Extrachromosomal circular DNA (eccDNA) generated from chromosomal DNA is found in all mammalian cells and increases with cell stress or aging. Studies of eccDNA structure and mode of formation provide insight into mechanisms of instability of the mammalian genome. Previous studies have suggested that eccDNA is generated through a process involving recombination between repetitive sequences. However, we observed that approximately one half of the small eccDNA fragments cloned from HeLa S3 cells were composed entirely of nonrepetitive or low-copy DNA sequences. We analyzed four of these fragments by polymerase chain reaction and nucleotide sequencing and found that they were complete eccDNAs. We then screened a human genomic library with the eccDNAs to isolate the complementary chromosomal sequences. Comparing the recombination junctions within the eccDNAs with the chromosomal sequences from which they were derived revealed that nonhomologous recombination was involved in their formation. One of the eccDNAs was composed of two separate sequences from different parts of the genome. These results suggest that rejoining of ends of fragmented DNA is responsible for the generation of a substantial portion of the eccDNAs found in HeLa S3 cells.

Human herpesvirus 6 (HHV-6) and HHV-7 are closely related T-lymphotropic betaherpesviruses which share a common genomic organization and are composed of a single unique component (U) that is bounded by direct repeats (DRL and DRR). In HHV-6, a sequences have been identified at each end of the DR motifs, resulting in the arrangement aDRLa-U-aDRRa. In order to determine whether determine whether HHV-7 contains similar a sequences, we have sequenced the DRL-U and U-DRR junctions of HHV-7 strain JI, together with the DRR.DRL junction from the head-to-tail concatamer that is generated during productive virus infection. In addition, we have sequenced the genomic termini of an independent isolate of HHV-7. As in HHV-6, a (GGGTTA)n motif identical to the human telomeric repeat sequence (TRS) was identified adjacent to, but not at, the genome termini of HHV-7. The left genome terminus and the U-DRR junction contained a homolog of the consensus herpesvirus packaging signal, pac-1, followed by short tandem arrays of TRSs separated by single copies of a second 6-bp repeat. This organization is similar to the arrangement found at U-DRR in HHV-6 but differs from it in that the TRS arrays are considerably shorter in HHV-7. The right genome terminus and the DRL-U junction contained a homolog of the consensus herpesvirus packaging signal, pac-2, followed by longer tandem arrays of TRSs separated by single copies of either a 6-bp or a 14-bp repeat. This arrangement is considerably more complex than the simple tandem array of TRSs that is present at the corresponding genomic location in HHV-6 and corresponds to a site of both inter- and intrastrain heterogeneity in HHV-7. The presence of TRSs in lymphotropic herpesviruses from humans (HHV-6 and HHV-7), horse (equine herpesvirus 2), and birds (Marek's disease virus) is striking and suggests that these sequences may have functional or structural significance.

Human herpesvirus 6 (HHV-6) is a T-lymphotropic herpesvirus, which infects almost all children by the age of two years and persists lifelong. Two distinct variants of HHV-6, HHV-6A and HHV-6B, have been described, and the latter has been shown to be a common cause of acute febrile illnesses in young children, including exanthem subitum (roseola). HHV-6 has also been associated with a number of neurological disorders, including encephalitis and seizures, and the virus has been postulated to play a role in acquired immunodeficiency syndrome (AIDS), multiple sclerosis (MS) and chronic fatigue immunodeficiency syndrome (CFIDS). This review provides a critical summary of research conducted on HHV-6.

Human herpesvirus 7 (HHV-7) DNA sequences colinear with the HHV-6 lytic-phase origin of DNA replication (oriLyt) were amplified by PCR. Plasmid constructs containing these sequences were replicated in HHV-7-infected cord blood mononuclear cells but not in HHV-6-infected cells. In contrast, plasmids bearing HHV-6 oriLyt were replicated in both HHV-6-and HHV-7-infected cells. Finally, the minimal HHV-7 DNA element necessary for replicator activity was mapped to a 600-bp region which contains two sites with high homology to the consensus binding site for the HHV-6 origin binding protein. At least one of these binding sites was shown to be essential for replicator function of HHV-7 oriLyt.

Chinese hamster ovary cells were synchronized at the G1/S-phase boundary of the cell cycle and pulse-labeled for 10 min with 125I-iododeoxyuridine 30 min after entering the S phase. Cell samples were harvested for freezing and 125I-decay accumulation at intervals ranging from 15 to 480 min after termination of labeling. The survival data showed a marked shift from cell killing characteristic of low-LET radiation to that more characteristic of killing by high-LET radiation with increasing intervals between DNA pulse-labeling and decay accumulation. Cells harvested and frozen within 1 h after pulse-labeling yielded a low-LET radiation survival response with a pronounced shoulder and a large D0 of up to 0.9 Gy. With longer chase periods the shoulder and the D0 decreased progressively, and cells harvested 5 h after pulse-labeling or later exhibited a high-LET survival response (D0: 0.13 Gy). Two interpretations for these findings are discussed. (1) If DNA is the sole target for radiation death, the results indicate that DNA maturation increases radiation damage to DNA or reduces damage repair. (2) If radiation cell death involves damage to higher-order structures in the cell nucleus, the findings suggest that newly replicated DNA is not attached to these structures during the initial low-LET period, but 125I starts to induce high-LET radiation effects as labeled DNA segments become associated with the target structure(s). On balance, or data favor the latter interpretation.