Kenji Kurokawa

The Initiator Function of DnaA Protein Is Negatively Regulated by the Sliding Clamp of the E. coli Chromosomal Replicase

The beta subunit of DNA polymerase III is essential for negative regulation of the initiator protein, DnaA. DnaA inactivation occurs through accelerated hydrolysis of ATP bound to DnaA; the resulting ADP-DnaA fails to initiate replication. The ability of beta subunit to promote DnaA inactivation depends on its assembly as a sliding clamp on DNA and must be accompanied by a partially purified factor, IdaB protein. DnaA inactivation in the presence of IdaB and DNA polymerase III is further stimulated by DNA synthesis, indicating close linkage between initiator inactivation and replication. In vivo, DnaA predominantly takes on the ADP form in a beta subunit-dependent manner. Thus, the initiator is negatively regulated by action of the replicase, a mechanism that may be key to effective control of the replication cycle.

Replication cycle-coordinated change of the adenine nucleotide-bound forms of DnaA protein in Escherichia coli

The ATP‐bound but not the ADP‐bound form of DnaA protein is active for replication initiation at the Escherichia coli chromosomal origin. The hydrolysis of ATP bound to DnaA is accelerated by the sliding clamp of DNA polymerase III loaded on DNA. Using a culture of randomly dividing cells, we now have evidence that the cellular level of ATP–DnaA is repressed to only ∼20% of the total DnaA molecules, in a manner depending on DNA replication. In a synchronized culture, the ATP–DnaA level showed oscillation that has a temporal increase around the time of initiation, and decreases rapidly after initiation. Production of ATP–DnaA depended on concomitant protein synthesis, but not on SOS response, Dam or SeqA. Regeneration of ATP–DnaA from ADP–DnaA was also observed. These results indicate that the nucleotide form shifts of DnaA are tightly linked with an epistatic cell cycle event and with the chromosomal replication system.

Evaluation of adult T‐cell leukemia/lymphoma incidence and its impact on non‐Hodgkin lymphoma incidence in southwestern Japan

The incidence of adult T‐cell leukemia/lymphoma (ATL) and its impact on that of total non‐Hodgkin lymphoma (NHL) were evaluated in Nagasaki, an area in southwestern Japan where human T‐cell lymphotropic virus type I (HTLV‐I) is endemic. The first study area comprised 4 towns located on the K Islands, which had a population of 26,870 in 1990. The overall HTLV‐I seroprevalence estimated from the serologic survey of 18,485 subjects was 16.2%. By using the data from the Nagasaki Prefectural Cancer Registry (NPCR) and reviewing clinical and laboratory information, we identified 40 cases of ATL and 35 cases of other NHL diagnosed between 1985 and 1995. The crude annual incidence of ATL among 100,000 HTLV‐I carriers aged 30 or older was estimated at 137.7 for men and 57.4 for women, with a significant sex difference after adjustment for age (rate ratio = 2.50, 95% confidence interval 1.32–4.73). The cumulative risk from 30 to 79 years of age was estimated at approximately 6.6% for men and 2.1% for women. Among the entire population, ATL accounted for 51 to 59% of the total NHL incidence, showing the strong impact of HTLV‐I infection. The second study area comprised the whole of Nagasaki Prefecture (total population in 1990 = 1.56 million). Between 1985 and 1995, 989 cases of ATL and 1,745 cases of other NHL were registered in the NPCR. The world age‐standardized annual incidence rate of ATL per 100,000 persons aged 30 or older was estimated at 10.5 for men and 6.0 for women, which accounted for approximately 37 to 41% of the total NHL incidence. Int. J. Cancer 85:319–324, 2000. ©2000 Wiley‐Liss, Inc.

Negative control of DNA replication by hydrolysis of ATP bound to DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli

DnaA protein, the initiation factor for chromosomal DNA replication in Escherichia coli, is activated by ATP. ATP bound to DnaA protein is slowly hydrolyzed to ADP, but the physiological role of ATP hydrolysis is unclear. We constructed, by site‐directed mutagenesis, mutated DnaA protein with lower ATPase activity, and we examined its function in vitro and in vivo. The ATPase activity of purified mutated DnaA protein (Glu204→Gln) decreased to one‐third that of the wild‐type DnaA protein. The mutation did not significantly affect the affinity of DnaA protein for ATP or ADP. The mutant dnaA gene showed lethality in wild‐type cells but not in cells growing independently of the function of oriC. Induction of the mutated DnaA protein in wild‐type cells caused an overinitiation of DNA replication. Our results lead to the thesis that the intrinsic ATPase activity of DnaA protein negatively regulates chromosomal DNA replication in E.coli cells.

DNA replication-coupled inactivation of DnaA protein in vitro: a role for DnaA arginine-334 of the AAA+ Box VIII motif in ATP hydrolysis.

The DnaA protein, which initiates chromosomal replication in Escherichia coli, is negatively regulated by both the sliding clamp of DNA polymerase III holoenzyme and the IdaB protein. We have found that, when the amount of minichromosome is limited in an in vitro replication system, minichromosomal replication‐stimulated hydrolysis of DnaA‐bound ATP yields the ADP‐bound inactive form. The number of sliding clamps formed during replication was at least five per minichromosome, which is 2.7‐fold higher than the number formed during incubation without replication. These results support the notion that coupling of DnaA‐ATP hydrolysis to DNA replication is the outcome of enhanced clamp formation. We have also found that the amino acid substitution R334H in DnaA severely inhibits the hydrolysis of bound ATP in vitro. Whereas ATP bound to wild‐type DnaA is hydrolysed in a DNA‐dependent intrinsic manner or in a sliding clamp‐dependent manner, ATP bound to DnaA R334H protein was resistant to hydrolysis under the same conditions. This arginine residue may be located in the vicinity where ATP binds, and therefore may play an essential role in ATP hydrolysis. This residue is highly conserved among DnaA homologues and also in the Box VIII motif of the AAA+ protein family.

High frequency of postnatal transmission of TT virus in infancy

Abstract.u2002DNA of TT virus (TTV), a novel human circovirus, was tested for in 116 mother-infant pairs who had participated in the adult T-cell leukemia prevention program (APP) in Nagasaki, Japan, and refrained from breast-feeding. By polymerase chain reaction with Okamoto’s seminested primers, 36 of the 115 (31%) mothers were positive. At the age of 6–8 months, 7 of 29 (24%) and 6 of 72 (8%) infants born to infected and uninfected mothers were positive, respectively (Pu2009=u20090.047; RR, 2.90). Maternal TTV DNA load did not correlate with infantile infections. Since 99 of 100 (99%) cord blood samples were negative and all the mothers refrained from breast-feeding, the infantile TTV transmission would not be intrauterine or milk-borne. Between 6–8 and 12–21 months of age, 4 of 12 (33%) and 5 of 22 (23%) children born to infected and uninfected mothers turned positive, respectively (NS). At 12–21 months of age, 8 of 21 (38%) and 12 of 32 (38%) children born to infected and uninfected mothers were positive, respectively (NS). These results indicate that the TTV infection prevails in children at a frequency comparative to that in their mothers within the first 2 years of life, regardless of the maternal TTV status.

Reproductive activity and survival of Culex pipiens pallens and Culex quinquefasciatus (Diptera: Culicidae) in Japan at high temperature.

Abstract The egg hatchability, insemination, and longevity of Japanese Culex pipiens pallens Coquillett and Japanese Culex quinquefasciatus Say were compared at 25 and 30°C. Egg hatchability was high in Cx. p. pallens at 25°C, but it was very low at 30°C because almost no females were inseminated at this temperature. In Cx. quinquefasciatus, the egg hatchability and insemination rates were very high, even at 30°C. The longevity of adult females and males was generally shorter in Cx. p. pallens than in Cx. quinquefasciatus at both temperatures. Because high temperatures may restrict the spread of Cx. p. pallens, we suggest that even if this species spreads to Okinawa, the possibility of it becoming established is very low.

A nested case–control study of risk factors for adult T-cell leukemia/lymphoma among human T-cell lymphotropic virus type-I carriers in Japan

Objectives: The purpose of this study was to investigate the serological risk factors for development of adult T-cell leukemia/lymphoma (ATL) among human T-cell lymphotropic virus type-I (HTLV-I) carriers. Methods: A nested case–control study was performed. The source population comprised 23,922 subjects who had either visited the outpatient clinic or who had received annual health check-ups at the K Hospital, Nagasaki, Japan, at least once during 1985–1996 (HTLV-I seroprevalence = 16.1%). Markers of HTLV-I infection were examined in stored sera from 29 incident cases of ATL diagnosed during 1985–1997, and 158 controls matched for sex, birth year, date of sample collection, and HTLV-I seropositivity (median follow-up = 6.4 years). Results: In exact conditional logistic regression analysis, high levels of soluble interleukin-2 receptor ( ≥ 500 U/ml) and high HTLV-I antibody titers (≥ 1024) were independently associated with an increased risk of developing ATL (Odds ratio 20.5, 95% confidence interval (CI) 4.5–194 and 2.9, 95% CI 0.98–9.5, respectively). The results remained essentially unchanged when the subjects were restricted to those whose histories were followed for two years or longer. Conclusions: These findings indicate that high soluble interleukin-2 receptor levels and high HTLV-I antibody titers are strong predictors of ATL among carriers of HTLV-I.

Trend in Chlamydia trachomatis infection among pregnant women in the past ten years in Japan: significance of Chlamydia trachomatis seroprevalence.

Background and Objectives: Chlamydia trachomatis infection is believed to be the most common bacterial sexually transmitted disease (STD) in industrialized countries. The objective of the current study was to assess the recent trend in the prevalence of C. trachomatis in Japan. Goal of this Study: To determine the trend in the seroprevalence for C. trachomatis among pregnant women in Nagasaki, Japan, during the past 10 years. Study Design: The seroprevalence for C. trachomatis of 9,652 pregnant women of various ages screened in 1996 and 1997 was compared with those of 275 and 297 stocked samples from 1987 and 1992, respectively. Serum antibodies to C. trachomatis were detected by the enzyme immunoassay. Prospective samples of 33 seropositive cases were also analyzed to determine kinetics of the serum antibody titer. Results: The seroprevalence has decreased in all age groups during the last 10 years. More than 70% of seropositive cases converted to be seronegative within 10 years. Conclusion: The prevalence of C. trachomatis has been decreasing among Japanese pregnant women.

Deleted HTLV-1 provirus in cord-blood samples of babies born to HTLV-1-carrier mothers

We screened 596 cord‐blood samples by nested short PCRs for the gag and pX regions of HTLV‐1 which are capable of detecting a single copy. The samples were derived from 449 and 147 babies born to seropositive and seronegative mothers respectively. Of these, 20 samples were positive in at least one of the PCRs: 9 (45%) were positive in both PCRs, but 10 and 1 samples in either the pX or the gag PCR respectively. These samples were tested further in nested long PCRs directed for gag‐pX, gag‐pol and pol‐pX regions capable of detecting 8, 1 and 2 copies respectively. Of 9 dually positive samples, 7 (77%) showed the predicted 6.2‐kbp band in the gag‐pX PCR; only 2 of them had the predicted band alone; 7 samples had discrete bands shorter than the predicted size. In the gag‐pol PCR, all 9 samples showed the predicted 2.2‐kbp band alone. In the pol‐pX PCR, 8/9 samples showed the predicted 4.2‐kbp band, including one with an additional 2.1‐kbp band, and the last a 1.0‐kbp band alone. Thus, all of the dually positive samples had proviruses harboring gag, pol and pX priming sites. In contrast, none of the 11 singly positive samples showed the predicted band in the gag‐pX PCR: 5 had no visible band, and the other 6 had shorter bands only. None of these 11 samples showed any positive signal in either gag‐pol or pol‐pX PCR. Our results suggest that HTLV‐1 proviruses in the cord blood are frequently defective. Int. J. Cancer 77:701–704, 1998.