Toshiki Tsurimoto

Structure-Function Relationship of the Eukaryotic DNA Replication Factor, Proliferating Cell Nuclear Antigen

Proliferating cell nuclear antigen (PCNA) is essential for eukaryotic DNA replication and functions as a processivity factor of DNA polymerase δ (pol δ). Due to the functional and structural similarity with the β-subunit of Escherichia coli DNA polymerase III, it has been proposed that PCNA would act as a molecular clamp during DNA synthesis. By site-directed mutagenesis and biochemical analyses, we have studied the functional domains of human PCNA required for stimulation of replication factor C (RF-C) ATPase and DNA synthesis by pol δ. Short deletions from either the N or C termini caused drastic changes in extraction and chromatographic behaviors, suggesting that both of these terminal regions are crucial to fold the tertiary structure of PCNA. The short C-terminal stretch from Lys254 to Glu256 is necessary for stimulation of RF-C ATPase activity, but not for stimulation of DNA synthesis by pol δ. Nine basic amino acids that are essential for activating DNA synthesis by pol δ are positioned at the internal α-helices of PCNA. This result is in good agreement with the observation that PCNA has a ring structure similar to the β-subunit and clamps a template DNA through this positively charged internal surface. Several other charged amino acids are also required to stimulate either RF-C ATPase or pol δ DNA synthesis. Some of them are positioned at loops which are exposed on one of the side surface of PCNA adjacent to the C-terminal loop. In addition, the β-sheets composing the intermolecular interface of the trimeric PCNA are important for interaction with pol δ. Therefore, the outer surface of PCNA has multiple functional surfaces which are responsible for the interaction with multiple factors. Furthermore, the two side surfaces seem to be functionally distinguishable, and this may determine the orientation of tracking PCNA along the DNA.

An in vitro system for screening anti-hepatitis b virus drugs

A human hepatoblastoma cell line (HB 611) that continuously synthesizes hepatitis B viral (HBV) DNA was grown in the presence of various inhibitors of DNA synthesis, and the DNA from the cells was analyzed by the Southern blotting method to examine selective inhibition of the viral DNA synthesis. Among those that showed selective inhibition, and interferons alpha and beta, acyclovir, and dideoxy cytidine were effective. This system should be useful for screening new antiviral agents against HBV.

Bacteriophage lambda initiators: Preparation from a strain that overproduces the O and P proteins

SummaryA recombinant plasmid was constructed which carries bacteriophage λ initiator genes O and P under control of tandemly arranged pL and pR promoters. These promoters were repressed by a thermosensitive repressor, cI857, at low temperature, but became active when the culture was incubated at 42° C. Upon elevation of the temperature, the O and P proteins were overproduced to the extent that they constituted several per cent of the total E. coli cellular proteins. Both the O and P proteins have been purified to apparent homogeneity, and were shown to consist of 298 and 233 amino acid residues, respectively. The amino acid composition and the terminal partial amino acid sequence of each protein were determined. Through these analyses, the locations of the O and P genes in the known λ DNA sequence were determined. The termination codon for the O gene overlaps with the initiation codon for the P gene. The purified O protein binds specifically to the replication origin of λ (λ ori) in accordance with our previous observations. The purified P protein inhibits an ATPase activity of dnaB protein.

Purification of bacteriophage λO protein that specifically binds to the origin of replication

SummaryBy means of a nitrocellulose filter binding assay, DNA binding activities among proteins fractionated from extracts of Escherichia coli carrying λdv have been surveyed. An activity was found that binds specifically to a fragment of 164 base pairs that specifies the λ replication origin (λ ori). This activity was not detected in an extract of cells not carrying the λdv plasmid. The activity was detected in extracts of cells carrying a hybrid plasmid in which the entire λ O gene had been cloned and placed under the control of the lac promoter. Deletion of a 60 base pair segment in the ‘amino-terminal region’ of the O gene abolished this activity, indicating that the λ ori binding protein is coded for by the λ O gene.The ori-specific binding protein was purified by five fractionation steps. The most purified preparation consists of a major polypeptide that migrates with a molecular weight of 32,000 in SDS-polyacrylamide gel electrophoresis. Binding of O protein to ori occurs in the absence of other protein aceous components.

Direction of transcription affects the replication mode of λ in an in vitro system

SummaryA set of artificial circular plasmids, named plasmoids, was constructed. They are about 1 kb in size and consist of a 178 bp λdv minimal DNA replication origin (ori) which has four direct repeats and the A+T-rich region conferring polarity to the ori fragment, a 775 bp DNA segment that codes for the CAT amino acid sequence and the 99 bp lac promoter (plac). They carry no other functional genes or genetic sites. The constructions involved various combinations of relative orientations of these components. These molecules do not replicate in vivo because they lack genes coding for initiation proteins, but they do replicate in an in vitro system (Fuller et al. 1981), and can be used for studies of interactions between transcription and replication. In these plasmoids, major transcription starts from the strong plac, and some weak unscheduled transcription starts from several other initiation sites. The major RNA synthesis was found to interfere with the unscheduled RNA synthesis, which was occurring on the opposite strand. The most active replication took place when the major RNA synthesis went through the λ origin region in the direction which occurs naturally in the λ genome. Under these conditions, DNA synthesis going against such transcription was less than that going along with the major transcription. When RNA synthesis through the λ origin region was in the opposite direction, DNA synthesis in the same direction was about half of that observed in the above case, whereas that going against transcription was very weak. Based on these observations, this paper discussed the interactions between the two transcription systems and between transcription and DNA synthesis.

Origin and Initiation Sites of λdv DNA Replication In Vitro

The sequence of λ DNA essential for the unique initiation of replication was analyzed in an in vitro replication system. Fragments of λ DNA were inserted into pBR322 and used as templates or were circularized in vitro in the absence of pBR322 and employed in the same tests. A 165-bp region left of the EcoRI site in the 0 gene of the λ genome was defined as the functional origin. This region, which we defined as the ori region, carries, in order, the 4 19-bp repeat sequences where the 0 protein binds (ori-repeats), an A+T-rich stretch, and a region that constitutes part of a large palindromic structure. Regions that have long been suspected to participate in λ DNA replication initiation, ice and oop were not required for the O, P-dependent λ-specific replication initiation. The λdv and the “ori region plus” recombinant Plasmids initiated DNA synthesis at or around this region, and the reaction depended on the presence of λ-coded initiators, O and P proteins.