005654 nucleoplasm | TAS Reactome:R-HSA-5653840 | ACCEPT | Summary: Reactome post-TLS completion of replication; supports nuclear localization. |
| GO:0005654 nucleoplasm | TAS Reactome:R-HSA-5690213 | ACCEPT | Summary: Reactome Polฮด/ฮต/ฮบ binding GG-NER site; supports nuclear localization. |
| GO:0005654 nucleoplasm | TAS Reactome:R-HSA-5690988 | ACCEPT | Summary: Reactome ERCC5 3'-incision in GG-NER; supports nuclear localization. |
| GO:0005654 nucleoplasm | TAS Reactome:R-HSA-5690997 | ACCEPT | Summary: Reactome GG-NER repair patch ligation; supports nuclear localization. |
| GO:0005654 nucleoplasm | TAS Reactome:R-HSA-5691001 | ACCEPT | Summary: Reactome NER repair synthesis by Polฮด/ฮต/ฮบ; supports nuclear localization. |
| GO:0005654 nucleoplasm | TAS Reactome:R-HSA-6782208 | ACCEPT | Summary: Reactome TC-NER repair synthesis; supports nuclear localization. |
| GO:0005654 nucleoplasm | TAS Reactome:R-HSA-6782211 | ACCEPT | Summary: Reactome Polฮด/ฮต/ฮบ binding TC-NER site; supports nuclear localization. |
| GO:0005654 nucleoplasm | TAS Reactome:R-HSA-6782224 | ACCEPT | Summary: Reactome ERCC5 3' incision in TC-NER; supports nuclear localization. |
| GO:0005654 nucleoplasm | TAS Reactome:R-HSA-6782227 | ACCEPT | Summary: Reactome TC-NER repair ligation; supports nuclear localization. |
| GO:0005654 nucleoplasm | TAS Reactome:R-HSA-69074 | ACCEPT | Summary: Reactome replication processive complex formation; supports nuclear localization. |
| GO:0005654 nucleoplasm | TAS Reactome:R-HSA-69116 | ACCEPT | Summary: Reactome Okazaki fragment formation; supports nuclear localization. |
| GO:0005654 nucleoplasm | TAS Reactome:R-HSA-69127 | ACCEPT | Summary: Reactome flap intermediate formation; supports nuclear localization. |
| GO:0005654 nucleoplasm | TAS Reactome:R-HSA-69140 | ACCEPT | Summary: Reactome RPA binding to flap; supports nuclear localization. |
| GO:0005654 nucleoplasm | TAS Reactome:R-HSA-69142 | ACCEPT | Summary: Reactome DNA2 recruitment to flap; supports nuclear localization. |
| GO:0005654 nucleoplasm | TAS Reactome:R-HSA-69144 | ACCEPT | Summary: Reactome RNA primer removal/RPA-DNA2 dissociation; supports nuclear localization. |
| GO:0005654 nucleoplasm | TAS Reactome:R-HSA-69152 | ACCEPT | Summary: Reactome remaining flap removal; supports nuclear localization. |
| GO:0005634 nucleus | TAS PMID:8530069 Cloning of the cDNAs for the small subunits of bovine and hu... | KEEP AS NON CORE | Summary: Correct but very general; nucleoplasm is the preferred specific localization. Supporting Evidence: PMID:8530069 cDNAs encoding the small subunit of bovine and human DNA polymerase delta have been cloned and sequenced. The predicted polypeptides, 50,885 and 51,289 Daltons, respectively, are 94% identical |
| GO:0006260 DNA replication | TAS PMID:8530069 Cloning of the cDNAs for the small subunits of bovine and hu... | ACCEPT | Summary: Core process; ACCEPT. Supporting Evidence: PMID:8530069 Cloning of the cDNAs for the small subunits of bovine and human DNA polymerase delta and chromosomal location of the human gene (POLD2). |
| GO:0006273 lagging strand elongation | TAS PMID:31449058 Polymerase ฮด deficiency causes syndromic immunodeficiency wi... | NEW | Summary: NEW annotation reflecting Polฮด's canonical role as the lagging-strand replicative polymerase; POLD2 contributes as a Polฮด subunit. Per PR #753 review feedback, evidence_type changed from IBA to TAS (IBA is reserved for the GO Consortium phylogenetic pipeline and must reference GO_REF:0000033, not a file). Supporting Evidence: PMID:31449058 Polymerase ฮด is essential for eukaryotic genome duplication and synthesizes DNA at both the leading and lagging strands. |
| GO:0019985 translesion synthesis | IDA PMID:24449906 Human Pol ฮถ purified with accessory subunits is active in tr... | NEW | Summary: NEW annotation. POLD2 is a constituent subunit of Pol ฮถ4 which performs translesion DNA synthesis. Captures the parent process of error-prone TLS. Supporting Evidence: PMID:24449906 The purified four-subunit Pol ฮถ4 (Rev3โRev7โPolD2โPolD3) is much more efficient and more processive at bypassing a 1,2-intrastrand d(GpG)-cisplatin cross-link than the two-subunit Pol ฮถ2 (Rev3โRev7). |
| GO:0006284 base-excision repair | TAS Reactome:R-HSA-110368 | NEW | Summary: NEW annotation. POLD2 is part of Polฮด which performs long-patch BER strand displacement synthesis; supported by Reactome BER pathway entries already annotated to POLD2 (CC nucleoplasm) and Thomas et al. 2023 (PMID:36861411) identifying POLD2 as a BER pathway gene. Supporting Evidence: PMID:36861411 Key proteins involved in the BER pathway, including APEX1/2, XRCC1, PARP1, POLD2, have been associated with chemoresistance across many cancer types. |
| GO:0006289 nucleotide-excision repair | TAS Reactome:R-HSA-5691001 | NEW | Summary: NEW annotation. POLD2 is part of Polฮด which performs NER repair synthesis (~27-30 nt patch) in both GG-NER and TC-NER pathways, as captured by multiple Reactome NER entries already linked to POLD2. Supporting Evidence: file:human/POLD2/POLD2-deep-research-falcon.md POLD2 functions in core genome duplication as part of Polฮด and also contributes to DNA repair-associated DNA synthesis. |
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Download this section (compressed HTML)Q: What is the mechanistic basis for the directionality of subunit assembly in Polฮด โ does POLD2 dimerize with POLD3 first and then dock onto POLD1, or does POLD1-POLD2 preassembly precede POLD3 incorporation?
Q: How is the partitioning of the POLD2-POLD3 dimer between Polฮด (with POLD1) and Polฮถ4 (with REV3-REV7) regulated in vivo, and does this stoichiometry change in response to replication stress or DNA damage?
Q: Does the POLD2 [4Fe-4S]-coordinating interface with the POLD1/REV3 C-terminal domain mediate redox-dependent regulation of PolฮดโPolฮถ switching, as proposed by Makarova et al. (PMID:22465957)?
Q: Are there isoform-specific or post-translationally modified pools of POLD2 dedicated to particular repair pathways (e.g., MMR vs. NER) versus bulk replication?
Experiment: Use rapid auxin-inducible degron (AID2) depletion of POLD2 in human cells coupled with iPOND/NCC-MS to define which replisome and repair complexes lose stoichiometry first, distinguishing assembly defects from catalytic loss.
Experiment: Solve the cryo-EM structure of the full human Polฮด4 holoenzyme on a primer-template with PCNA to define the POLD2-POLD1 and POLD2-POLD3 interfaces at near-atomic resolution and rationalize the D293N disease variant.
Experiment: Perform separation-of-function alleles of POLD2 that selectively disrupt the POLD2-POLD3 interface vs. the POLD2-POLD1 interface vs. the POLD2-REV3 interface, and test for distinct replication, repair, and TLS phenotypes (replication stress, mutagenesis, MiDAS, cisplatin sensitivity).
Experiment: Test whether POLD2 directly contacts the [4Fe-4S] cluster of POLD1 in vitro and whether its binding affinity to POLD1 vs. REV3 is modulated by the oxidation state of the cluster.
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