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Cell-free DNA (cfDNA) (e.g. fetal- or tumor-derived DNA) is DNA found in the blood circulation. It is now widely investigated as a biomarker for prenatal screening, tumor diagnosis, and tumor monitoring as “liquid biopsies”. However, the biological and biochemical aspects of cfDNA remain unclear. Although cfDNA is considered to be mainly derived from dead cells, information is scarce as to whether it is apoptotic or necrotic and what kinds of endonucleases or DNases are involved. We induced in vivo hepatocyte necrosis and apoptosis in mice deficient in DNase1L3 (also named DNase γ) and/or caspase-activated DNase (CAD) genes with acetaminophen overdose and anti-Fas antibody treatments. We found that (i) DNase1L3 was the endonuclease responsible for generating cfDNA in acetaminophen-induced hepatocyte necrosis and (ii) CAD and DNase1L3 cooperated in producing cfDNA for anti-Fas mediated hepatocyte apoptosis.

Although chromatin condensation is a well-known hallmark of apoptosis, the generation mechanism has not been clarified. Histone H1, a positively-charged abundant nuclear protein, is located in the linker region of chromatin. There are several Histone H1 subtypes that are encoded by variant genes. Using serial histone H1-deletion mutant cells established from the chicken B-cell leukemia line DT40, we found that apoptotic chromatin condensation was decreased in relation to histone H1 protein level and that the chromatin in nuclei prepared from the live null mutant cells had a high accessibility of DNases and transposase. This indicated that linker histone H1 was the general chromatin condensation factor and that the loss of histone H1 generated open chromatin in both apoptotic and live cells.

Blood DNases hack the NET Neutrophil extracellular traps (NETs) are lattices of processed chromatin decorated with select secreted and cytoplasmic proteins that trap and neutralize microbes. However, their inappropriate release may do more harm than good by promoting inflammation and thrombosis. Jiménez-Alcázar et al. report that two deoxyribonucleases (DNases), DNASE1 and DNASE1L3, have partially redundant roles in degrading NETs in the circulation (see the Perspective by Gunzer). Knockout mice lacking these enzymes were unable to tolerate chronic neutrophilia, quickly dying after blood vessels were occluded by NET clots. Furthermore, the damage unleashed by clots during septicemia was enhanced when these DNases were absent. Science , this issue p. 1202 ; see also p. 1126

Serum endonucleases are essential for degrading the chromatin released from dead cells and preventing autoimmune diseases such as systemic lupus erythematosus. Serum DNase I is known as the major endonuclease, but recently, another endonuclease, DNase γ/DNase I-like 3, gained attention. However, the precise role of each endonuclease, especially that of DNase γ, remains unclear. In this study, we distinguished the activities of DNase γ from those of DNase I in mouse serum and concluded that both cooperated in degrading DNA during necrosis: DNase γ functions as the primary chromatolytic activity, causing internucleosomal DNA fragmentation, and DNase I as the secondary one, causing random DNA digestion for its complete degradation. These results were confirmed by two in vivo experimental mouse models, in which necrosis was induced, acetaminophen-induced hepatic injury and streptozotocin-induced β-cell necrosis models. We also determined that DNase γ functions as a backup endonuclease for caspase-activated DNase (CAD) in the secondary necrosis phase after γ-ray-induced apoptosis in vivo.

High-mobility group box 1 (HMGB1) protein is the major component of the nonhistone nuclear protein group and is involved in nucleosome stabilization and transcription regulation. HMGB1 has recently been focused on as a proinflammatory cytokine associated with various inflammatory diseases and as a target of anti-inflammatory therapy. Mastitis, a serious inflammatory disease of dairy cows, is caused by infection of the mammary gland and has detrimental effects on the quantity and quality of milk. By detecting the presence of HMGB1 in milk, we investigated the correlation between HMGB1 concentration and the severity of bovine mastitis, which was determined using the California Mastitis Test and somatic cell count (SCC). We detected a substantial amount of HMGB1 in mastitic milk but not in the milk from normal cows. We used the Spearman rank correlation coefficient to assess the relationship between HMGB1 concentration and SCC and found a significant correlation (n = 12, r = 0.975). Thus, we confirmed the positive correlation between HMGB1 concentration and SCC in milk, i.e., the severity of mastitis, which suggested that HMGB1 in milk is a new indicator of bovine mastitis.

The internucleosomal cleavage of genomic DNA is a biochemical hallmark of apoptosis. DNase gamma, a Mg2+/Ca2+-dependent endonuclease, has been suggested to be one of the apoptotic endonucleases, but its biochemical characteristic has not been fully elucidated. Here, using recombinant DNase gamma, we showed that DNase gamma is a Mg2+/Ca2+-dependent single-stranded DNA nickase and has a high activity at low ionic strength. Under higher ionic strength, such as physiological buffer conditions, the endonuclease activity of DNase gamma is restricted, but its activity is enhanced in the presence of linker histone H1, which explains DNA cleavage at linker regions of apoptotic nuclei.

Rolling circle amplification (RCA) of plasmid DNA using random hexamers and bacteriophage phi29 DNA polymerase is an increasingly applied technique for amplifying template DNA for DNA sequencing. We analyzed this RCA reaction at a single-molecular level by atomic force microscopy (AFM) and found that multibranched amplified products containing tandem repeats of a circle unit are formed within 1 h. We also used the RCA product of a GFP expression vector for the protein expression in cells, and found that the crude RCA product from one bacterial colony is sufficient for the GFP expression. Thus, the RCA reaction is useful in amplifying DNA for both DNA sequencing and protein expression.

The gene product of XRCC4 has been implicated in both V(D)J recombination and the more general process of double strand break repair (DSBR). To date its role in these processes is unknown. Here, we describe biochemical characteristics of the murine XRCC4 protein. XRCC4 expressed in insect cells exists primarily as a disulfide-linked homodimer, although it can also form large multimers. Recombinant XRCC4 is phosphorylated during expression in insect cells. XRCC4 phosphorylation in Sf9 cells occurs on serine, threonine, and tyrosine residues. We also investigated whether XRCC4 interacts with the other factor known to be requisite for both V(D)J recombination and DSBR, the DNA-dependent protein kinase. We report that XRCC4 is an efficient in vitro substrate of DNA-PK and another unidentified serine/ threonine protein kinase(s). Both DNA-PK dependent and independent phosphorylation of XRCC4 in vitro occurs only on serine and threonine residues within the COOH-terminal 130 amino acids, a region of the molecule that is not absolutely required for XRCC4's DSBR function. Finally, recombinant XRCC4 facilitates Ku binding to DNA, promoting assembly of DNA-PK and complexing with DNA-PK bound to DNA. These data are consistent with the hypothesis that XRCC4 functions as an alignment factor in the DNA-PK complex.

Apoptosis and necrosis, two major forms of cell death, can be distinguished morphologically and biochemically. Internucleosomal DNA fragmentation (INDF) is a biochemical hallmark of apoptosis, and caspase-activated DNase (CAD), also known as DNA fragmentation factor 40 kDa (DFF40), is one of the major effector endonucleases. DNase γ, a Mg(2+)/Ca(2+)-dependent endonuclease, is also known to generate INDF but its role among other apoptosis-associated endonucleases in cell death is unclear. Here we show that (i) INDF occurs even during necrosis in cell lines, primary cells, and in tissues of mice in vivo, and (ii) DNase γ, but not CAD, is the effector endonuclease for INDF in cells undergoing necrosis. These results document a previously unappreciated role for INDF in necrosis and define its molecular basis.

Defective expression of the Ku80 gene has been implicated as underlying the V(D)J recombination and DNA double-strand break repair defects in the xrs-6 Chinese hamster ovary cell line. We now show that the mutation in the Ku80 gene Involves a G to A transition 15 bp upstream of exon 2. This mutation creates a new splice acceptor site which results in the generation of a Ku80 transcript that cannot encode a functional product due a 13 nucleotide insertion and a resulting frameshlft.

Murine cells homozygous for the severe combined immune deficiency mutation ( scid ) and V3 mutant hamster cells fall into the same complementation group and show similar defects in V(D)J recombination and DNA double-stranded break repair. Here we show that both cell types lack DNA-dependent protein kinase (DNA-PK) activity owing to defects in DNA-PK cs , the catalytic subunit of this enzyme. Furthermore, we demonstrate that yeast artificial chromosomes containing the DNA-PK cs , gene complement both the DNA repair and recombination deficiencies of V3 cells, and we conclude that DNA-PK cs , is encoded by the XRCC7 gene. As DNA-PK binds to DNA ends and is activated by these structures, our findings provide novel insights into V(D)J recombination and DNA repair processes.

Somatic hypermutation (SHM) diversifies the rearranged immunoglobulin variable (V) region gene in B cells, contributing to affinity maturation of antibodies. It is believed that SHM is generated either by direct replication or by error-prone repair systems resolving V region DNA lesions caused directly or indirectly by cytidine deaminase AID. In accord with a part of these mechanisms, it was reported that SHM is associated with staggered double-strand DNA breaks (DSBs) occurring in the rearranged V regions. However, endonucleases responsible for the DSBs remain elusive. Here we show that DNase gamma, a member of DNase I family endonucleases, contributes to the generation of SHM including point mutation, and nucleotide insertion and deletion in chicken DT40 B cell line. DNase gamma also contributes to the generation of staggered DSBs in the rearranged V region. These results raise a possibility that DNase gamma is involved in the V gene mutation machinery.

Here, we describe the non-redundant roles of caspase-activated DNase (CAD) and DNasegamma during apoptosis in the immature B-cell line WEHI-231. These cells induce DNA-ladder formation and nuclear fragmentation by activating CAD during cytotoxic drug-induced apoptosis. Moreover, these apoptotic manifestations are accompanied by inhibitor of CAD (ICAD) cleavage and are abrogated by the constitutive expression of a caspase-resistant ICAD mutant. No such nuclear changes occur during oxidative stress-induced necrosis, indicating that neither CAD nor DNasegamma functions under necrotic conditions. Interestingly, the DNA-ladder formation and nuclear fragmentation induced by B-cell receptor ligation occur in the absence of ICAD cleavage and are not significantly affected by the ICAD mutant. Both types of nuclear changes are preceded by the upregulation of DNasegamma expression and are strongly suppressed by 4-(4,6-dichloro-[1, 3, 5]-triazin-2-ylamino)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-benzoic acid (DR396), which is a specific inhibitor of DNasegamma. Our results suggest that DNasegamma provides an alternative mechanism for inducing nuclear changes when the working apoptotic cascade is unsuitable for CAD activation.

It is suggested that the formation of the switch (S) region RNA-DNA hybrid and the subsequent generation of higher-order chromatin structures including R-loop initiate a class switch recombination of the immunoglobulin gene. The primary factor of this recombination is the S-region derived noncoding RNA. However, the biochemical character of this guanine-rich (G-rich) transcript is poorly understood. The present study was performed to analyze the structure of this G-rich RNA using atomic force microscope (AFM). The in vitro transcribed S-region RNA was spread on a mica plate, air-dried and observed by non-contact mode AFM in air. The G-rich transcripts tend to aggregate on the template DNA and to generate a higher-order RNA-DNA complex. However, the transcripts that incorporated guanine analogues as substitutes for guanine neither aggregated nor generated higher-order structures. Incorporation of guanine analogues in transcribed RNA partially disrupts hydrogen bonds related to guanine, such as Watson-Crick GC-base pair and Hoogsteen bond GG-base pair. Thus, aggregation of S-region RNA and generation of the higher-order RNA-DNA complex are attributed to hydrogen bonds of guanine.

A cDNA encoding a rat Smubp-2 has been cloned from a lambdagt11 library by South-Western blot screening using a 50-bp tannic acid responsive element [J. Biol. Chem. 273 (1998) 12499] of the mouse mammary tumor virus (MMTV) promoter region as a probe. The full-length cDNA encodes a protein with a predicted size of 108 kDa. Northern blot analysis revealed that the gene expression of Smubp-2 is comparatively high in testis, moderate in brain, and low in other tissues. The recombinant Smubp-2 protein was expressed as a GST- or Trx-fusion protein in Escherichia coli and purified by affinity column chromatography. Gel mobility shift competition analysis indicated that the recombinant Smubp-2 protein binds to region II (containing the ACTG-motif) in the 50-bp element in the MMTV promoter. A transient transfection assay of the Smubp-2 expression vector with MMTV promoter-containing Luciferase (Luc) reporter plasmids into mouse cells suggested that Smubp-2 is a negative transcription factor. Furthermore, the MMTV promoter activity was suppressed in cells expressing high levels of Smubp-2. Insertion of the 50-bp element upstream of the SV40 promoter negatively responded to the induced expression of Smubp-2. These results suggest that the negative transcriptional effect of Smubp-2 arises from its binding to the 50-bp element located in the MMTV promoter region.

Immunoglobulin heavy-chain (IgH) class switch recombination (CSR) is initiated by DNA breakage in the switch (S) region featuring tandem repetitive nucleotide sequences. Various studies have demonstrated that S-region transcription and splicing proceed to genomic recombination and are indispensable for CSR in vivo, although the precise molecular mechanism is largely unknown. Here, we show the novel physical property of the in vitro transcribed S-region RNA by direct visualization using an atomic force microscope (AFM). The S-region sense RNA, but not the antisense RNA, forms a persistent hybrid with the template plasmid DNA and changes the plasmid conformation from supercoil to open circle in the presence of spermidine. In addition, the S-region transcripts generate globular forms and are assembled on the template DNA into a large aggregate that may stall replication and increase the recombinogenicity of the S-region DNA.

XRCC4 is a generally expressed protein of 334 amino acids that is involved in the repair of DNA double-strand breaks and in V(D)J recombination, but its function is unknown. In this study, we have used a mutational approach and the yeast two-hybrid method to perform an initial characterization of this protein. We show that the XRCC4 protein is located in the nucleus. We also demonstrate that several potential phosphorylation sites are not required for XRCC4 function in a transient V(D)J recombination assay. In addition, we show that XRCC4 forms a homodimer in vivo with the homodimerization domain being located within amino acids 115-204. Finally, we define a core domain of XRCC4 that functions in V(D)J recombination and comprises amino acids 18-204. Potential functions of XRCC4 are discussed.

The human RAD51 protein is a homologue of the bacteria RecA and yeast RAD51 proteins that are involved in homologous recombination and DNA repair. RAD51 interacts with proteins involved in recombination and also with tumor suppressor proteins p53 and breast cancer susceptibility gene 1 (BRCA1). We have used the yeast two-hybrid system to clone murine cDNA sequences that encode two RAD51-associated molecules, RAB22 and RAB163. RAB163 encodes the C-terminal portion of mouse BRCA2, the homologue of the second breast cancer susceptibility gene protein in humans, demonstrating an in vitro association between RAD51 and BRCA2. RAB22 is a novel gene product that also interacts with RAD51 in vitro . To detect RAD51 interactions in vivo , we developed a transient nuclear focus assay that was used to demonstrate a complete colocalization of RAB22 with RAD51 in large nuclear foci.

The radiosensitive mutant xrs-6, derived from Chinese hamster ovary cells, is defective in DNA double-strand break repair and in ability to undergo V(D)J recombination. The human XRCC5 DNA repair gene, which complements this mutant, is shown here through genetic and biochemical evidence to be the 80-kilodalton subunit of the Ku protein. Ku binds to free double-stranded DNA ends and is the DNA-binding component of the DNA-dependent protein kinase. Thus, the Ku protein is involved in DNA repair and in V(D)J recombination, and these results may also indicate a role for the Ku-DNA-dependent protein kinase complex in those same processes.

We have cloned the cDNA encoding the human homologue of S mu bp-2, which binds to single-stranded DNA with 5'-phosphorylated guanine-rich sequences related to the immunoglobulin mu chain switch (S mu) region. The deduced amino acid sequences of the mouse and human S mu bp-2 are 76.5% homologous and contain motifs conserved among helicases. We have identified a domain essential for DNA binding at residues 638-786. The binding domain is less conserved (63% homologous) than the putative catalytic domain of N-terminal half containing most of the helicase motifs (85% homologous). The human and mouse S mu bp-2 have similar, although slightly different, binding specificities. Although the mouse S mu bp-2 preferentially binds to the mouse S mu motif (GGGGT), the human S mu bp-2 binds equally well to the human (GGGCT) and mouse S mu motifs. The human S mu bp-2 gene was mapped to chromosome 11 q13.2-q13.4 by in situ hybridization.

We have isolated the cDNA encoding a binding protein to the sequence motif of the immunoglobulin S mu region by the southwestern method. The binding protein designated S mu bp-2 specifically binds to 5'-phosphorylated single-stranded DNA containing 5'-G and GGGG stretches. The amino acid sequence deduced from the cDNA sequence showed that the S mu bp-2 belongs to the putative helicase superfamily which is involved in replication, recombination and repair. Expression of S mu bp-2 mRNA is ubiquitous and augmented in spleen cells stimulated with lipopolysaccharide and interleukin 4 which also induce class switching. The S mu bp-2 gene is conserved among vertebrates. Possible involvement of S mu bp-2 in class switching is discussed.

The SSK41 cell is an immunoglobulin IgM+ human neoplastic B-cell line that switches to IgG+ cells (SSK gamma) spontaneously. We isolated a derivative of SSK41 (SSKWB) that expressed both IgM and IgG. Studies on the Ig heavy chain gene organization have shown that the SSKWB cell had two identical VDJ genes on the same chromosome; one linked to the C mu gene and the other to the C gamma 1 gene, both of which are transcribed to produce mu- and gamma-mRNAs with the same VDJ sequence. We also isolated the two switch variants derived from the SSKWB cell by cell sorter: 1G (IgG+) and 11M (IgM+). The 1G cells contained two populations; one had a similar genetic organization as SSK gamma and expressed only IgG1, and the other carried the same genetic organization as the SSKWB cell but produced aberrantly spliced mu-chain mRNA, in which the hydrophobic signal sequence exon is directly joined to the C mu exon. Te 11M cell deleted the VDJ-C gamma 1 segment of the SSKWB cell and expressed IgM. These results raise the interesting possibility of another mechanism for class switching.

In three lymphoma cell lines carrying t(14;18), named FL-18, FL-218, and FL-318, the genomic organization of IgH gene was compared with the expression of bcl-2 gene; the t(14;18) of the FL-18 cells occurred downstream from the major breakpoint cluster region (mbr) of a bcl-2 gene, and that of the FL-218 and FL-318 cells within the mbr. The FL- 318 expressed the normal-sized bcl-2 transcript of 8.5-kb mRNA having the noncoding region 3 to the mbr, which was found in the FL-18, and the FL-218 lacking the intact bcl-2 gene did not. This finding suggests that in t(14;18)-positive lymphoma having the breakpoint within the mbr, transcription of the nontranslocated bcl-2 allele is not necessarily silent. In addition, the FL-218 and FL-318 expressed aberrant bcl-2 transcripts and heterogenous IgH transcripts lacking the VH region, and the bcl-2 transcripts each comigrated with parts of the sterile IgH mRNAs. The FL-318, which did not exhibit switch recombination on either IgH allele, contained abundant amounts of l gamma mRNAs, a prerequisite for the recombination into the C gamma locus. One of the I-mRNA species comigrated with the aberrant bcl-2 transcript. The FL-18 and FL-218 lacking the I gamma mRNAs had completed switch recombination of both IgH alleles. This result raises a possibility that deregulated bcl-2 transcription caused by t(14;18) is capable of playing a role in class switch recombination of IgH gene.

A human neoplastic B cell line SSK41 that expresses IgM on its surface switches spontaneously to IgG-producing cells. The SSK41 line contains a single immunoglobulin heavy-chain locus, the constant region (C) genes of which retain the germline configuration. The IgG-producing SSK41 line was purified by sorting, and shown to have undergone S-S recombination with deletion of the C mu gene. This line produced secretory and membrane-bound forms of gamma-chain mRNA. From cDNA libraries of a mixed population of IgM+/IgG+ SSK41 cells, we have isolated cDNA clones encoding the mature membrane-bound and secretory forms of the mu and gamma 1 heavy chains, all of which share the same variable region sequence. cDNA clones containing the mature gamma 3 chain were identified as well. We also isolated cDNA clones containing C gamma 1 and C gamma 3 sterile transcripts from the SSK41 line. These sterile transcripts contained additional exon sequences designated 'I' which were localized upstream of the C gamma 1 and C gamma 3 switch regions and homologous to murine counterparts. The I sequences were precisely spliced to the 5' ends of the corresponding C gamma exon sequences. These features of germline CH transcripts, i.e. the isotype specificity to class switching, location of exons, and sequences per se, are highly conserved between man and mouse.

We isolated the chromosomal gene for mouse interleukin-5 (IL-5) from genomic libraries using a cloned mouse IL-5 cDNA as probe. Nucleotide sequence determination of the IL-5 gene and its flanking regions showed that the gene was composed of four exons. The sequence homologies of the exons 1, 2, 3, and 4 of the human and murine IL-5 genes are 70, 79, 85, and 77%, respectively. TATA-like and CAAT-like sequences were found 25 and 52 base-pairs, respectively, upstream of the transcription initiation site which was identified by S1 mapping analysis. The nucleotide sequences of the mouse and human IL-5 genes were about 70% homologous in the 5'-flanking region extending to 200 base-pairs upstream of the transcription initiation site, beyond which the homology declined rapidly. The sequence surrounding 50 base-pairs upstream of the initiation site was conserved among genes for other lymphokines: such as mouse granulocyte/macrophage colony stimulating factor, mouse IL-2, and mouse IL-4.

We have studied immunoglobulin double-isotype expression in a transgenic mouse (TG.SA) in which expression of the endogenous immunoglobulin heavy chain locus is almost completely excluded by a nonallelic rearranged human mu transgene. By flow-cytometric analyses, we have shown that a small, but significant, portion (about 4%) of transgenic spleen cells expresses human mu (transgene) and mouse gamma (endogenous) chains when cultured in vitro with bacterial lipopolysaccharide and interleukin 4. By using amplification of cDNA by the polymerase chain reaction, followed by cloning and sequencing of the amplified cDNA fragment, we have demonstrated expression of trans-mRNA consisting of the transgenic variable and endogenous constant (gamma 1) region sequences. Such trans-mRNA could be produced by either switch recombination or trans-splicing between the transgene and endogenous sterile gamma 1-gene transcripts. These results indicate that trans-splicing might be a possible mechanism for the immunoglobulin double-isotype expression in normal B lymphocytes that have not rearranged the second expressed constant region gene.

We isolated the chromosomal gene for human interleukin-5 (IL-5) from human genomic libraries using a cloned human IL-5 cDNA as probe. Nucleotide sequence determination of the IL-5 gene and its flanking regions showed that the gene consisted of four exons and three introns. TATA-like and CAAT-like sequences reside 26 and 73 base pairs, respectively, upstream of the transcription initiation site identified by S1 mapping analysis. The 5'-flanking region of the IL-5 gene has sequences homologous with those of the corresponding regions of the genes for human granulocyte/macrophage colony-stimulating factor, murine IL-4, human interferon-gamma, and human IL-2. The intron-exon organization and location of the cysteine residue suggest that the IL-5 gene is phylogenetically related to the genes for the granulocyte/macrophage colony-stimulating factor, IL-4, and IL-2, although their amino acid sequences are not significantly conserved.

Three new human lymphokines, interleukin-5, BSF-2 and BSF-MP6, were shown to be active in the enhancement of the IL-2 receptor expression on T cells, although they do not stimulate growth of the T cells.