The promoting effect and mechanism of MAD2L2 on stemness maintenance and malignant progression in glioma.

BACKGROUND: Glioblastoma, the most common primary malignant tumor of the brain, is associated with poor prognosis. Glioblastoma cells exhibit high proliferative and invasive properties, and glioblastoma stem cells (GSCs) have been shown to play a crucial role in the malignant behavior of glioblastoma cells. This study aims to investigate the molecular mechanisms involved in GSCs maintenance and malignant progression. METHODS: Bioinformatics analysis was performed based on data from public databases to explore the expression profile of Mitotic arrest deficient 2 like 2 (MAD2L2) and its potential function in glioma. The impact of MAD2L2 on glioblastoma cell behaviors was assessed through cell viability assays (CCK8), colony formation assays, 5-Ethynyl-2'-deoxyuridine (EDU) incorporation assays, scratch assays, and transwell migration/invasion assays. The findings from in vitro experiments were further validated in vivo using xenograft tumor model. GSCs were isolated from the U87 and LN229 cell lines through flow cytometry and the stemness characteristics were verified by immunofluorescence staining. The sphere-forming ability of GSCs was examined using the stem cell sphere formation assay. Bioinformatics methods were conducted to identified the potential downstream target genes of MAD2L2, followed by in vitro experimental validation. Furthermore, potential upstream transcription factors that regulate MAD2L2 expression were confirmed through chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays. RESULTS: The MAD2L2 exhibited high expression in glioblastoma samples and showed significant correlation with patient prognosis. In vitro and in vivo experiments confirmed that silencing of MAD2L2 led to decreased proliferation, invasion, and migration capabilities of glioblastoma cells, while decreasing stemness characteristics of glioblastoma stem cells. Conversely, overexpression of MAD2L2 enhanced these malignant behaviors. Further investigation revealed that MYC proto-oncogene (c-MYC) mediated the functional role of MAD2L2 in glioblastoma, which was further validated through a rescue experiment. Moreover, using dual-luciferase reporter gene assays and ChIP assays determined that the upstream transcription factor E2F-1 regulated the expression of MAD2L2. CONCLUSION: Our study elucidated the role of MAD2L2 in maintaining glioblastoma stemness and promoting malignant behaviors through the regulation of c-MYC, suggesting its potential as a therapeutic target.

Bridging Gaps in HDR Improvement: The Role of MAD2L2, SCAI, and SCR7.

This study aimed to enhance homology-directed repair (HDR) efficiency in CRISPR/Cas-mediated genome editing by targeting three key factors regulating the balance between HDR and non-homologous end joining (NHEJ): MAD2L2, SCAI, and Ligase IV. In order to achieve this, a cellular model using mutated eGFP was designed to monitor HDR events. Results showed that MAD2L2 knockdown and SCR7 treatment significantly improved HDR efficiency during Cas9-mediated HDR repair of the mutated eGFP gene in the HEK293T cell line. Fusion protein Cas9-SCAI did not improve HDR. This study is the first to demonstrate that MAD2L2 knockdown during CRISPR-mediated gene editing in HEK293T cells can increase precise correction by up to 10.2 times. The study also confirmed a moderate but consistent effect of SCR7, an inhibitor of Ligase IV, which increased HDR by 1.7 times. These findings provide valuable insights into improving HDR-based genome editing efficiency.

Role of the WNT/ß-catenin/ZKSCAN3 Pathway in Regulating Chromosomal Instability in Colon Cancer Cell lines and Tissues.

Zinc finger protein with KRAB and SCAN domains 3 (ZKSCAN3) acts as an oncogenic transcription factor in human malignant tumors, including colon and prostate cancer. However, most of the ZKSCAN3-induced carcinogenic mechanisms remain unknown. In this study, we identified ZKSCAN3 as a downstream effector of the oncogenic Wnt/ß-catenin signaling pathway, using RNA sequencing and ChIP analyses. Activation of the Wnt pathway by recombinant Wnt gene family proteins or the GSK inhibitor, CHIR 99021 upregulated ZKSCAN3 expression in a ß-catenin-dependent manner. Furthermore, ZKSCAN3 upregulation suppressed the expression of the mitotic spindle checkpoint protein, Mitotic Arrest Deficient 2 Like 2 (MAD2L2) by inhibiting its promoter activity and eventually inducing chromosomal instability in colon cancer cells. Conversely, deletion or knockdown of ZKSCAN3 increased MAD2L2 expression and delayed cell cycle progression. In addition, ZKSCAN3 upregulation by oncogenic WNT/ß-catenin signaling is an early event of the adenoma-carcinoma sequence in colon cancer development. Specifically, immunohistochemical studies (IHC) were performed using normal (NM), hyperplastic polyps (HPP), adenomas (AD), and adenocarcinomas (AC). Their IHC scores were considerably different (61.4 in NM; 88.4 in HPP; 189.6 in AD; 246.9 in AC). In conclusion, ZKSCAN3 could be responsible for WNT/ß-catenin-induced chromosomal instability in colon cancer cells through the suppression of MAD2L2 expression.

Analysis of Ser/Thr Kinase HASPIN-Interacting Proteins in the Spermatids.

HASPIN is predominantly expressed in spermatids, and plays an important role in cell division in somatic and meiotic cells through histone H3 phosphorylation. The literature published to date has suggested that HASPIN may play multiple roles in cells. Here, 10 gene products from the mouse testis cDNA library that interact with HASPIN were isolated using the two-hybrid system. Among them, CENPJ/CPAP, KPNA6/importin alpha 6, and C1QBP/HABP1 were analyzed in detail for their interactions with HASPIN, with HASPIN phosphorylated C1QBP as the substrate. The results indicated that HASPIN is involved in spermatogenesis through the phosphorylation of C1QBP in spermatids, and also may be involved in the formation of centrosomes.

Genetic Variants in Double-Strand Break Repair Pathway Genes to Predict Platinum-Based Chemotherapy Prognosis in Patients With Lung Cancer.

Objective: The purpose of this study was to investigate the associations of genetic variants in double-strand break (DSB) repair pathway genes with prognosis in patients with lung cancer treated with platinum-based chemotherapy. Methods: Three hundred ninety-nine patients with lung cancer who received platinum-based chemotherapy for at least two cycles were included in this study. A total of 35 single nucleotide polymorphisms (SNPs) in DSB repair, base excision repair (BER), and nucleotide excision repair (NER) repair pathway genes were genotyped, and were used to evaluate the overall survival (OS) and the progression-free survival (PFS) of patients who received platinum-based chemotherapy using Cox proportional hazard models. Results: The PFS of patients who carried the MAD2L2 rs746218 GG genotype was shorter than that in patients with the AG or AA genotypes (recessive model: p = 0.039, OR = 5.31, 95% CI = 1.09-25.93). Patients with the TT or GT genotypes of TNFRSF1A rs4149570 had shorter OS times than those with the GG genotype (dominant model: p = 0.030, OR = 0.57, 95% CI = 0.34-0.95). We also investigated the influence of age, gender, histology, smoking, stage, and metastasis in association between SNPs and OS or PFS in patients with lung cancer. DNA repair gene SNPs were significantly associated with PFS and OS in the subgroup analyses. Conclusion: Our study showed that variants in MAD2L2 rs746218 and TNFRSF1A rs4149570 were associated with shorter PFS or OS in patients with lung cancer who received platinum-based chemotherapy. These variants may be novel biomarkers for the prediction of prognosis of patients with lung cancer who receive platinum-based chemotherapy.

Disrupting the MAD2L2-Rev1 Complex Enhances Cell Death upon DNA Damage.

DNA-damaging chemotherapy agents such as cisplatin have been the first line of treatment for cancer for decades. While chemotherapy can be very effective, its long-term success is often reduced by intrinsic and acquired drug resistance, accompanied by chemotherapy-resistant secondary malignancies. Although the mechanisms causing drug resistance are quite distinct, they are directly connected to mutagenic translesion synthesis (TLS). The TLS pathway promotes DNA damage tolerance by supporting both replication opposite to a lesion and inaccurate single-strand gap filling. Interestingly, inhibiting TLS reduces both cisplatin resistance and secondary tumor formation. Therefore, TLS targeting is a promising strategy for improving chemotherapy. MAD2L2 (i.e., Rev7) is a central protein in TLS. It is an essential component of the TLS polymerase zeta (ζ), and it forms a regulatory complex with Rev1 polymerase. Here we present the discovery of two small molecules, c#2 and c#3, that directly bind both in vitro and in vivo to MAD2L2 and influence its activity. Both molecules sensitize lung cancer cell lines to cisplatin, disrupt the formation of the MAD2L2-Rev1 complex and increase DNA damage, hence underlining their potential as lead compounds for developing novel TLS inhibitors for improving chemotherapy treatments.

Mitotic Arrest-Deficient 2 Like 2 (MAD2L2) Interacts with Escherichia coli Effector Protein EspF.

Enteropathogenic (EPEC) and Enterohemorrhagic (EHEC) Escherichia coli are considered emerging zoonotic pathogens of worldwide distribution. The pathogenicity of the bacteria is conferred by multiple virulence determinants, including the locus of enterocyte effacement (LEE) pathogenicity island, which encodes a type III secretion system (T3SS) and effector proteins, including the multifunctional secreted effector protein (EspF). EspF sequences differ between EPEC and EHEC serotypes in terms of the number and residues of SH3-binding polyproline-rich repeats and N-terminal localization sequence. The aim of this study was to discover additional cellular interactions of EspF that may play important roles in E. coli colonization using the Yeast two-hybrid screening system (Y2H). Y2H screening identified the anaphase-promoting complex inhibitor Mitotic Arrest-Deficient 2 Like 2 (MAD2L2) as a host protein that interacts with EspF. Using LUMIER assays, MAD2L2 was shown to interact with EspF variants from EHEC O157:H7 and O26:H11 as well as EPEC O127:H6. MAD2L2 is targeted by the non-homologous Shigella effector protein invasion plasmid antigen B (IpaB) to halt the cell cycle and limit epithelial cell turnover. Therefore, we postulate that interactions between EspF and MAD2L2 serve a similar function in promoting EPEC and EHEC colonization, since cellular turnover is a key method for bacteria removal from the epithelium. Future work should investigate the biological importance of this interaction that could promote the colonization of EPEC and EHEC E. coli in the host.

A comprehensive molecular study identified 12 complementation groups with 56 novel FANC gene variants in Indian Fanconi anemia subjects.

Fanconi anemia (FA) is a rare autosomal or X-linked genetic disorder characterized by chromosomal breakages, congenital abnormalities, bone marrow failure (BMF), and cancer. There has been a discovery of 22 FANC genes known to be involved in the FA pathway. This wide number of pathway components makes molecular diagnosis challenging for FA. We present here the most comprehensive molecular diagnosis of FA subjects from India. We observed a high frequency (4.42 ± 1.5 breaks/metaphase) of chromosomal breakages in 181 FA subjects. The major clinical abnormalities observed were skin pigmentation (70.2%), short stature (46.4%), and skeletal abnormalities (43.1%), along with a few minor clinical abnormalities. The combination of Sanger sequencing and Next Generation Sequencing could molecularly characterize 164 (90.6%) FA patients and identified 12 different complementation groups [FANCA (56.10%), FANCG (16.46%), FANCL (12.80%), FANCD2 (4.88%), FANCJ (2.44%), FANCE (1.22%), FANCF (1.22%), FANCI (1.22%), FANCN (1.22%), FANCC (1.22%), FANCD1 (0.61%) and FANCB (0.61%)]. A total of 56 novel variants were identified in our cohort, including a hotspot variant: a deletion of exon 27 in the FANCA gene and a nonsense variant at c.787 C>T in the FANCG gene. Our comprehensive molecular findings can aid in the stratification of molecular investigation in the diagnosis and management of FA patients.

Mitotic syndicates Aurora Kinase B (AURKB) and mitotic arrest deficient 2 like 2 (MAD2L2) in cohorts of DNA damage response (DDR) and tumorigenesis.

Aurora Kinase B (AURKB) and Mitotic Arrest Deficient 2 Like 2 (MAD2L2) are emerging anticancer therapeutic targets. AURKB and MAD2L2 are the least well studied members of their protein families, compared to AURKA and MAD2L1. Both AURKB and MAD2L2 play a critical role in mitosis, cell cycle checkpoint, DNA damage response (DDR) and normal physiological processes. However, the oncogenic roles of AURKB and MAD2L2 in tumorigenesis and genomic instability have also been reported. DDR acts as an arbitrator for cell fate by either repairing the damage or directing the cell to self-destruction. While there is strong evidence of interphase DDR, evidence of mitotic DDR is just emerging and remains largely unelucidated. To date, inhibitors of the DDR components show effective anti-cancer roles. Contrarily, long-term resistance towards drugs that target only one DDR target is becoming a challenge. Targeting interactions between protein-protein or protein-DNA holds prominent therapeutic potential. Both AURKB and MAD2L2 play critical roles in the success of mitosis and their emerging roles in mitotic DDR cannot be ignored. Small molecule inhibitors for AURKB are in clinical trials. A few lead compounds towards MAD2L2 inhibition have been discovered. Targeting mitotic DDR components and their interaction is emerging as a potent next generation anti-cancer therapeutic target. This can be done by developing small molecule inhibitors for AURKB and MAD2L2, thereby targeting DDR components as anti-cancer therapeutic targets and/or targeting mitotic DDR. This review focuses on AURKB and MAD2L2 prospective synergy to deregulate the p53 DDR pathway and promote favourable conditions for uncontrolled cell proliferation.

MDA-MB-157 Cell Line Presents High Levels of MAD2L2 and Dysregulated Mitosis.

BACKGROUND/AIM: Accurate regulation of the spindle assembly checkpoint (SAC) and anaphase promoting complex/cyclosome (APC/C) are essential for the correct execution of mitosis. In this work, we focused on MAD2L2 (REV7), a central translesion (TLS) protein, which also functions as a mitotic regulator by inhibiting APC/C in prometaphase. MATERIALS AND METHODS: Using bioinformatics analysis, live cell imaging and APC/C protein binding and degradation assays, we explored the influence of MAD2L2 over-expression in breast cancer. RESULTS: A significant over-expression of MAD2L2 was found in triple negative breast cancers (TNBC), compared to other breast cancers, correlating to poor patient prognosis. We also identified significant over-expression of MAD2L2 in the MDA-MB-157 triple negative (TN) cell line. A high percentage of MDA-MB-157 cells failed to complete mitosis and died during mitosis or shortly after. In addition, these cells completed mitosis at a significantly slower rate than control cells. MDA-MB-157 cells present high levels of mitotic slippage upon nocodazole treatment and acute dysregulation in APC/C function and substrate degradation. Moreover, silencing of MAD2L2 in the MDA-MB-157 cell line improved mitotic phenotypes. CONCLUSION: MAD2L2 over-expression supports the carcinogenic phenotype of MDA-MB-157 cells by promoting uncontrolled mitosis.

Targeting CARM1 in ovarian cancer with EZH2 and PARP inhibitors.

Coactivator-associated arginine methyltransferase 1 (CARM1)-expressing high-grade serous ovarian cancers are characterized by poor prognosis and limited therapeutic options. Here we discuss a novel therapeutic strategy to target CARM1-expressing ovarian cancer based on a combination of poly (ADP-ribose) polymerase (PARP) and enhancer of zeste homology 2 (EZH2) inhibitors.

CDH1 binds MAD2L2 in a Rev1-like pattern.

MAD2L2 (i.e. Rev7) is a central regulatory protein important in several processes, such as translesion synthesis (TLS), DNA damage response and mitosis. In TLS, MAD2L2 binds Rev3 to form Pol zeta (ζ) and promotes formation of the Pol ζ- REV1 complex allowing extension beyond distorted DNA structures. MAD2L2 is also part of the heterotetrameric shieldin complex that regulates DNA repair at sites of damage, where similarly to TLS, it bridges the interaction between SHLD2 and SHLD3. Lastly, during mitosis, MAD2L2 prevents premature activation of the anaphase promoting complex/cyclosome (APC/C), by sequestering its activator, CDH1. MAD2L2 exits in a 'closed' active conformation binding Rev3 and Rev1, or SHLD2 and SHLD3, and an 'open' inactive conformation, with no binding partners. Moreover, Pol ζ- REV1 forms a homodimer using a protein-protein interaction (PPI) domain comprised of a central αC helix, promoting Rev3-MAD2L2 interaction and C-terminus ß-sheets, enabling Rev1-MAD2L2 interaction. While the role of MAD2L2 in TLS is well established, molecular details regarding the CDH1-MAD2L2 interaction and MAD2L2 homodimerization are still missing. Here we demonstrate, in a human cell line, using a series of MAD2L2 mutants, that MAD2L2's C-terminus interface is essential for the CDH1-MAD2L2 binding as well as for homodimerization. In addition, we show that CDH1 interacts with MAD2L2 in a Rev1-like pattern, using the same C-terminus residues on MAD2L2 which Rev1 binds. Thus, identification of CDH1 as an additional Rev1-like binding protein strengthens the versatility of MAD2L2 as a regulatory protein and emphasizes the complexity involved in MAD2L2's preferential complex formation.

EZH2 Inhibition Sensitizes CARM1-High, Homologous Recombination Proficient Ovarian Cancers to PARP Inhibition.

In response to DNA double-strand breaks, MAD2L2-containing shieldin complex plays a critical role in the choice between homologous recombination (HR) and non-homologous end-joining (NHEJ)-mediated repair. Here we show that EZH2 inhibition upregulates MAD2L2 and sensitizes HR-proficient epithelial ovarian cancer (EOC) to poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitor in a CARM1-dependent manner. CARM1 promotes MAD2L2 silencing by driving the switch from the SWI/SNF complex to EZH2 through methylating the BAF155 subunit of the SWI/SNF complex on the MAD2L2 promoter. EZH2 inhibition upregulates MAD2L2 to decrease DNA end resection, which increases NHEJ and chromosomal abnormalities, ultimately causing mitotic catastrophe in PARP inhibitor treated HR-proficient cells. Significantly, EZH2 inhibitor sensitizes CARM1-high, but not CARM-low, EOCs to PARP inhibitors in both orthotopic and patient-derived xenografts.

Structural basis for shieldin complex subunit 3-mediated recruitment of the checkpoint protein REV7 during DNA double-strand break repair.

Shieldin complex subunit 3 (SHLD3) is the apical subunit of a recently-identified shieldin complex and plays a critical role in DNA double-strand break repair. To fulfill its function in DNA repair, SHLD3 interacts with the mitotic spindle assembly checkpoint protein REV7 homolog (REV7), but the details of this interaction remain obscure. Here, we present the crystal structures of REV7 in complex with SHLD3's REV7-binding domain (RBD) at 2.2-2.3 Å resolutions. The structures revealed that the ladle-shaped RBD in SHLD3 uses its N-terminal loop and C-terminal α-helix (αC-helix) in its interaction with REV7. The N-terminal loop exhibited a structure similar to those previously identified in other REV7-binding proteins, and the less-conserved αC-helix region adopted a distinct mode for binding REV7. In vitro and in vivo binding analyses revealed that the N-terminal loop and the αC-helix are both indispensable for high-affinity REV7 binding (with low-nanomolar affinity), underscoring the crucial role of SHLD3 αC-helix in protein binding. Moreover, binding kinetics analyses revealed that the REV7 "safety belt" region, which plays a role in binding other proteins, is essential for SHLD3-REV7 binding, as this region retards the dissociation of the RBD from the bound REV7. Together, the findings of our study reveal the molecular basis of the SHLD3-REV7 interaction and provide critical insights into how SHLD3 recognizes REV7.

REV7 has a dynamic adaptor region to accommodate small GTPase RAN/Shigella IpaB ligands, and its activity is regulated by the RanGTP/GDP switch.

REV7, also termed mitotic arrest-deficient 2-like 2 (MAD2L2 or MAD2B), acts as an interaction module in a broad array of cellular pathways, including translesion DNA synthesis, cell cycle control, and nonhomologous end joining. Numerous REV7 binding partners have been identified, including the human small GTPase Ras-associated nuclear protein (RAN), which acts as a potential upstream regulator of REV7. Notably, the Shigella invasin IpaB hijacks REV7 to disrupt cell cycle control to prevent intestinal epithelial cell renewal and facilitate bacterial colonization. However, the structural details of the REV7-RAN and REV7-IpaB interactions are mostly unknown. Here, using fusion protein and rigid maltose-binding protein tagging strategies, we determined the crystal structures of these two complexes at 2.00-2.35 Å resolutions. The structures revealed that both RAN and IpaB fragments bind the "safety belt" region of REV7, inducing rearrangement of the C-terminal ß-sheet region of REV7, conserved among REV7-related complexes. Of note, the REV7-binding motifs of RAN and IpaB each displayed some unique interactions with REV7 despite sharing consensus residues. Structural alignments revealed that REV7 has an adaptor region within the safety belt region that can rearrange secondary structures to fit a variety of different ligands. Our structural and biochemical results further indicated that REV7 preferentially binds GTP-bound RAN, implying that a GTP/GDP-bound transition of RAN may serve as the molecular switch that controls REV7's activity. These results provide insights into the regulatory mechanism of REV7 in cell cycle control, which may help with the development of small-molecule inhibitors that target REV7 activity.

MAD2L2 inhibits colorectal cancer growth by promoting NCOA3 ubiquitination and degradation.

Nuclear receptor coactivator 3 (NCOA3) is a transcriptional coactivator that has elevated expression in multiple tumor types, including colorectal cancer (CRC). However, the molecular mechanisms that regulate the tumorigenic functions of NCOA3 in CRC remain largely unknown. In this study, we aimed to discover and identify the novel regulatory proteins of NCOA3 and explore their mechanisms of action. Immunoprecipitation (IP) coupled with mass spectrometry (IP-MS) analysis was used to detect, identify, and verify the proteins that interacted with NCOA3 in CRC cells. The biological functions of the candidate proteins and the underlying molecular mechanism were investigated in CRC cells and mouse model in vitro and in vivo. The clinical significance of NCOA3 and its interaction partner protein in CRC patients was also studied. We identified mitotic arrest deficient 2-like protein 2 (MAD2L2, also known as MAD2B or REV7), with two signal peptide sequences of LIPLK and EVYPVGIFQK, to be an interaction partner of NCOA3. Overexpression of MAD2L2 suppressed the proliferation, migration, and clonogenicity of CRC cells by inducing the degradation of NCOA3. The mechanism study showed that increased MAD2L2 expression in CRC cells activated p38, which was required for the phosphorylation of NCOA3 that led to its ubiquitination and degradation by the proteasome. Moreover, we found that MAD2L2 predicted favorable prognosis in CRC patients. We have discovered a novel role of MAD2L2 in the regulation of NCOA3 degradation and proposed that MAD2L2 serves as a tumor suppressor in CRC.

Dynamic feature of mitotic arrest deficient 2-like protein 2 (MAD2L2) and structural basis for its interaction with chromosome alignment-maintaining phosphoprotein (CAMP).

Mitotic arrest deficient 2-like protein 2 (MAD2L2), also termed MAD2B or REV7, is involved in multiple cellular functions including translesion DNA synthesis (TLS), signal transduction, transcription, and mitotic events. MAD2L2 interacts with chromosome alignment-maintaining phosphoprotein (CAMP), a kinetochore-microtubule attachment protein in mitotic cells, presumably through a novel "WK" motif in CAMP. Structures of MAD2L2 in complex with binding regions of the TLS proteins REV3 and REV1 have revealed that MAD2L2 has two faces for protein-protein interactions that are regulated by its C-terminal region; however, the mechanisms underlying the MAD2L2-CAMP interaction and the mitotic role of MAD2L2 remain unknown. Here we have determined the structures of human MAD2L2 in complex with a CAMP fragment in two crystal forms. The overall structure of the MAD2L2-CAMP complex in both crystal forms was essentially similar to that of the MAD2L2-REV3 complex. However, the residue interactions between MAD2L2 and CAMP were strikingly different from those in the MAD2L2-REV3 complex. Furthermore, structure-based interaction analyses revealed an unprecedented mechanism involving CAMP's WK motif. Surprisingly, in one of the crystal forms, the MAD2L2-CAMP complex formed a dimeric structure in which the C-terminal region of MAD2L2 was swapped and adopted an immature structure. The structure provides direct evidence for the dynamic nature of MAD2L2 structure, which in turn may have implications for the protein-protein interaction mechanism and the multiple functions of this protein. This work is the first structural study of MAD2L2 aside from its role in TLS and might pave the way to clarify MAD2L2's function in mitosis.

Human Pol ζ purified with accessory subunits is active in translesion DNA synthesis and complements Pol η in cisplatin bypass.

DNA polymerase ζ (Pol ζ) is a eukaryotic B-family DNA polymerase that specializes in translesion synthesis and is essential for normal embryogenesis. At a minimum, Pol ζ consists of a catalytic subunit Rev3 and an accessory subunit Rev7. Mammalian Rev3 contains >3,000 residues and is twice as large as the yeast homolog. To date, no vertebrate Pol ζ has been purified for biochemical characterization. Here we report purification of a series of human Rev3 deletion constructs expressed in HEK293 cells and identification of a minimally catalytically active human Pol ζ variant. With a tagged form of an active Pol ζ variant, we isolated two additional accessory subunits of human Pol ζ, PolD2 and PolD3. The purified four-subunit Pol ζ4 (Rev3-Rev7-PolD2-PolD3) is much more efficient and more processive at bypassing a 1,2-intrastrand d(GpG)-cisplatin cross-link than the two-subunit Pol ζ2 (Rev3-Rev7). We show that complete bypass of cisplatin lesions requires Pol η to insert dCTP opposite the 3' guanine and Pol ζ4 to extend the primers.