Dimerization-dependent serine protease activity of FAM111A prevents replication fork stalling at topoisomerase 1 cleavage complexes.

FAM111A, a serine protease, plays roles in DNA replication and antiviral defense. Missense mutations in the catalytic domain cause hyper-autocleavage and are associated with genetic disorders with developmental defects. Despite the enzyme's biological significance, the molecular architecture of the FAM111A serine protease domain (SPD) is unknown. Here, we show that FAM111A is a dimerization-dependent protease containing a narrow, recessed active site that cleaves substrates with a chymotrypsin-like specificity. X-ray crystal structures and mutagenesis studies reveal that FAM111A dimerizes via the N-terminal helix within the SPD. This dimerization induces an activation cascade from the dimerization sensor loop to the oxyanion hole through disorder-to-order transitions. Dimerization is essential for proteolytic activity in vitro and for facilitating DNA replication at DNA-protein crosslink obstacles in cells, while it is dispensable for autocleavage. These findings underscore the role of dimerization in FAM111A's function and highlight the distinction in its dimerization dependency between substrate cleavage and autocleavage.

Intra-S phase checkpoint kinase Chk1 dissociates replication proteins Treslin and TopBP1 through multiple mechanisms during replication stress.

Replication stress impedes DNA polymerase progression causing activation of the ataxia telangiectasia and Rad3-related signaling pathway, which promotes the intra-S phase checkpoint activity through phosphorylation of checkpoint kinase 1 (Chk1). Chk1 suppresses replication origin firing, in part, by disrupting the interaction between the preinitiation complex components Treslin and TopBP1, an interaction that is mediated by TopBP1 BRCT domain-binding to two cyclin-dependent kinase (CDK) phosphorylation sites, T968 and S1000, in Treslin. Two nonexclusive models for how Chk1 regulates the Treslin-TopBP1 interaction have been proposed in the literature: in one model, these proteins dissociate due to a Chk1-induced decrease in CDK activity that reduces phosphorylation of the Treslin sites that bind TopBP1 and in the second model, Chk1 directly phosphorylates Treslin, resulting in dissociation of TopBP1. However, these models have not been formally examined. We show here that Treslin T968 phosphorylation was decreased in a Chk1-dependent manner, while Treslin S1000 phosphorylation was unchanged, demonstrating that T968 and S1000 are differentially regulated. However, CDK2-mediated phosphorylation alone did not fully account for Chk1 regulation of the Treslin-TopBP1 interaction. We also identified additional Chk1 phosphorylation sites on Treslin that contributed to disruption of the Treslin-TopBP1 interaction, including S1114. Finally, we showed that both of the proposed mechanisms regulate origin firing in cancer cell line models undergoing replication stress, with the relative roles of each mechanism varying among cell lines. This study demonstrates that Chk1 regulates Treslin through multiple mechanisms to promote efficient dissociation of Treslin and TopBP1 and furthers our understanding of Treslin regulation during the intra-S phase checkpoint.

Functions and evolution of FAM111 serine proteases.

Proteolysis plays fundamental and regulatory roles in diverse cellular processes. The serine protease FAM111A (FAM111 trypsin-like peptidase A) emerged recently as a protease involved in two seemingly distinct processes: DNA replication and antiviral defense. FAM111A localizes to nascent DNA and plays a role at the DNA replication fork. At the fork, FAM111A is hypothesized to promote DNA replication at DNA-protein crosslinks (DPCs) and protein obstacles. On the other hand, FAM111A has also been identified as a host restriction factor for mutants of SV40 and orthopoxviruses. FAM111A also has a paralog, FAM111B, a serine protease with unknown cellular functions. Furthermore, heterozygous missense mutations in FAM111A and FAM111B cause distinct genetic disorders. In this review, we discuss possible models that could explain how FAM111A can function as a protease in both DNA replication and antiviral defense. We also review the consequences of FAM111A and FAM111B mutations and explore possible mechanisms underlying the diseases. Additionally, we propose a possible explanation for what drove the evolution of FAM111 proteins and discuss why some species have two FAM111 proteases. Altogether, studies of FAM111 proteases in DNA repair, antiviral defense, and genetic diseases will help us elucidate their functions and the regulatory mechanisms.

MYC regulates ribosome biogenesis and mitochondrial gene expression programs through its interaction with host cell factor-1.

The oncoprotein transcription factor MYC is a major driver of malignancy and a highly validated but challenging target for the development of anticancer therapies. Novel strategies to inhibit MYC may come from understanding the co-factors it uses to drive pro-tumorigenic gene expression programs, providing their role in MYC activity is understood. Here we interrogate how one MYC co-factor, host cell factor (HCF)-1, contributes to MYC activity in a human Burkitt lymphoma setting. We identify genes connected to mitochondrial function and ribosome biogenesis as direct MYC/HCF-1 targets and demonstrate how modulation of the MYC-HCF-1 interaction influences cell growth, metabolite profiles, global gene expression patterns, and tumor growth in vivo. This work defines HCF-1 as a critical MYC co-factor, places the MYC-HCF-1 interaction in biological context, and highlights HCF-1 as a focal point for development of novel anti-MYC therapies.

Tumours form when cells lose control of their growth. Usually, cells produce signals that control how much and how often they divide. But if these signals become faulty, cells may grow too quickly or multiply too often. For example, a group of proteins known as MYC proteins activate growth genes in a cell, but too much of these proteins causes cells to grow uncontrollably. With one third of all cancer deaths linked to excess MYC proteins, these molecules could be key targets for anti-cancer drugs. However, current treatments fail to target these proteins. One option for treating cancers linked to MYC proteins could be to target proteins that work alongside MYC proteins, such as the protein HCF-1, which can attach to MYC proteins. To test if HCF-1 could be a potential drug target, Popay et al. first studied how HCF-1 and MYC proteins interacted using specific cancer cells grown in the laboratory. This revealed that when the two proteins connected, they activated genes that trigger rapid cell growth. When these cancer cells were then injected into mice, tumours quickly grew. However, when the MYC and HCF-1 attachments in the cancer cells were disrupted, the tumours shrunk. This suggests that if anti-cancer drugs were able to target HCF-1 proteins, they could potentially reduce or even reverse the growth of tumours. While further research is needed to identify drug candidates, these findings reveal a promising target for treating tumours that stem from over-abundant MYC proteins.

DNA-protein crosslinks from environmental exposure: Mechanisms of formation and repair.

Many environmental carcinogens cause DNA damage, which can result in mutations and other alterations in genomic DNA if not repaired promptly. Because of the bulkiness of the lesions, DNA-protein crosslinks (DPCs) are one of the types of toxic DNA damage with potentially deleterious consequences. Despite the importance of DPCs, how cells remove these complex DNA adducts has been incompletely understood. However, major progress in the DPC repair field over the past 5 years now supports the view that cells are equipped with multiple mechanisms to cope with DPCs. Here, we first provide an overview of environmental substances that induce DPCs, describing the sources of exposure and mechanisms of DPC formation. We then review current models of DPC repair and discuss their significance for environmental carcinogens.

FAM111A protects replication forks from protein obstacles via its trypsin-like domain.

Persistent protein obstacles on genomic DNA, such as DNA-protein crosslinks (DPCs) and tight nucleoprotein complexes, can block replication forks. DPCs can be removed by the proteolytic activities of the metalloprotease SPRTN or the proteasome in a replication-coupled manner; however, additional proteolytic mechanisms may exist to cope with the diversity of protein obstacles. Here, we show that FAM111A, a PCNA-interacting protein, plays an important role in mitigating the effect of protein obstacles on replication forks. This function of FAM111A requires an intact trypsin-like protease domain, the PCNA interaction, and the DNA-binding domain that is necessary for protease activity in vivo. FAM111A, but not SPRTN, protects replication forks from stalling at poly(ADP-ribose) polymerase 1 (PARP1)-DNA complexes trapped by PARP inhibitors, thereby promoting cell survival after drug treatment. Altogether, our findings reveal a role of FAM111A in overcoming protein obstacles to replication forks, shedding light on cellular responses to anti-cancer therapies.

Tyrosine Phosphorylation of Mitochondrial Creatine Kinase 1 Enhances a Druggable Tumor Energy Shuttle Pathway.

How mitochondrial metabolism is altered by oncogenic tyrosine kinases to promote tumor growth is incompletely understood. Here, we show that oncogenic HER2 tyrosine kinase signaling induces phosphorylation of mitochondrial creatine kinase 1 (MtCK1) on tyrosine 153 (Y153) in an ABL-dependent manner in breast cancer cells. Y153 phosphorylation, which is commonly upregulated in HER2+ breast cancers, stabilizes MtCK1 to increase the phosphocreatine energy shuttle and promote proliferation. Inhibition of the phosphocreatine energy shuttle by MtCK1 knockdown or with the creatine analog cyclocreatine decreases proliferation of trastuzumab-sensitive and -resistant HER2+ cell lines in culture and in xenografts. Finally, we show that cyclocreatine in combination with the HER2 kinase inhibitor lapatinib reduces the growth of a trastuzumab-resistant HER2+ patient-derived xenograft. These findings suggest that activation of the phosphocreatine energy shuttle by MtCK1 Y153 phosphorylation creates a druggable metabolic vulnerability in cancer.

Spartan deficiency causes accumulation of Topoisomerase 1 cleavage complexes and tumorigenesis.

Germline mutations in SPRTN cause Ruijs-Aalfs syndrome (RJALS), a disorder characterized by genome instability, progeria and early onset hepatocellular carcinoma. Spartan, the protein encoded by SPRTN, is a nuclear metalloprotease that is involved in the repair of DNA-protein crosslinks (DPCs). Although Sprtn hypomorphic mice recapitulate key progeroid phenotypes of RJALS, whether this model expressing low amounts of Spartan is prone to DPC repair defects and spontaneous tumors is unknown. Here, we showed that the livers of Sprtn hypomorphic mice accumulate DPCs containing Topoisomerase 1 covalently linked to DNA. Furthermore, these mice exhibited DNA damage, aneuploidy and spontaneous tumorigenesis in the liver. Collectively, these findings provide evidence that partial loss of Spartan impairs DPC repair and tumor suppression.

BRCA1-associated protein 1 (BAP1) deubiquitinase antagonizes the ubiquitin-mediated activation of FoxK2 target genes.

BRCA1-associated protein 1 (BAP1), which is frequently mutated in cancer, functions as a deubiquitinase (DUB) for histone H2A. Although BAP1 interacts with a transcriptional regulator, HCF-1, and transcription factors FoxK1 and FoxK2, how BAP1 controls gene expression remains unclear. This study investigates the importance of BAP1 DUB activity and the interactions with FoxK2 and HCF-1 in the regulation of FoxK2 target genes. We show that FoxK2 recruits BAP1 to the target genes through the forkhead-associated domain, which interacts with Thr(P)-493 on BAP1. BAP1, in turn, recruits HCF-1, thereby forming a ternary complex in which BAP1 bridges FoxK2 and HCF-1. BAP1 represses FoxK2 target genes, and this effect requires BAP1 DUB activity but not interaction with HCF-1. Importantly, BAP1 depletion causes up-regulation of FoxK2 target genes only in the presence of the Ring1B-Bmi1 complex, an E3 ubiquitin ligase for histone H2A, indicating an antagonizing role of BAP1 against Ring1B-Bmi1. Our findings suggest that BAP1 deficiency causes increased expression of target genes in a Ring1B-Bmi1-dependent manner.

Spartan deficiency causes genomic instability and progeroid phenotypes.

Spartan (also known as DVC1 and C1orf124) is a PCNA-interacting protein implicated in translesion synthesis, a DNA damage tolerance process that allows the DNA replication machinery to replicate past nucleotide lesions. However, the physiological relevance of Spartan has not been established. Here we report that Spartan insufficiency in mice causes chromosomal instability, cellular senescence and early onset of age-related phenotypes. Whereas complete loss of Spartan causes early embryonic lethality, hypomorphic mice with low amounts of Spartan are viable. These mice are growth retarded and develop cataracts, lordokyphosis and cachexia at a young age. Cre-mediated depletion of Spartan from conditional knockout mouse embryonic fibroblasts results in impaired lesion bypass, incomplete DNA replication, formation of micronuclei and chromatin bridges and eventually cell death. These data demonstrate that Spartan plays a key role in maintaining structural and numerical chromosome integrity and suggest a link between Spartan insufficiency and progeria.

Regulation of error-prone translesion synthesis by Spartan/C1orf124.

Translesion synthesis (TLS) employs low fidelity polymerases to replicate past damaged DNA in a potentially error-prone process. Regulatory mechanisms that prevent TLS-associated mutagenesis are unknown; however, our recent studies suggest that the PCNA-binding protein Spartan plays a role in suppression of damage-induced mutagenesis. Here, we show that Spartan negatively regulates error-prone TLS that is dependent on POLD3, the accessory subunit of the replicative DNA polymerase Pol δ. We demonstrate that the putative zinc metalloprotease domain SprT in Spartan directly interacts with POLD3 and contributes to suppression of damage-induced mutagenesis. Depletion of Spartan induces complex formation of POLD3 with Rev1 and the error-prone TLS polymerase Pol ζ, and elevates mutagenesis that relies on POLD3, Rev1 and Pol ζ. These results suggest that Spartan negatively regulates POLD3 function in Rev1/Pol ζ-dependent TLS, revealing a previously unrecognized regulatory step in error-prone TLS.

Spartan/C1orf124 is important to prevent UV-induced mutagenesis.

Uninterrupted replication across damaged DNA is critical to prevent replication fork collapse and resulting double-strand DNA breaks. Rad18-mediated PCNA ubiquitination is a crucial event that triggers a number of downstream pathways important for lesion bypass. Here, we report characterization of Spartan, an evolutionarily conserved protein containing a PCNA-interacting peptide motif, called a PIP box, and a UBZ4 ubiquitin-binding domain. Spartan is a nuclear protein and forms DNA damage-induced foci that colocalize with markers for stalled DNA replication. Focus formation of Spartan requires its PIP-box and the UBZ4 domain and is dependent on Rad18 and the PCNA ubiquitination site, indicating that Spartan is recruited to ubiquitinated PCNA. Spartan depletion results in increased mutagenesis during replication of UV-damaged DNA. Taken together, our data suggest that Spartan is recruited to sites of stalled replication via ubiquitinated PCNA and plays an important role to prevent mutations associated with replication of damaged DNA.

Human KIAA1018/FAN1 localizes to stalled replication forks via its ubiquitin-binding domain.

Genome maintenance pathways correct aberrations in DNA that would be deleterious to the organism. A crucial element of many genome maintenance processes is the ability to degrade DNA that either contains errors or obscures useful substrates for recombination and/or repair by means of nucleases. We have examined a putative nuclease that has heretofore been unreported, KIAA1018/FAN1. This protein contains a predicted ubiquitin-binding zinc finger domain (UBZ) near its N-terminus and an endonuclease-like fold near its C-terminus. Here we describe that FAN1 is a nuclear protein and forms DNA-damage-induced foci, which appear to be at stalled replication forks as denoted by RPA colocalization. Localization of FAN1 to sites of damage is dependent upon its UBZ domain. In addition, knockdown of FAN1 by RNA interference leads to increased sensitivity to interstrand crosslinking agents and accumulation of abnormal chromosomes. FAN1 may be an important new player in the maintenance of genome stability.

The deubiquitinating enzyme BAP1 regulates cell growth via interaction with HCF-1.

The deubiquitinating enzyme BRCA1-associated protein 1 (BAP1) possesses growth inhibitory activity and functions as a tumor suppressor. In this study we report that BAP1 also plays positive roles in cell proliferation. BAP1 depletion by RNAi inhibits cell proliferation as does overexpression of a dominant negative mutant of BAP1. Mass spectrometry analyses of copurified proteins revealed that BAP1 is associated with factors involved in chromatin modulation and transcriptional regulation. We show that the interaction with host cell factor-1 (HCF-1), a cell-cycle regulator composed of HCF-1N and HCF-1C, is critical for the BAP1-mediated growth regulation. We found that HCF-1N is modified with Lys-48-linked polyubiquitin chains on its Kelch domain. The HCF-1 binding motif of BAP1 is required for interaction with HCF-1N and mediates deubiquitination of HCF-1N by BAP1. The importance of the BAP1-HCF-1 interaction is underscored by the fact that growth suppression by the dominant negative BAP1 mutant is entirely dependent on the HCF-1 binding motif. These results suggest that BAP1 regulates cell proliferation by deubiquitinating HCF-1.

UBE2T, the Fanconi anemia core complex, and FANCD2 are recruited independently to chromatin: a basis for the regulation of FANCD2 monoubiquitination.

The Fanconi anemia (FA) nuclear core complex and the E2 ubiquitin-conjugating enzyme UBE2T are required for the S phase and DNA damage-restricted monoubiquitination of FANCD2. This constitutes a key step in the FA tumor suppressor pathway, and much attention has been focused on the regulation at this point. Here, we address the importance of the assembly of the FA core complex and the subcellular localization of UBE2T in the regulation of FANCD2 monoubiquitination. We establish three points. First, the stable assembly of the FA core complex can be dissociated of its ability to function as an E3 ubiquitin ligase. Second, the actual E3 ligase activity is not determined by the assembly of the FA core complex but rather by its DNA damage-induced localization to chromatin. Finally, UBE2T and FANCD2 access this subcellular fraction independently of the FA core complex. FANCD2 monoubiquitination is therefore not regulated by multiprotein complex assembly but by the formation of an active E2/E3 holoenzyme on chromatin.

APC/C--the master controller of origin licensing?

DNA replication must be tightly controlled to prevent initiation of a second round of replication until mitosis is complete. So far, components of the pre-replicative complex (Cdt1, Cdc6 and geminin) were considered key players in this regulation. In a new study, Machida and Dutta have shown that depletion of Emi1 caused cells to replicate their DNA more than once per cell cycle 1. This effect was dependent on the ability of Emi1 to inhibit the APC/C. In addition to its role in regulating entry into mitosis, oscillation of APC/C activity regulates pre-RC formation: high APC/C activity in late M/G1 allows pre-RC formation and low APC/C activity in S/G2 prevents pre-RC formation for a second time thereby preventing rereplication. Each redundant pathway to prevent rereplication is dependent on regulating one of the pre-RC components, and all of the pathways are co-regulated by Emi1 through the APC/C. In this commentary we discuss how this new role of Emi1 adds to our understanding of the regulation of replication initiation. We also review the literature to analyze whether APC/C has a role in regulating endoreduplication (a normal state of polyploidy in some differentiated cells). Similarly a role of premature APC/C activation in genomic instability of tumors is discussed.

The APC/C inhibitor, Emi1, is essential for prevention of rereplication.

Emi1 (early mitotic inhibitor) inhibits APC/C (anaphase-promoting complex/cyclosome) activity during S and G2 phases, and is believed to be required for proper mitotic entry. We report that Emi1 plays an essential function in cell proliferation by preventing rereplication. Rereplication seen after Emi1 depletion is due to premature activation of APC/C that results in destabilization of geminin and cyclin A, two proteins shown here to play redundant roles in preventing rereplication in mammalian cells. Geminin is known to inhibit the replication initiation factor Cdt1. The rereplication block by cyclin A is mediated through its association with S and G2/M cyclin-dependent kinases (Cdks), Cdk2 and Cdk1, suggesting that phosphorylation of proteins by cyclin A-Cdk is responsible for the block. Rereplication upon Emi1 depletion activates the DNA damage checkpoint pathways. These data suggest that Emi1 plays a critical role in preserving genome integrity by blocking rereplication, revealing a previously unrecognized function of this inhibitor of APC/C.

Targeted comparative RNA interference analysis reveals differential requirement of genes essential for cell proliferation.

Differences in the genetic and epigenetic make up of cell lines have been very useful for dissecting the roles of specific genes in the biology of a cell. Targeted comparative RNAi (TARCOR) analysis uses high throughput RNA interference (RNAi) against a targeted gene set and rigorous quantitation of the phenotype to identify genes with a differential requirement for proliferation between cell lines of different genetic backgrounds. To demonstrate the utility of such an analysis, we examined 257 growth-regulated genes in parallel in a breast epithelial cell line, MCF10A, and a prostate cancer cell line, PC3. Depletion of an unexpectedly high number of genes (25%) differentially affected proliferation of the two cell lines. Knockdown of many genes that spare PC3 (p53-) but inhibit MCF10A (p53+) proliferation induces p53 in MCF10A cells. EBNA1BP2, involved in ribosome biogenesis, is an example of such a gene, with its depletion arresting MCF10A at G1/S in a p53-dependent manner. TARCOR is thus useful for identifying cell type-specific genes and pathways involved in proliferation and also for exploring the heterogeneity of cell lines. In particular, our data emphasize the importance of considering the genetic status, when performing siRNA screens in mammalian cells.

UBE2T is the E2 in the Fanconi anemia pathway and undergoes negative autoregulation.

The Fanconi anemia pathway is required for the efficient repair of damaged DNA. A key step in this pathway is the monoubiquitination of the FANCD2 protein by the ubiquitin ligase (E3) composed of Fanconi anemia core complex proteins. Here, we show that UBE2T is the ubiquitin-conjugating enzyme (E2) essential for this pathway. UBE2T binds to FANCL, the ubiquitin ligase subunit of the Fanconi anemia core complex, and is required for the monoubiquitination of FANCD2 in vivo. DNA damage in UBE2T-depleted cells leads to the formation of abnormal chromosomes that are a hallmark of Fanconi anemia. In addition, we show that UBE2T undergoes automonoubiquitination in vivo. This monoubiquitination is stimulated by the presence of the FANCL protein and inactivates UBE2T. Therefore, UBE2T is the E2 in the Fanconi anemia pathway and has a self-inactivation mechanism that could be important for negative regulation of the Fanconi anemia pathway.