Extracellular vesicles (EVs) such as exosomes and microvesicles serve as messengers of intercellular network, allowing exchange of cellular components between cells. EVs carry lipids, proteins, and RNAs derived from their producing cells, and have potential as biomarkers specific to cell types and even cellular states. However, conventional methods (such as ultracentrifugation or polymeric precipitation) for isolating EVs have disadvantages regarding purity and feasibility. Here, we have developed a novel method for EV purification by using Tim4 protein, which specifically binds the phosphatidylserine displayed on the surface of EVs. Because the binding is Ca2+-dependent, intact EVs can be easily released from Tim4 by adding Ca2+ chelators. Tim4 purification, which we have applied to cell conditioned media and biofluids, is capable of yielding EVs of a higher purity than those obtained using conventional methods. The lower contamination found in Tim4-purified EV preparations allows more EV-specific proteins to be detected by mass spectrometry, enabling better characterization and quantification of different EV populations’ proteomes. Tim4 protein can also be used as a powerful tool for quantification of EVs in both ELISA and flow cytometry formats. Thus, the affinity of Tim4 for EVs will find abundant applications in EV studies.

Antibodies against the receptor-binding-domain of the SARS-CoV-2 spike protein prevent SARS-CoV-2 infection. However, the effects of antibodies against other spike protein domains are largely unknown. Here, we screened a series of anti-spike monoclonal antibodies from COVID-19 patients, and found that some of antibodies against the N-terminal-domain (NTD) induced the open conformation of receptor binding domain (RBD) and thus enhanced the binding capacity of the spike protein to ACE2 and infectivity of SARS-CoV-2. Mutational analysis revealed that all the infectivity-enhancing antibodies recognized a specific site on the NTD. Structural analysis demonstrated that all the infectivity-enhancing antibodies bound to NTD in a similar manner. The antibodies against this infectivity-enhancing site were detected at high levels in severe patients. Moreover, we identified antibodies against the infectivity-enhancing site in uninfected donors, albeit at a lower frequency. These findings demonstrate that not only neutralizing antibodies but also enhancing antibodies are produced during SARS-CoV-2 infection.

The yeast Saccharomyces cerevisiae Cdc7p/Dbf4p protein kinase complex was purified to near homogeneity from insect cells. The complex efficiently phosphorylated yeast Mcm2p and less efficiently the remaining Mcm proteins or other replication proteins. Significantly, when pretreated with alkaline phosphatase, Mcm2p became completely inactive as a substrate, suggesting that it must be phosphorylated by other protein kinase(s) to be a substrate for the Cdc7p/Dbf4p complex. Mutant Cdc7p/Dbf4p complexes containing either Cdc7-1p or Dbf4-1ϳ5p were also partially purified from insect cells and characterized in vitro. Furthermore, the autonomously replicating sequence binding activity of various dbf4 mutants was also analyzed. These studies suggest that the autonomously replicating sequencebinding and Cdc7p protein kinase activation domains of Dbf4p collaborate to form an active Cdc7p/Dbf4p complex and function during S phase in S. cerevisiae. It is shown that Rad53p phosphorylates the Cdc7p/Dbf4p complex in vitro and that this phosphorylation greatly inhibits the kinase activity of Cdc7p/Dbf4p. This result suggests that Rad53p controls the initiation of chromosomal DNA replication by regulating the protein kinase activity associated with the Cdc7p/Dbf4p complex.Initiation of chromosomal DNA replication and cell cycle progression are tightly regulated in eukaryotes. In the yeast Saccharomyces cerevisiae, several cdc (cell division cycle) mutants that block initiation of chromosomal DNA replication have been isolated and characterized (1, 2), for example, cdc28, cdc4, cdc6, and cdc7. The stepwise assembly of proteins at origins of DNA replication is a crucial part of regulating entry into S phase. Two key factors that mediate such cell cycle regulation are Cdc6 protein level and availability and the presence of an active cyclin-dependent kinase (Cdk) (see Ref. 3 for review). The origin recognition complex is bound to origins of DNA replication at all stages of the cell cycle in S. cerevisiae (4 -6). However, Cdc6 is not recruited to origins until late in M phase and is required for the association of the Mcm2-7 family proteins at origins to form a prereplicative complex (6 -8).The Cdc6 protein-dependent stage of the assembly reaction is inhibited by active Clb-Cdks (6, 9, 10). Because Cdc6 protein is synthesized only from late M phase until late G1 (11), prereplicative complexes can only be assembled during this period of the cell cycle. S phase cyclin-Cdk (Cdc28p/Clb5p or Cdc28p/ Clb6p) activity is required for the chromatin association of Cdc45p just before the initiation of chromosomal DNA replication (12). The previous results demonstrated that Cdc28 protein-Clb kinase is required throughout S phase to activate origins when they are scheduled to fire (13).The Cdc7/Dbf4 complex is a Cdk-like protein kinase (see Refs. 14 and 15 for review) that is also required for entry into S phase at a very late stage. CDC7 transcript levels are constant throughout the cell cycle, whereas DBF4 transcription is cell cycle regulated a...

AbstractmRNA-based vaccines provide effective protection against most common SARS-CoV-2 variants. However, identifying likely breakthrough variants is critical for future vaccine development. Here, we found that the Delta variant completely escaped from anti-N-terminal domain (NTD) neutralizing antibodies, while increasing responsiveness to anti-NTD infectivity-enhancing antibodies. Although Pfizer-BioNTech BNT162b2-immune sera neutralized the Delta variant, when four common mutations were introduced into the receptor binding domain (RBD) of the Delta variant (Delta 4+), some BNT162b2-immune sera lost neutralizing activity and enhanced the infectivity. Unique mutations in the Delta NTD were involved in the enhanced infectivity by the BNT162b2-immune sera. Sera of mice immunized by Delta spike, but not wild-type spike, consistently neutralized the Delta 4+ variant without enhancing infectivity. Given the fact that a Delta variant with three similar RBD mutations has already emerged according to the GISAID database, it is necessary to develop vaccines that protect against such complete breakthrough variants.

DNA polymerases ␦ and ⑀ (pol ␦ and ⑀) are the major replicative polymerases and possess 3-5 proofreading exonuclease activities that correct errors arising during DNA replication in the yeast Saccharomyces cerevisiae. This study measures the fidelity of the holoenzyme of wild-type pol ⑀, the 3-5 exonuclease-deficient pol2-4, a ؉1 frameshift mutator for homonucleotide runs, pol2C1089Y, and pol2C1089Y pol2-4 enzymes using a synthetic 30-mer primer/100-mer template. The nucleotide substitution rate for wild-type pol ⑀ was 0.47 ؋ 10 ؊5for G:G mismatches, 0.15 ؋ 10 ؊5 for T:G mismatches, and less than 0.01 ؋ 10 ؊5 for A:G mismatches. The accuracy for A opposite G was not altered in the exonucleasedeficient pol2-4 pol ⑀; however, G:G and T:G misincorporation rates increased 40-and 73-fold, respectively. The pol2C1089Y pol ⑀ mutant also exhibited increased G:G and T:G misincorporation rates, 22-and 10-fold, respectively, whereas A:G misincorporation did not differ from that of wild type. Since the fidelity of the double mutant pol2-4 pol2C1089Y was not greatly decreased, these results suggest that the proofreading 3-5 exonuclease activity of pol2C1089Y pol ⑀ is impaired even though it retains nuclease activity and the mutation is not in the known exonuclease domain.The yeast Saccharomyces cerevisiae has three DNA polymerases, (pol ␣, ␦, and ⑀), which are required for cell growth, chromosomal DNA replication (1), and DNA double-strand break repair (2). pol ␣ has four subunits (Pol1 (Cdc17), Pol10, Pri1, and Pri2) and is involved primarily in the initiation of DNA replication and priming of Okazaki fragments. pol ␦ and pol ⑀ are required during synthesis of the leading and lagging DNA strands at the replication fork; they bind at/or near replication origins and move along DNA with the replication fork (3, 4). The precise roles of pol ␦ and pol ⑀ during leading and lagging strand DNA synthesis have not yet been defined; however, genetic and biochemical evidence suggest that lagging strand DNA synthesis is carried out by pol ␣ and pol ␦ (5, 6).pol ␦ of S. cerevisiae has three subunits (Pol3 (Cdc2), Hys2 (Pol31) (7,8), and Pol32 (8)), which are homologues of Schizosaccharomyces pombe Pol3, Cdc1, and Cdc27, respectively (9). S. pombe pol ␦ has one additional subunit, Cmt1 (9). Purified yeast pol ␦ requires accessory factors including PCNA 1 and RFC to catalyze processive DNA synthesis (8). pol ␦ possesses a 3Ј-5Ј exonuclease activity, which performs proofreading/editing during DNA synthesis (10, 11).The S. cerevisiae pol ⑀ is also a multi-subunit protein complex that includes Pol2, Dpb2, Dpb3, and Dpb4 and like pol ␦ has a 3Ј-5Ј exonuclease activity (1, 12). pol ⑀ is a highly processive enzyme (12)(13)(14). Although pol ⑀ requires PCNA and RFC complex to catalyze processive DNA synthesis on singly primed single-stranded viral DNA (13), these cofactors may not be required for processive DNA synthesis in vivo. Pol2 is the catalytic subunit of pol ⑀ and is encoded by the POL2 gene (15), which is essential for cell growth and re...

In enterohemorrhagic Escherichia coli, Shiga toxin is produced by lysogenic prophages. We have isolated the prophage VT2-Sa that is responsible for production of Shiga toxin type 2 protein, and determined the complete nucleotide sequence of this phage DNA. The entire DNA sequence consisted of 60,942 bp, exhibiting marked similarity to the 933W phage genome. However, several differences were observed in the immunity and replication regions, where cI, cII, cIII, N, cro, O, and P genes were present: Predicted amino acid sequences of N, cI, cro, O and P in the VT2-Sa genome did not show significant similarity to the counterparts of the 933W genome; however its cI showed higher similarity to lambda. Furthermore, O and P closely resembled those of phage HK022. These observations suggest that the various degrees of homology observed in the immunity and replication regions of VT2-Sa could have resulted from frequent recombination events among the lambdoid phages, and that these regions play a key role as a functional unit for phage propagation in competition with other lambdoid phages.

The continuity of duplex DNA is generally considered a prerequisite for chromosome continuity. However, as previously shown in yeast as well as human cells, the introduction of a double-strand break (DSB) does not generate a chromosome break (CRB) in yeast or human cells. The transition from DSB to CRB was found to be under limited control by the tethering function of the RAD50/ MRE11/XRS2 (MRX) complex. Using a system for differential fluorescent marking of both sides of an endonuclease-induced DSB in single cells, we found that nearly all DSBs are converted to CRBs in cells lacking both exonuclease 1 (EXO1) activity and MRX complex. Thus, it appears that some feature of exonuclease processing or resection at a DSB is critical for maintaining broken chromosome ends in close proximity. In addition, we discovered a thermal sensitive (cold) component to CRB formation in an MRX mutant that has implications for chromosome end mobility and/or end-processing.

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