PTIP Regulates 53BP1 and SMC1 at the DNA Damage Sites

Although PTIP is implicated in the DNA damage response, through interactions with 53BP1, the function of PTIP in the DNA damage response remain elusive. Here, we show that RNF8 controls DNA damage-induced nuclear foci formation of PTIP, which in turn regulates 53BP1 localization to the DNA damage sites. In addition, SMC1, a substrate of ATM, could not be phosphorylated at the DNA damage sites in the absence of PTIP. The PTIP-dependent pathway is important for DNA double strand breaks repair and DNA damage-induced intra-S phase checkpoint activation. Taken together, these results suggest that the role of PTIP in the DNA damage response is downstream of RNF8 and upstream of 53BP1. Thus, PTIP regulates 53BP1-dependent signaling pathway following DNA damage.The DNA damage response pathways are signal transduction pathways with DNA damage sensors, mediators, and effectors, which are essential for maintaining genomic stability (1–3). Following DNA double strand breaks, histone H2AX at the DNA damage sites is rapidly phosphorylated by ATM/ATR/DNAPK (4–10), a family homologous to phosphoinositide 3-kinases (11, 12). Subsequently, phospho-H2AX (γH2AX) provides the platform for accumulation of a larger group of DNA damage response factors, such as MDC1, BRCA1, 53BP1, and the MRE11·RAD50·NBS1 complex (13, 14), at the DNA damage sites. Translocalization of these proteins to the DNA double strand breaks (DSBs)³ facilitates DNA damage checkpoint activation and enhances the efficiency of DNA damage repair (14, 15).Recently, PTIP (Pax2 transactivation domain-interacting protein, or Paxip) has been identified as a DNA damage response protein and is required for cell survival when exposed to ionizing radiation (IR) (1, 16–18). PTIP is a 1069-amino acid nuclear protein and has been originally identified in a yeast two-hybrid screening as a partner of Pax2 (19). Genetic deletion of the PTIP gene in mice leads to early embryonic lethality at embryonic day 8.5, suggesting that PTIP is essential for early embryonic development (20). Structurally, PTIP contains six tandem BRCT (BRCA1 carboxyl-terminal) domains (16–18, 21). The BRCT domain is a phospho-group binding domain that mediates protein-protein interactions (17, 22, 23). Interestingly, the BRCT domain has been found in a large number of proteins involved in the cellular response to DNA damages, such as BRCA1, MDC1, and 53BP1 (7, 24–29). Like other BRCT domain-containing proteins, upon exposure to IR, PTIP forms nuclear foci at the DSBs, which is dependent on its BRCT domains (16–18). By protein affinity purification, PTIP has been found in two large complexes. One includes the histone H3K4 methyltransferase ALR and its associated cofactors, the other contains DNA damage response proteins, including 53BP1 and SMC1 (30, 31). Further experiments have revealed that DNA damage enhances the interaction between PTIP and 53BP1 (18, 31).To elucidate the DNA damage response pathways, we have examined the upstream and downstream partners of PTIP. Here, we report that PTIP is downstream of RNF8 and upstream of 53BP1 in response to DNA damage. Moreover, PTIP and 53BP1 are required for the phospho-ATM association with the chromatin, which phosphorylates SMC1 at the DSBs. This PTIP-dependent pathway is involved in DSBs repair.     

Dbf4-Cdc7 Phosphorylation of Mcm2 Is Required for Cell Growth

The Dbf4-Cdc7 kinase (DDK) is required for the activation of the origins of replication, and DDK phosphorylates Mcm2 in vitro. We find that budding yeast Cdc7 alone exists in solution as a weakly active multimer. Dbf4 forms a likely heterodimer with Cdc7, and this species phosphorylates Mcm2 with substantially higher specific activity. Dbf4 alone binds tightly to Mcm2, whereas Cdc7 alone binds weakly to Mcm2, suggesting that Dbf4 recruits Cdc7 to phosphorylate Mcm2. DDK phosphorylates two serine residues of Mcm2 near the N terminus of the protein, Ser-164 and Ser-170. Expression of mcm2-S170A is lethal to yeast cells that lack endogenous MCM2 (mcm2Δ); however, this lethality is rescued in cells harboring the DDK bypass mutant mcm5-bob1. We conclude that DDK phosphorylation of Mcm2 is required for cell growth.The Cdc7 protein kinase is required throughout the yeast S phase to activate origins (1, 2). The S phase cyclin-dependent kinase also activates yeast origins of replication (3–5). It has been proposed that Dbf4 activates Cdc7 kinase in S phase, and that Dbf4 interaction with Cdc7 is essential for Cdc7 kinase activity (6). However, it is not known how Dbf4-Cdc7 (DDK)² acts during S phase to trigger the initiation of DNA replication. DDK has homologs in other eukaryotic species, and the role of Cdc7 in activation of replication origins during S phase may be conserved (7–10).The Mcm2-7 complex functions with Cdc45 and GINS to unwind DNA at a replication fork (11–15). A mutation of MCM5 (mcm5-bob1) bypasses the cellular requirements for DBF4 and CDC7 (16), suggesting a critical physiologic interaction between Dbf4-Cdc7 and Mcm proteins. DDK phosphorylates Mcm2 in vitro with proteins purified from budding yeast (17, 18) or human cells (19). Furthermore, there are mutants of MCM2 that show synthetic lethality with DBF4 mutants (6, 17), suggesting a biologically relevant interaction between DBF4 and MCM2. Nevertheless, the physiologic role of DDK phosphorylation of Mcm2 is a matter of dispute. In human cells, replacement of MCM2 DDK-phosphoacceptor residues with alanines inhibits DNA replication, suggesting that Dbf4-Cdc7 phosphorylation of Mcm2 in humans is important for DNA replication (20). In contrast, mutation of putative DDK phosphorylation sites at the N terminus of Schizosaccharomyces pombe Mcm2 results in viable cells, suggesting that phosphorylation of S. pombe Mcm2 by DDK is not critical for cell growth (10).In budding yeast, Cdc7 is present at high levels in G₁ and S phase, whereas Dbf4 levels peak in S phase (18, 21, 22). Furthermore, budding yeast DDK binds to chromatin during S phase (6), and it has been shown that Dbf4 is required for Cdc7 binding to chromatin in budding yeast (23, 24), fission yeast (25), and Xenopus (9). Human and fission yeast Cdc7 are inert on their own (7, 8), but Dbf4-Cdc7 is active in phosphorylating Mcm proteins in budding yeast (6, 26), fission yeast (7), and human (8, 10). Based on these data, it has been proposed that Dbf4 activates Cdc7 kinase in S phase and that Dbf4 interaction with Cdc7 is essential for Cdc7 kinase activity (6, 9, 18, 21–24). However, a mechanistic analysis of how Dbf4 activates Cdc7 has not yet been accomplished. For example, the multimeric state of the active Dbf4-Cdc7 complex is currently disputed. A heterodimer of fission yeast Cdc7 (Hsk1) in complex with fission yeast Dbf4 (Dfp1) can phosphorylate Mcm2 (7). However, in budding yeast, oligomers of Cdc7 exist in the cell (27), and Dbf4-Cdc7 exists as oligomers of 180 and 300 kDa (27).DDK phosphorylates the N termini of human Mcm2 (19, 20, 28), human Mcm4 (10), budding yeast Mcm4 (26), and fission yeast Mcm6 (10). Although the sequences of the Mcm N termini are poorly conserved, the DDK sites identified in each study have neighboring acidic residues. The residues of budding yeast Mcm2 that are phosphorylated by DDK have not yet been identified.In this study, we find that budding yeast Cdc7 is weakly active as a multimer in phosphorylating Mcm2. However, a low molecular weight form of Dbf4-Cdc7, likely a heterodimer, has a higher specific activity for phosphorylation of Mcm2. Dbf4 or DDK, but not Cdc7, binds tightly to Mcm2, suggesting that Dbf4 recruits Cdc7 to Mcm2. DDK phosphorylates two serine residues of Mcm2, Ser-164 and Ser-170, in an acidic region of the protein. Mutation of Ser-170 is lethal to yeast cells, but this phenotype is rescued by the DDK bypass mutant mcm5-bob1. We conclude that DDK phosphorylation of Ser-170 of Mcm2 is required for budding yeast growth.     

Dual Nuclease and Helicase Activities of Helicobacter pylori AddAB Are Required for DNA Repair, Recombination, and Mouse Infectivity

Helicobacter pylori infection of the human stomach is associated with disease-causing inflammation that elicits DNA damage in both bacterial and host cells. Bacteria must repair their DNA to persist. The H. pylori AddAB helicase-exonuclease is required for DNA repair and efficient stomach colonization. To dissect the role of each activity in DNA repair and infectivity, we altered the AddA and AddB nuclease (NUC) domains and the AddA helicase (HEL) domain by site-directed mutagenesis. Extracts of Escherichia coli expressing H. pylori addA^NUCB or addAB^NUC mutants unwound DNA but had approximately half of the exonuclease activity of wild-type AddAB; the addA^NUCB^NUC double mutant lacked detectable nuclease activity but retained helicase activity. Extracts with AddA^HELB lacked detectable helicase and nuclease activity. H. pylori with the single nuclease domain mutations were somewhat less sensitive to the DNA-damaging agent ciprofloxacin than the corresponding deletion mutant, suggesting that residual nuclease activity promotes limited DNA repair. The addA^NUC and addA^HEL mutants colonized the stomach less efficiently than the wild type; addB^NUC showed partial attenuation. E. coli ΔrecBCD expressing H. pylori addAB was recombination-deficient unless H. pylori recA was also expressed, suggesting a species-specific interaction between AddAB and RecA and also that H. pylori AddAB participates in both DNA repair and recombination. These results support a role for both the AddAB nuclease and helicase in DNA repair and promoting infectivity.Infection of the stomach with Helicobacter pylori causes a variety of diseases including gastritis, peptic ulcers, and gastric cancer (1). A central feature of the pathology of these conditions is the establishment of a chronic inflammatory response that acts both on the host and the infecting bacteria (2). Both epithelial (3, 4) and lymphoid (5, 6) cells in the gastric mucosa of infected individuals release DNA-damaging agents that can introduce double-stranded (ds)² breaks into the bacterial chromosome (7). The ds breaks must be repaired for the bacteria to survive and establish chronic colonization of the stomach. Homologous recombination is required for the faithful repair of DNA damage and bacterial survival. Alteration of the expression of one of a series of cell surface proteins on H. pylori occurs by an apparent gene conversion of babA, the frequency of which is reduced in repair-deficient strains (8, 9). This change in the cell surface, which may allow H. pylori to evade the host immune response, is a second means by which recombination can promote efficient colonization of the stomach by H. pylori.The initiation or presynaptic steps of recombination at dsDNA breaks in most bacteria involves the coordinated action of nuclease and helicase activities provided by one of two multisubunit enzymes, the AddAB and RecBCD enzymes (10). Escherichia coli recBCD null mutants have reduced cell viability, are hypersensitive to DNA-damaging agents, and are homologous recombination-deficient (11–14). Similarly, H. pylori addA and addB null mutants are hypersensitive to DNA-damaging agents, have reduced frequencies of babA gene conversion, and colonize the stomach of mice less efficiently than wild-type strains (8).The activities of RecBCD enzyme from E. coli (15–19) and AddAB from H. pylori (8) or Bacillus subtilis (20–23) indicate some common general features of the presynaptic steps of DNA repair. In the case of E. coli, repair begins when the RecBCD enzyme binds to a dsDNA end and unwinds the DNA using its ATP-dependent helicase activities (17, 24). Single-stranded (ss) DNA produced during unwinding, with or without accompanying nuclease, is coated with RecA protein (16, 25). This recombinogenic substrate engages in strand exchange with a homologous intact duplex to form a joint molecule. Joint molecules are thought to be converted into intact, recombinant DNA either by replication or by cutting and ligation of exchanged strands (26).Although the AddAB and RecBCD enzymes appear to play similar roles in promoting recombination and DNA repair, they differ in several ways. RecBCD is a heterotrimer, composed of one copy of the RecB, RecC, and RecD gene products (27), whereas AddAB has two subunits, encoded by the addA and addB genes (21, 28). The enzyme subunit(s) responsible for helicase activity can be inferred from the presence of conserved protein domains or the activity of purified proteins. AddA, RecB, and RecD are superfamily I helicases with six highly conserved helicase motifs, including the conserved Walker A box found in many enzymes that bind ATP (29–32). A Walker A box is defined by the consensus sequence (G/A)XXGXGKT (X is any amino acid (29). RecBCD enzymes in which the conserved Lys in this motif is changed to Gln have a reduced affinity for ATP binding (33, 34) and altered helicase activity (17, 35–37).A nuclease domain with the conserved amino acid sequence LDYK is found in RecB, AddA, AddB, and many other nucleases (38). The conserved Asp plays a role in Mg²⁺ binding at the active site; Mg²⁺ is required for nuclease activity (39). The recB1080 mutation, which changes codon 1080 from the conserved Asp in this motif to Ala, eliminates nuclease activity (39).We have recently shown that addA and addB deletion mutants are hypersensitive to DNA-damaging agents and impaired in colonization of the mouse stomach compared with wild-type strains (8). To determine the roles of the individual helicase and nuclease activities of H. pylori AddAB in DNA repair and infectivity, we used site-directed mutagenesis to inactivate the conserved nuclease domains of addA and addB and the conserved ATPase (helicase) domain of AddA. Here, we report that loss of the AddAB helicase is sufficient to impair H. pylori DNA repair and infectivity and, when the genes are expressed in E. coli, homologous recombination. AddAB retains partial activity in biochemical and genetic assays when either of the two nuclease domains is inactivated but loses all detectable nuclease activity when both domains are inactivated. Remarkably, H. pylori AddAB can produce recombinants in E. coli only in the presence of H. pylori RecA, suggesting a species-specific interaction in which AddAB facilitates the production of ssDNA-coated with RecA protein. Our results show that both the helicase and nuclease activities are required for the biological roles of H. pylori AddAB.     

Reconstitution of Rad53 Activation by Mec1 through Adaptor Protein Mrc1

Upon DNA replication stress, stalled DNA replication forks serve as a platform to recruit many signaling proteins, leading to the activation of the DNA replication checkpoint. Activation of Rad53, a key effector kinase in the budding yeast Saccharomyces cerevisiae, is essential for stabilizing DNA replication forks during replication stress. Using an activity-based assay for Rad53, we found that Mrc1, a replication fork-associated protein, cooperates with Mec1 to activate Rad53 directly. Reconstitution of Rad53 activation using purified Mec1 and Mrc1 showed that the addition of Mrc1 stimulated a more than 70-fold increase in the ability of Mec1 to activate Rad53. Instead of increasing the catalytic activity of Mec1, Mrc1 was found to facilitate the phosphorylation of Rad53 by Mec1 via promotion of a stronger enzyme-substrate interaction between them. Further, the conserved C-terminal domain of Mrc1 was found to be required for Rad53 activation. These results thus provide insights into the role of the adaptor protein Mrc1 in activating Rad53 in the DNA replication checkpoint.Faithful replication of the genome is important for the survival of all organisms. During DNA replication, replication stress can arise from a variety of situations, including intrinsic errors made by DNA polymerases, difficulties in replicating repeated DNA sequences, and failures to repair damaged DNA caused by either endogenous oxidative agents or exogenous mutagens such as UV light and DNA-damaging chemicals (1–3). In eukaryotes, there is an evolutionarily conserved DNA replication checkpoint that becomes activated in response to DNA replication stress. It helps to stabilize DNA replication forks, block late replication origin firing, and delay mitosis and ultimately helps recovery from stalled replication forks after DNA repair (4–7). Defects in the DNA replication checkpoint could result in elevated genomic instabilities, cancer development, or cell death (8, 9).Aside from replicating the genome, the DNA replication forks also provide a platform to assemble many signaling proteins that function in the DNA replication checkpoint. In the budding yeast Saccharomyces cerevisiae, Mec1, an ortholog of human ATR,² is a phosphoinositide 3-kinase-like kinase (PIKK) involved in sensing stalled DNA replication forks. Mec1 forms a protein complex with Ddc2 (ortholog of human ATRIP). The Mec1-Ddc2 complex is recruited to stalled replication forks through replication protein A (RPA)-coated single-stranded DNA (10, 11). The Mec3-Rad17-Ddc1 complex, a proliferating cell nuclear antigen (PCNA)-like checkpoint clamp and ortholog of the human 9-1-1 complex, was shown to be loaded onto the single- and double-stranded DNA junction of the stalled replication forks by the clamp loader Rad24-RFC complex (12). Once loaded, the Mec3-Rad17-Ddc1 complex stimulates Mec1 kinase activity (13). Dbp11 and its homolog TopBP1 in vertebrates are known components of the replication machinery (14). In addition to regulating the initiation of DNA replication, they were found to play a role in the DNA replication checkpoint (15–17). They interact with the 9-1-1 complex and directly stimulate Mec1/ATR activity in vitro (18–20). Thus, the assembly of multiple protein complexes at stalled DNA replication forks appears to facilitate activation of the DNA replication checkpoint (13, 18).Mrc1 (for mediator of replication checkpoint) was originally identified to be important for cells to respond to hydroxyurea in S. cerevisiae and Schizosaccharomyces pombe (21, 22). Mrc1 is a component of the DNA replisome and travels with the replication forks along chromosome during DNA synthesis (23–25). Deletion of MRC1 causes defects in DNA replication, indicating its role in the normal progression of DNA replication (23). Interestingly, when DNA replication is blocked by hydroxyurea, Mrc1 undergoes Mec1- and Rad3 (S. pombe ortholog of Mec1)-dependent phosphorylation (21, 22). In S. cerevisiae, mutations of Mrc1 at the (S/T)Q sites, which are consensus phosphorylation sites of the Mec1/ATR family kinases, abolishes hydroxyurea-induced Mrc1 phosphorylation in vivo, suggesting a direct phosphorylation of Mrc1 by Mec1 (21, 22).Rad53 and Cds1, homologs of human Chk2, are the major effector kinases in the DNA replication checkpoints in S. cerevisiae and S. pombe, respectively. Activation of Rad53 is a hallmark of DNA replication checkpoint activation and is important for the maintenance of DNA replication forks in response to DNA replication stress (5, 6). Thus, it is important to understand how Rad53 activity is controlled. Interestingly, mutation of all the (S/T)Q sites of Mrc1 not only abolishes the phosphorylation of Mrc1 by Mec1 but also compromises hydroxyurea-induced Rad53 activation in S. cerevisiae (21). Similarly, mutation of the TQ sites of Mrc1 in S. pombe was shown to abolish the binding between Cds1 and Mrc1 as well as Cds1 activation (22). Further, mutation of specific TQ sites of Mrc1 in S. pombe abolishes its binding to Cds1 in vitro and the activation of Cds1 in vivo (26). Thus, Mec1/Rad3-dependent phosphorylation of Mrc1 is responsible for Mrc1 binding to Rad53/Cds1, which is essential for Rad53/Cds1 activation.An intriguing property of the Chk2 family kinases is their ability to undergo autophosphorylation and activation in the absence of other proteins in vitro (27, 28). First, autophosphorylation of a conserved threonine residue in the activation loop of Chk2 family kinase was found to be an essential part of their activation processes (26, 29–31). Second, a direct and trans-phosphorylation of the N-terminal TQ sites of the Chk2 family kinases by the Mec1/ATR family kinases is also important for their activation in vivo. Analogous to the requirement of N-terminal TQ site phosphorylation of Chk2 by ATR in human (32), the activation of Rad53/Cds1 in vivo requires phosphorylation of TQ sites in their N termini by Mec1/Rad3 (33, 34).Considering that Mec1, Mrc1, and many other proteins are recruited at stalled DNA replication forks and have been shown to be involved in DNA replication checkpoint activation, a key question remains unresolved: what is the minimal system that is capable of activating Rad53 directly? Given the direct physical interaction between Mrc1 and Rad53 and the requirement of Mrc1 and Mec1 in vivo, it is likely that they both play a role in Rad53 activation. Furthermore, what is the molecular mechanism of Rad53 activation by its upstream activators? To address these questions, a faithful reconstitution of the activation of Rad53 using purified proteins is necessary. In this study, we developed an activity-based assay consisting of the Dun1 kinase, a downstream substrate of Rad53, and Sml1, as a substrate of Dun1, to quantitatively measure the activity of Rad53. Using this coupled kinase assay from Rad53 to Dun1 and then to Sml1, we screened for Mrc1 and its associated factors to see whether they could directly activate Rad53 in vitro. Our results showed that Mec1 and Mrc1 collaborate to constitute a minimal system in direct activation of Rad53.     

Aberrant Splice Variants of HAS1 (Hyaluronan Synthase 1) Multimerize with and Modulate Normally Spliced HAS1 Protein: A POTENTIAL MECHANISM PROMOTING HUMAN CANCER*

Most human genes undergo alternative splicing, but aberrant splice forms are hallmarks of many cancers, usually resulting from mutations initiating abnormal exon skipping, intron retention, or the introduction of a new splice sites. We have identified a family of aberrant splice variants of HAS1 (the hyaluronan synthase 1 gene) in some B lineage cancers, characterized by exon skipping and/or partial intron retention events that occur either together or independently in different variants, apparently due to accumulation of inherited and acquired mutations. Cellular, biochemical, and oncogenic properties of full-length HAS1 (HAS1-FL) and HAS1 splice variants Va, Vb, and Vc (HAS1-Vs) are compared and characterized. When co-expressed, the properties of HAS1-Vs are dominant over those of HAS1-FL. HAS1-FL appears to be diffusely expressed in the cell, but HAS1-Vs are concentrated in the cytoplasm and/or Golgi apparatus. HAS1-Vs synthesize detectable de novo HA intracellularly. Each of the HAS1-Vs is able to relocalize HAS1-FL protein from diffuse cytoskeleton-anchored locations to deeper cytoplasmic spaces. This HAS1-Vs-mediated relocalization occurs through strong molecular interactions, which also serve to protect HAS1-FL from its otherwise high turnover kinetics. In co-transfected cells, HAS1-FL and HAS1-Vs interact with themselves and with each other to form heteromeric multiprotein assemblies. HAS1-Vc was found to be transforming in vitro and tumorigenic in vivo when introduced as a single oncogene to untransformed cells. The altered distribution and half-life of HAS1-FL, coupled with the characteristics of the HAS1-Vs suggest possible mechanisms whereby the aberrant splicing observed in human cancer may contribute to oncogenesis and disease progression.About 70–80% of human genes undergo alternative splicing, contributing to proteomic diversity and regulatory complexities in normal development (1). About 10% of mutations listed so far in the Human Gene Mutation Database (HGMD) of “gene lesions responsible for human inherited disease” were found to be located within splice sites. Furthermore, it is becoming increasingly apparent that aberrant splice variants, generated mostly due to splicing defects, play a key role in cancer. Germ line or acquired genomic changes (mutations) in/around splicing elements (2–4) promote aberrant splicing and aberrant protein isoforms.Hyaluronan (HA)³ is synthesized by three different plasma membrane-bound hyaluronan synthases (1, 2, and 3). HAS1 undergoes alternative and aberrant intronic splicing in multiple myeloma, producing truncated variants termed Va, Vb, and Vc (5, 6), which predicted for poor survival in a cohort of multiple myeloma patients (5). Our work suggests that this aberrant splicing arises due to inherited predispositions and acquired mutations in the HAS1 gene (7). Cancer-related, defective mRNA splicing caused by polymorphisms and/or mutations in splicing elements often results in inactivation of tumor suppressor activity (e.g. HRPT2 (8, 9), PTEN (10), MLHI (11–14), and ATR (15)) or generation of dominant negative inhibitors (e.g. CHEK2 (16) and VWOX (17)). In breast cancer, aberrantly spliced forms of progesterone and estrogen receptors are found (reviewed in Ref. 3). Intronic mutations inactivate p53 through aberrant splicing and intron retention (18). Somatic mutations with the potential to alter splicing are frequent in some cancers (19–25). Single nucleotide polymorphisms in the cyclin D1 proto-oncogene predispose to aberrant splicing and the cyclin D1b intronic splice variant (26–29). Cyclin D1b confers anchorage independence, is tumorogenic in vivo, and is detectable in human tumors (30), but as yet no clinical studies have confirmed an impact on outcome. On the other hand, aberrant splicing of HAS1 shows an association between aberrant splice variants and malignancy, suggesting that such variants may be potential therapeutic targets and diagnostic indicators (19, 31–33). Increased HA expression has been associated with malignant progression of multiple tumor types, including breast, prostate, colon, glioma, mesothelioma, and multiple myeloma (34). The three mammalian HA synthase (HAS) isoenzymes synthesize HA and are integral transmembrane proteins with a probable porelike structural assembly (35–39). Although in humans, the three HAS genes are located on different chromosomes (hCh19, hCh8, and hCh16, respectively) (40), they share a high degree of sequence homology (41, 42). HAS isoenzymes synthesize a different size range of HA molecules, which exhibit different functions (43, 44). HASs contribute to a variety of cancers (45–55). Overexpression of HASs promotes growth and/or metastatic development in fibrosarcoma, prostate, and mammary carcinoma, and the removal of the HA matrix from a migratory cell membrane inhibits cell movement (45, 53). HAS2 confers anchorage independence (56). Our work has shown aberrant HAS1 splicing in multiple myeloma (5) and Waldenstrom''s macroglobulinemia (6). HAS1 is overexpressed in colon (57), ovarian (58), endometrial (59), mesothelioma (60), and bladder cancers (61). A HAS1 splice variant is detected in bladder cancer (61).Here, we characterize molecular and biochemical characteristics of HAS1 variants (HAS1-Vs) (5), generated by aberrant splicing. Using transient transfectants and tagged HAS1 family constructs, we show that HAS1-Vs differ in cellular localization, de novo HA localization, and turnover kinetics, as compared with HAS1-FL, and dominantly influence HAS1-FL when co-expressed. HAS1-Vs proteins form intra- and intermolecular associations among themselves and with HAS1-FL, including covalent interactions and multimer formation. HAS1-Vc supports vigorous cellular transformation of NIH3T3 cells in vitro, and HAS1-Vc-transformed NIH3T3 cells are tumorogenic in vivo.     

FANCI Binds Branched DNA and Is Monoubiquitinated by UBE2T-FANCL

FANCI is integral to the Fanconi anemia (FA) pathway of DNA damage repair. Upon the occurrence of DNA damage, FANCI becomes monoubiquitinated on Lys-523 and relocalizes to chromatin, where it functions with monoubiquitinated FANCD2 to facilitate DNA repair. We show that FANCI and its C-terminal fragment possess a DNA binding activity that prefers branched structures. We also demonstrate that FANCI can be ubiquitinated on Lys-523 by the UBE2T-FANCL pair in vitro. These findings should facilitate future efforts directed at elucidating molecular aspects of the FA pathway.Fanconi anemia (FA)⁴ is characterized by developmental defects, bone marrow failure, and a strong predisposition to cancer. FA cells exhibit exquisite sensitivity to DNA cross-linking agents and marked genomic instability, indicative of a failure to repair damaged DNA (1–3). Thirteen FA proteins have been identified, of which eight, FANC-A, -B, -C, -E, -F, -G, -L, and -M, form part of a nuclear core complex that is required to monoubiquitinate two other FA proteins, FANCD2 and FANCI. When monoubiquitinated, FANCD2 and FANCI become chromatin-associated in foci that contain various factors, including the RAD51 recombinase BRCA2 (also known as FANCD1) and PALB2 (also called FANCN), which mediate DNA repair via RAD51-catalyzed homologous recombination (4).Monoubiquitination of FANCD2 appears to be a key event for proper repair of exogenous DNA damage but also occurs during an unperturbed S phase, likely in response to stalled replication forks (4–7). FANCD2 monoubiquitination depends on the E3 ligase activity of FANCL (8) and on the E2 ubiquitin-conjugating enzyme, UBE2T (9). In vitro, FANCL and UBE2T can monoubiquitinate chicken FANCD2 (10).FANCI was identified recently as a target protein for the ATM/ATR kinase. FANCI is also monoubiquitinated, in a manner that is dependent on the FA core complex (11). In cells, a fraction of FANCD2 and FANCI associates in a complex. Moreover, the amount and monoubiquitination of these two FA proteins are co-dependent in human cells, i.e. the quantity and monoubiquitination of FANCD2 are diminished in FANCI-deficient cells and vice versa (11–14). These observations suggest that FANCI and FANCD2 form a complex integral to cellular DNA repair capacity. Mutating the ubiquitinated target lysine of FANCI (Lys-523) renders cells sensitive to DNA damage and impairs the assembly of DNA damage-induced nuclear foci of FANCD2 and FANCI (11, 14). Herein, we document studies that reveal several biochemical attributes of FANCI, including DNA binding, and its monoubiquitination, that are relevant for understanding the biological role of this key FA protein.