Specific terminal DNA replication sequence of X chromosomes in different tissues of a live-born triploid infant

Using the thymidine pulse method, DNA replication kinetics were studied on cells derived from cartilage, gonad, lymphocytes, and skin of a live-born triploid (69,XXY) infant with typical clinical findings. Replication studies showed that 3% of the lymphocytes had one early and one late replicating X, and 97% of the lymphocytes, and cartilage, gonad, and skin cells had two early replicating X's. Asynchronous DNA replication between the two early replicating X's was observed in all tissues (range 25-40%). The predominant terminal replication sequence of X chromosomes from chondrocytes, gonad, and skin fibroblast differed from that of the lymphocytes. Thus, a tissue-specific DNA replication pattern of the early-replicating X chromosome may be present. In every tissue, the last band to complete DNA replication was Xq21. Polymorphisms of metaphase chromosomes of parents and the patient were studied by Q-banding. The possible origin of the extra haploid set of chromosomes is discussed.     

Analyzing DNA strand compositional asymmetry to identify candidate replication origins of Borrelia burgdorferi linear and circular plasmids

The Lyme disease agent Borrelia burgdorferi has a genome composed of a linear chromosome and a series of linear and circular plasmids. We previously mapped the oriC of the linear chromosome to the center of the molecule, where a pronounced switch in CG skew occurs. In this study, we analyzed B. burgdorferi plasmid sequences for AT and CG skew in an effort to similarly identify plasmid replication origins. Cumulative skew diagrams of the plasmids suggested that they, like the linear chromosome, replicate bidirectionally from an internal origin. The B. burgdorferi linear chromosome contains homologs to partitioning protein genes soj and spoOJ, which are closely linked to oriC at the minimum cumulative skew point of the 1-Mb molecule. A soj/parA homolog also maps to cumulative skew minima of the B. burgdorferi linear and circular plasmids, further suggesting that these regions contain the replication origin. The heterogeneity in these genes and in the nucleotide sequences of the putative origin regions could account for the mutual compatibility of the multiple DNA elements in B. burgdorferi.     

Mitochondrial DNA of Chlamydomonas reinhardtii: the structure of the ends of the linear 15.8-kb genome suggests mechanisms for DNA replication

The mitochondrial genome of Chlamydomonas reinhardtii is a linear double-stranded DNA of 15.8 kb. With the exception of the termini its DNA sequence has been published. Here we describe the unique structure of the two termini determined from cloned fragments or, for the very terminal sequences, by the Maxam and Gilbert method after 5 labeling of uncloned terminal fragments. The 15.8-kb DNA is characterized by terminal inverted repeats of 531 or 532 bp in length including long 3 extensions. The 3 single-stranded extensions of the left and right ends are non-complementary, identical in sequence, and comprise 39 to 41 nucleotides. Remarkably, the linear genome possesses in addition an internal 86-bp repeat of the two outermost sequences. The unusual structure of the 15.8-kb DNA termini is compared with those of other linear mitochondrial DNAs. Possible mechanisms of 15.8-kb DNA replication are discussed.Dedicated to Prof Dr. Piotr Slonimski on the occasion of this 70th birthday     

A novel ring-like complex of Xenopus proteins essential for the initiation of DNA replication

We have identified Xenopus homologs of the budding yeast Sld5 and its three interacting proteins. These form a novel complex essential for the initiation of DNA replication in Xenopus egg extracts. The complex binds to chromatin in a manner dependent on replication licensing and S-phase CDK. The chromatin binding of the complex and that of Cdc45 are mutually dependent and both bindings require Xenopus Cut5, the yeast homolog of which interacts with Sld5. On replicating chromatin the complex interacts with Cdc45 and MCM, putative components of replication machinery. Electron microscopy further reveals that the complex has a ring-like structure. These results suggest that the complex plays an essential role in the elongation stage of DNA replication as well as the initiation stage.     

The linear plasmid pSA3239 is essential for the replication of the Streptomyces lavendulae subsp. lavendulae CCM 3239 chromosome

We previously reported the complete genome of Streptomyces lavendulae subsp. lavendulae CCM 3239, containing the linear chromosome and the large linear plasmid pSA3239. Although the chromosome exhibited replication features characteristic for the archetypal end-patching replication, it lacked the tap/tpg gene pair for two proteins essential for this process. However, this archetypal tpgSa-tapSa operon is present in pSA3239. Complete genomic sequence of the S. lavendulae Del-LP strain lacking this plasmid revealed the circularization of its chromosome with a large deletion of both arms. These results suggest an essential role of pSA3239-encoded TapSa/TpgSa in the end-patching replication of the chromosome.     

Antibodies specific for the phi 29 terminal protein inhibit the initiation of DNA replication in vitro

The phi 29 DNA-terminal protein serves as a primer for the initiation of DNA replication by covalently binding the first nucleotide in the DNA chain. Two distinct antibodies were used for functional analysis of this protein. One antibody was raised against sonicated phi 29 DNA-protein complex isolated from phage virions (anti-TP). The other antibody was raised against a conjugate of bovine serum albumin and a synthetic peptide corresponding to the carboxy-terminal of the phi 29 terminal protein (anti-gp3C), which was predicted from the nucleotide sequence of phi 29 DNA. Both antibodies react with native phi 29 terminal protein as determined by immunoprecipitation and enzyme-linked immunosorbent assay. Both antibodies specifically inhibit the complex-forming reaction between the phi 29 terminal protein and dAMP, the first nucleotide of phi 29 DNA.     

ATPase/helicase motif mutants of Escherichia coli PriA protein essential for recombination-dependent DNA replication

BACKGROUNDS: PriA protein, a DEXH-type helicase with C2C2 zinc-finger motifs, plays essential roles in RecA-dependent modes of Escherichia coli chromosomal DNA replication, namely inducible and constitutive stable DNA replication (iSDR and cSDR respectively, which may be initiated from a D-loop or R-loop structure), and in repair of double-stranded DNA breaks generated by various genotoxic agents or spontaneously during the course of DNA replication. However, the roles of ATPase/DNA helicase activities in functions of PriA are not well understood. RESULTS: We have generated and characterized mutants of PriA protein carrying amino acid substitutions in its conserved ATPase/DNA helicase motifs, namely the Walker A, B and QXXGRXGR motifs. All these mutants were deficient in ATP hydrolysis and DNA helicase activities, but showed wild-type levels of D-loop DNA binding, except for the Walker B mutant which showed reduced DNA binding activity, suggesting that the helicase motifs are not directly involved in the DNA binding activity of PriA protein. They also rescued the low viability and UV-sensitivity of priA null cells. However, they did not rescue iSDR or cSDR-alternative modes of chromosomal DNA replication of the E. coli genome dependent on recombination functions-to the full extent. CONCLUSIONS: ATPase/DNA helicase activities of PriA protein are required for full-level DNA synthesis in recombination-dependent modes of DNA replication in E. coli.     

Binding of SV40 a protein to the BK virus origin of DNA replication

The SV40 A protein (T antigen) binds to the putative origin of replication of the related BK virus (BKV). Protection studies with dimethyl sulfate identify multiple pentanucleotide contact sites in three distinct binding regions. In addition to the consensus family of recognition sequences, 5′-(G > T)(A > G)GGC-3′, previously identified in the origin of SV40, BKV contains a contact 5′-AAGGC-3′ site. As in the case of SV40, each binding region contains recognition sequences in different arrangements. The high affinity region I contains two sites arranged as direct repetitions covering a span of 17 base pairs (bp). In the intermediate affinity region II, four pentanucleotides are oriented as inverted repetitions with a span of 23 bp. Low affinity region III has a single contact site that can direct binding of the A protein. These different arrangements of DNA contact sites determine different patterns of protein binding and DNase protection in the three regions. The findings support a model of A protein binding to DNA previously proposed for the SV40 origin and establish a basis for future studies on regulatory phenomena at the BKV origin.     

Identification of the protein firmly bound to the ends of bacteriophage phi 29 DNA.

The linear DNA duplex of the Bacillus subtilis phage φ29 is firmly associated at its ends with protein which is essential for transfectivity. The DNA-protein complex was isolated from phage particles (subsequently labeled with ¹²⁵I by lactoperoxidase-catalyzed iodination) and also from phage-infected cells labeled in vivo with [³⁵S]methionine. The purified complexes were digested with DNase I and phosphodiesterase and the proteins were analyzed by electrophoresis in discontinuous-pH SDS-polyacrylamide gels and in SDS-urea gels of high cross-linkage. The main protein, peak from both sources of DNA has a molecular weight of about 31,000. Occasionally, low-molecular weight protein was observed which probably represents fragments of the larger protein. Digestion of φ29 DNA with the restriction endonuclease EcoRl and analysis of purified terminal restriction fragments indicated that proteins of the same molecular weight bind firmly at each end of the DNA molecule. We postulate that the 31,000-dalton protein may be specified by gene 3, the product of which affects both transfection and DNA replication.     

PHYTOCLOCK 1 encoding a novel GARP protein essential for the Arabidopsis circadian clock

Previously, we screened 50 000 seedlings of Arabidopsis thaliana carrying a P(GI)::LUC+ bioluminescence reporter gene mutagenized with ethylmethanesulfonate for mutants with phenotypes of extensively altered circadian rhythms, and identified three loci, PHYTOCLOCK 1 (PCL1), PCL2 and PCL3, whose mutations cause arrhythmia. Here we succeeded to clone the PCL1 gene and show that the PCL1 gene encodes a novel DNA binding protein belonging to the GARP protein family and is essential for a functional clock oscillator in A. thaliana. The PCL1 gene satisfies the requirements for the clock oscillator gene: (i) pcl1 null mutations caused arrhythmia in multiple circadian outputs, including expression of potential clock genes TOC1, CCA1 and LHY, and flowering lacked a photoperiodic response; (ii) PCL1 expression showed circadian rhythm in both constant light and constant dark; (iii) over-expression of the PCL1 gene gradually caused arrhythmicity in all the multiple circadian outputs examined; and (iv) the PCL1 gene controlled its own expression via negative feedback. Therefore, the PCL1 gene is the clock oscillator gene essential to the generation of clock oscillation in the higher plant.     

GTPase activity is not essential for the interferon-inducible MxA protein to inhibit the replication of hepatitis B virus

Multiple studies have established that GTPase activity is critical for MxA to act against RNA viruses. Recently, it was shown that MxA can also restrict the replication of hepatitis B virus (HBV), a DNA virus, but the requirements for GTPase activity in inhibition of HBV by MxA remain unknown. Here, we report that GTPase-defective mutants (K83A, T103A, and L612K) can downregulate extracellullar HBsAg and HBeAg and reduce the expression of extra- and intracellular HBV DNA in HepG2 cells to levels similar to that achieved by wild-type MxA. Furthermore, TMxA and T103, two nuclear forms of wild-type MxA and a GTPase-defective mutant (T103A) could only slightly decrease the expression of extra- and intracellular HBV DNA in HepG2 cells. In conclusion, GTPase activity is not essential for MxA protein to inhibit HBV replication, and MxA may have only a minimal effect on the replicative cycle of HBV in the nucleus.     

Nucleotide sequence of a gene essential for viral DNA replication in the baculovirus Autographa californica nuclear polyhedrosis virus

The nucleotide sequence of the 60.1- to 65.5-m.u. region of Autographa californica nuclear polyhedrosis virus (AcMNPV) was determined. Seven large open reading frames were identified. Two open reading frames potentially encoding gene products of 143 and 38 kDa were found in the counterclockwise direction upstream of the p6.9 gene. Four additional open reading frames were found in the opposite direction. Analysis of the predicted amino acid sequence of the 143-kDa gene revealed a potential leucine zipper motif, a putative nuclear localization signal, and seven amino acid motifs previously identified in a number of proteins involved in NTP binding and DNA/RNA unwinding. The mutation in a DNA replication defective temperature-sensitive mutant was fine mapped to the carboxy terminus of the ORF1(p143) gene. Sequence analysis of the mutation site identified a single base change of a guanine to an adenine, resulting in the substitution of a methionine for valine. This mutation resides seven amino acids downstream of the putative NTP-binding motif of the ORF1(p143) gene product and results in a DNA negative mutant. Together these data strongly suggest that the ORF1(p143) gene product is a baculovirus helicase.     

The terminal protein of a linear mitochondrial plasmid is encoded in the N-terminus of the DNA polymerase gene in white-rot fungus Pleurotus ostreatus

The gene structure and expression of the linear mitochondrial plasmids of the white-rot fungus Pleurotus ostreatus, pMLP1 and pMLP2, were analyzed. Cleavage by proteinase K and exonucleases indicated that the 5′ ends of pMLP1 and pMLP2 DNAs were associated with terminal proteins. Nucleotide sequencing of the entire pMLP1 DNA revealed that it consists of 9,879 bp with terminal inverted repeat (TIR) sequences of 381 bp. The end sequence of TIR in pMLP1 is 3′-CCCCC-5′, similar to those of Escherichia coli phage PRD1. The pMLP1 plasmid harbors two long open reading frames (ORF1 and ORF2) and at least one minor ORF (mORF1). The deduced product of ORF1 is homologous to RNA polymerases of yeast mitochondria and several bacteriophages, whereas that of ORF2 is homologous to the protein-primed DNA polymerases of family B type. The mORF1 encodes a highly basic protein, most likely a TIR-binding protein, with no apparent sequence homology in the database. Expression of the predicted gene products from pMLP1 in mitochondria was demonstrated by Western blot analysis using antibodies against various expressed regions of pMLP1 ORFs. A plasmid-free strain, generated by curing with ethidium bromide, did not express any of these gene products. Terminal proteins of 70 kDa (TP1) and 73 kDa (TP2) were identified from pMLP1 and pMLP2, respectively. Western blot analysis indicated that TP1 was generated from the N-terminal half of the full-length product of ORF2 encoding a putative DNA polymerase. Received: 9 February 2000 / Accepted: 18 July 2000     

The Saccharomyces telomere-binding protein Cdc13p interacts with both the catalytic subunit of DNA polymerase alpha and the telomerase-associated est1 protein

Saccharomyces telomeres consist of approximately 350 bp of C(1-3)A/TG(1-3) DNA. Most of this approximately 350 bp is replicated by standard, semiconservative DNA replication. After conventional replication, the C(1-3)A strand is degraded to generate a long single strand TG(1-3) tail that can serve as a substrate for telomerase. Cdc13p is a single strand TG(1-3) DNA-binding protein that localizes to telomeres in vivo. Genetic data suggest that the Cdc13p has multiple roles in telomere replication. We used two hybrid analysis to demonstrate that Cdc13p interacted with both the catalytic subunit of DNA polymerase alpha, Pol1p, and the telomerase RNA-associated protein, Est1p. The association of these proteins was confirmed by biochemical analysis using full-length or nearly full-length proteins. Point mutations in either CDC13 or POL1 that reduced the Cdc13p-Pol1p interaction resulted in telomerase mediated telomere lengthening. Over-expression of the carboxyl terminus of Est1p partially suppressed the temperature sensitive lethality of a cdc13-1 strain. We propose that Cdc13p's interaction with Est1p promotes TG(1-3) strand lengthening by telomerase and its interaction with Pol1p promotes C(1-3)A strand resynthesis by DNA polymerase alpha.     

Cooperativity at a distance promoted by the combined action of two replication initiator proteins and a DNA bending protein at the replication origin of pSC101

We have investigated the interaction of the host-encoded DNA bending protein IHF, the host-encoded initiator DnaA, and the plasmid-encoded initiator RepA with the replication origin of pSC101. We have discovered that DNA bending induced by IHF in vitro promoted the interaction of DnaA protein with two physically separated binding sites called dnaAs and dnaAw. This cooperative interaction at a distance, most probably, caused looping out of the ihf site. We have also discovered that RepA protein binding to its cognate sites promoted enhanced binding of DnaA protein to the physically distant dnaAs site, probably also by DNA looping. The addition of RepA to a binding reaction containing IHF and DnaA further enhanced the binding of DnaA protein to the dnaAs site. Thus, the three DNA-binding proteins interacted with the origin, generating a higher order structure in vitro. On the basis of the results of the known requirement of all three proteins for replication initiation, we have proposed a model for the structure of a preinitiation complex at the replication origin.