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Role of bacterial chaperones in DNA replication.

Studies on the involvement of chaperone proteins in DNA replication have been limited to a few replication systems, belonging primarily to the prokaryotic world. The insights gained from these studies have substantially contributed to our understanding of the eukaryotic DNA replication process as well. The finding that molecular chaperones can activate some initiation proteins before DNA synthesis has led to the more general suggestion that molecular chaperones can influence the DNA-binding activity of many proteins, including transcriptional factors involved in cell regulatory systems. The DnaK/DnaJ/GrpE molecular chaperone system became a paradigm of our understanding of fundamental processes, such as protein folding, translocation, selective proteolysis and autoregulation of the heat-shock response. Studies on the Clp ATPase family of molecular chaperones will help to define the nature of signals involved in chaperone-dependent proteins' refolding and the degradation of misfolded proteins.

Bacteria↗

Implications of DNA replication for eukaryotic gene expression.

DNA replication has a key role in many developmental processes. Recent progress in understanding events at the replication fork suggests mechanisms for both establishing and maintaining programs of eukaryotic gene activity. In this review, I discuss the consequences of replication fork passage for preexisting chromatin structures and describe how the mechanism of nucleosome assembly at the replication fork may facilitate the formation of either transcriptionally active or repressed chromatin.

Animals↗

Role of the terminal repeat GAGC trimer, the major Rep78 binding site, in adeno-associated virus DNA replication.

The adeno-associated virus (AAV) terminal repeats (TR) are cis required, and the AAV encoded Rep78 protein is trans required, for AAV DNA replication. The Rep78 protein recognizes and interacts with at least three regions within the TR DNA. The major binding site, with the highest affinity for Rep78 binding, is within the TR stem (nt 36-16) and includes the 'core' GAGC trimer (GAGC3, nt 33-22; Fig. 2) sequence. In this study mutations were made within the GAGC trimer and these mutants assayed for their ability to allow for AAV double stranded (ds DNA, prepackaging DNA replication), and single stranded DNA (ss DNA, due to virion packaging) replication. Here, it is shown that when the two inside GAGC motifs are mutated, with only motif no. 1 left intact (see Fig. 2), the resulting AAV (mutA) genome was significantly defective for both ds DNA (17% of wild type) and ss DNA (9%). If the TRs contained only the two outside motifs intact (mutB), motifs no. 1 and 2, the AAV genome had a significant but reduced level of both ds (50%) and ss (34%) DNA replication. Finally, if only the middle motif no. 2 was mutated, with motifs no. 1 and 3 left intact (mutC), the resulting DNA replication for both ds and ss forms was essentially wild type (80% that of wild type). These data suggest that the GAGC trimer plays a role in AAV DNA replication, and that GAGC motif no. 3 is the most important of the three motifs for both ds and ss DNA replication.

Base Sequence↗

Cell cycle-dependent and DNA damage-inducible nuclear localization of FEN-1 nuclease is consistent with its dual functions in DNA replication and repair.

Flap endonuclease-1 (FEN-1), a 43-kDa protein, is a structure-specific and multifunctional nuclease. It plays important roles in RNA primer removal of Okazaki fragments during DNA replication, DNA base excision repair, and maintenance of genome stability. Three functional motifs of the enzyme were proposed to be responsible for its nuclease activities, interaction with proliferating cell nuclear antigen, and nuclear localization. In this study, we demonstrate in HeLa cells that a signal located at the C terminus (the nuclear localization signal (NLS) motif) facilitates nuclear localization of the enzyme during S phase of the cell cycle and in response to DNA damage. Truncation of the NLS motif prevents migration of the protein from the cytoplasm to the nucleus, while having no effect on the nuclease activities and its proliferating cell nuclear antigen interaction capability. Site-directed mutagenesis further revealed that a mutation of the KRK cluster to three alanine residues completely blocked the localization of FEN-1 into the nucleus, whereas mutagenesis of the KKK cluster led to a partial defect of nuclear localization in HeLa cells without observable phenotype in yeast. Therefore, the KRKXXXXXXXXKKK motif may be a bipartite NLS driving the protein into nuclei. Yeast RAD27Delta cells transformed with human mutant M(krk) survived poorly upon methyl methanesulfonate treatment or when they were incubated at an elevated temperature.

Cell Cycle↗

Analysis of an Autographa californica nucleopolyhedrovirus lef-11 knockout: LEF-11 is essential for viral DNA replication.

The Autographa californica nucleopolyhedrovirus (AcMNPV) lef-11 gene was previously identified by transient late expression assays as a gene important for viral late gene expression. The lef-11 gene was not previously identified as necessary for DNA replication in transient origin-dependent plasmid DNA replication assays. To examine the role of lef-11 in the context of the infection cycle, we generated a deletion of the lef-11 gene by recombination in an AcMNPV genome propagated as a BACmid in Escherichia coli. The resulting AcMNPV lef-11-null BACmid (vAc(lef11KO)) was unable to propagate in cell culture, although a "repair" AcMNPV BACmid (vAc(lef11KO-REP)), which was generated by transposition of the lef-11 gene into the polyhedrin locus of the vAc(lef11KO) BACmid, was able to replicate in a manner similar to wild-type or control AcMNPV viruses. Thus, the lef-11 gene is essential for viral replication in Sf9 cells. The vAc(lef11KO) BACmid was examined to determine if the defect in viral replication resulted from a defect in DNA replication or from a defect in late transcription. The lef-11-null BACmid and control BACmids were transfected into Sf9 cells, and viral DNA replication was monitored. The viral DNA genome of the lef-11-null BACmid (vAc(lef11KO)) was not amplified, whereas replication and amplification of the genomes of the repair BACmid (vAc(lef11KO-REP)), wild-type AcMNPV, and a nonpropagating gp64-null control BACmid (vAc(GUSgp64KO)) were readily detected. Northern blot analysis of transcripts from selected early, late, and very late genes showed that late and very late transcription was absent in cells transfected with the lef-11-null BACmid. Thus, in contrast to prior studies using transient replication and late expression assays, studies of a lef-11-null BACmid indicate that LEF-11 is required for viral DNA replication during the infection cycle.

Animals↗

Simian virus 40 DNA replication in vitro: purification and characterization of replication factors from mouse cells.

We have previously developed simian virus 40 (SV40) DNA replication system in vitro (Ariga and Sugano, J. Virol. 48, 481, 1983). This system is composed of human HeLa or mouse FM3A nuclear extract and cytoplasmic extract of SV40 infected CosI cells. Here FM3A nuclear extract was fractionated by DEAE Sephacel and single-stranded DNA cellulose chromatography into three components required for accurate in vitro SV40 DNA replication. One fraction (A fraction) contained DNA polymerase-primase, and the second component (B fraction) contained DNA topoisomerase. Third component was further purified to near homogenuity using DEAE-Sephacel, single-stranded DNA cellulose, and glycerol gradient centrifugation. The purified protein (named factor I) bound to the origin containing fragment of SV40 DNA. The factor I enhanced the initiation of SV40 DNA replication catalyzed by SV40 infected CosI cytoplasm alone. When all four fractions consisting of A, B fractions, factor I, and SV40 infected CosI cytoplasm were mixed together, the system was reconstituted, meaning that initiation and subsequent elongation were completed to generate the full sized daughter molecules.

Animals↗

Genetic analysis of the enhancer requirements for polyomavirus DNA replication in mice.

In this report, we describe the first systematic analysis of the genetic requirements for polyomavirus (Py) enhancer-activated viral DNA replication during the acute phase of infection in mice. Four mutants were made which substituted XhoI sites for conserved enhancer consensus sequences (adenovirus type 5 E1A, c-fos, simian virus 40, and a glucocorticoidlike consensus sequence). Viral DNA replication in infected mouse organs was measured by DNA blot analysis. Only the loss of the glucocorticoidlike consensus sequence element significantly reduced Py DNA replication in the kidneys, the primary target organ for viral replication. The loss of the c-fos, adenovirus type 5 E1A, or simian virus 40 consensus sequences, however, expanded organ-specific viral DNA replication, relative to wild-type Py, by allowing high-level replication in the pancreas or heart or both. Analysis of Py variants selected for replication in undifferentiated embryonal carcinoma cell lines (PyF441, PyF111) showed that there was little change in levels of viral DNA replication in kidneys and other organs as compared with those in the wild-type virus. If the entire B enhancer is deleted, only low overall levels of viral replication are observed. Wild-type levels of replication in the kidneys can be reconstituted by addition of a single domain from within the A enhancer (nucleotides 5094 to 5132) to the B enhancer deletion virus, suggesting that a single domain from the A enhancer can functionally substitute for the entire B enhancer. This also indicates that the determinants for kidney-specific replication are not found in the B enhancer.

Animals↗

Identification of the Xenopus laevis homolog of Saccharomyces cerevisiae DNA2 and its role in DNA replication.

The DNA2 gene of Saccharomyces cerevisiae is essential for growth and appears to be required for a late stage of chromosomal DNA replication. S. cerevisiae Dna2p (ScDna2p) is a DNA helicase and also a nuclease. We have cloned and sequenced the homologous gene from Xenopus (Xenopus Dna2). Xenopus Dna2p (XDna2p) is 32% identical to ScDna2p, and the similarity extends over the entire length, including but not limited to the five conserved helicase motifs. XDna2p is even more closely related (60% identical) to a partial human cDNA. The Xenopus Dna2 (XDna2) gene was able to complement an S. cerevisiae dna2-1 mutant strain for growth at the nonpermissive temperature, suggesting that XDna2p is a functional as well as a structural homolog of the yeast protein. Recombinant XDna2p was expressed in insect cells and purified. Like the ScDna2p purified from yeast, it is a single-stranded DNA endonuclease and a DNA-dependent ATPase, suggesting that both of these activities are part of the essential function of Dna2p. However, unlike ScDna2p from yeast, recombinant XDna2p showed no DNA helicase activity. When XDna2 was immunodepleted from interphase egg extracts, chromosomal DNA replication was almost completely inhibited. From the size of the residually synthesized DNA from the XDna2-depleted egg extracts, it seems that initiation of DNA replication may be impaired. This interpretation is also supported by the normal DNA replication of M13 single-stranded DNA in the XDna2-depleted egg extracts.

Amino Acid Sequence↗

Nucleotide sequence of the DNA replication origin for human papovavirus BKV: sequence and structural homology with SV40.

DNA and RNA sequencing techniques were used to obtain the sequence surrounding the origin of DNA replication for human papovavirus BKV. The structure is characterized by a true palindrome of 17 residues followed by two sets of symmetrical sequences and a stretch of 20 AT residues. Within the two symmetrical sequences is a segment containing a strong purine bias, 23 of 26 nucleotides. These structures are similar, if not identical, to those found in the region of the SV40 replication, origin. Within the homologous DNA segments, 60-80% of the BKV and SV40 nucleotides are the same. The remarkable similarity of BKV and SV40 sequences containing the origins of DNA replication would appear to confirm our previous suggestion of an evolutionary relationship between the two genomes. In addition, topological similarities between these sequences suggest the possibility of certain structural requirements for bidirectional replication origins in these superhelical DNAs.

BK Virus↗

Adenovirus DNA replication in vitro: characterization of a protein covalently linked to nascent DNA strands.

The 5' terminus of each nascent daughter strand of adenovirus DNA replicated in vitro is covalently linked to a protein with an apparent Mr of 80,000. This protein may represent a precursor to the 55,000-dalton protein known to be linked to the 5' ends of mature adenovirus DNA strands. Partial proteolysis experiments indicate that the 80,000-dalton and 55,000-dalton proteins are structurally related. Furthermore, both proteins are attached to DNA by the same linkage: a phosphodiester bond between the beta-OH of a serine residue in the protein and the 5'-OH of the terminal deoxycytidine residue of the DNA. The role of the 80,000-dalton protein in adenovirus DNA replication is not yet clear, although one reasonable possibility is that it serves as the primer for daughter strand synthesis.

Adenoviruses, Human↗

Analysis of the tobacco chloroplast DNA replication origin (oriB) downstream of the 23 S rRNA gene.

We have mapped the origin of DNA replication (oriB) downstream of the 23 S rRNA gene in each copy of the inverted repeat (IR) of tobacco chloroplast DNA between positions 130,502 and 131,924 (IR(A)) by a combination of approaches. In vivo chloroplast DNA replication intermediates were examined by two-dimensional agarose gel electrophoresis. Extended arc patterns suggestive of replication intermediates containing extended single-stranded regions were observed with the 4.29 kb SspI fragment and an overlapping EcoRI fragment from one end of the inverted repeat, while only simple Y patterns were observed with a 3.92 kb BamHI-KpnI fragment internal to the SspI fragment. Other restriction fragments of tobacco chloroplast DNA besides those at the oriA region also generated only simple Y patterns in two-dimensional agarose gels. Several chloroplast DNA clones from this region were tested for their ability to support in vitro DNA replication using a partially purified chloroplast protein fraction. Templates with a deletion of 154 bp from the SspI to the BamHI sites near the end of the inverted repeat resulted in a considerable loss of in vitro DNA replication activity. These results support the presence of a replication origin at the end of the inverted repeat. The 5' end of nascent DNA from the replication displacement loop was identified at position 130,697 for IR(A) (111,832 for IR(B)) by primer extension. A single major product insensitive to alkali and RNase treatment was observed and mapped to the base of a stem-loop structure which contains one of two neighboring BamHI sites near the end of each inverted repeat. This provides the first precise determination of the start site of DNA synthesis from oriB. Adjacent DNA fragments containing the stem-loop structure and the 5' region exhibit sequence-specific gel mobility shift activity when incubated with the replication protein fraction, suggesting the presence of multiple binding sites.

Base Sequence↗

Oncogene v-jun modulates DNA replication.

Cell transformation leads to alterations in both transcription and DNA replication. Activation of transcription by the expression of a number of transforming oncogenes is mediated by the transcription factor AP1 (Herrlich & Ponta, 1989; Imler & Wasylyk, 1989). AP1 is a composite transcription factor, consisting of members of the jun and fos gene-families. c-jun and c-fos are progenitors of oncogenes, suggestion that an important transcriptional event in cell transformation is altered activity of AP1, which may arise either indirectly by oncogene expression or directly by structural modification of AP1. We report here that the v-jun oncogene and its progenitor c-jun, as fusion proteins with the lex-A-repressor DNA binding domain, can activate DNA replication from the Polyoma virus (Py) origin of replication, linked to the lex-A operator. The transcription-activation region of v-jun is required for activation of replication. When excess v-jun is expressed in the cell, replication is inhibited or 'squelched'. These results suggest that one consequence of deregulated jun activity could be altered DNA replication and that there are similarities in the way v-jun activates replication and transcription.

Base Sequence↗

Intermediates of adeno-associated virus DNA replication in vitro.

Intermediates of adeno-associated virus type 2 (AAV) DNA replication in an in vitro assay have been characterized. The assay involves rescue and replication of an AAV insert in pBR322. Intermediates were shown to be duplex molecules in which at least one terminus was in the extended configuration, in contrast to the hairpinned ends seen after rescue in the absence of AAV DNA replication. Also present were linear double-stranded dimers, which were characterized as either head-to-head or tail-to-tail tandems; no head-to-tail dimers were detected. The results are in accord with the current model of AAV DNA replication.

DNA Replication↗

DNA replication origins: from sequence specificity to epigenetics.

Site-specific initiation of DNA replication is a conserved function in all organisms. In Escherichia coli and Saccharomyces cerevisiae, DNA replication origins are sequence specific, but in multicellular organisms, origins are not so clearly defined. In this article, I present a model of origin specification by epigenetic mechanisms that allows the establishment of stable chromatin domains, which are characterized by autonomous replication. According to this model, origins of DNA replication help to establish domains of gene expression for the generation of cell diversity.

Animals↗

Role of the first round of DNA replication in reprogramming gene expression in the preimplantation mouse embryo.

The first round of DNA replication has been proposed to provide a window of opportunity for maternally-derived transcription factors to gain access to their cognate cis-binding DNA sequences and thereby reprogram the pattern of gene expression that occurs during the 2-cell stage. Using high-resolution, two-dimensional electrophoresis of metabolically radio-labeled polypeptides, we report the expression of a group of several polypeptides whose synthesis in the 2-cell embryo is due to transcription (i.e., alpha-amanitin-sensitive) and depends on the first round of DNA replication (i.e., aphidicolin-sensitive). We also describe the synthesis of another subset of alpha-amanitin-sensitive polypeptides whose expression does not require the first round of DNA replication (i.e., aphidicolin-insensitive). These results are consistent with the proposed role of the first round of DNA replication in reprogramming the pattern of gene expression.

Amanitins↗

Role for a Drosophila Myb-containing protein complex in site-specific DNA replication.

There is considerable interest in the developmental, temporal and tissue-specific patterns of DNA replication in metazoans. Site-specific DNA replication at the chorion loci in Drosophila follicle cells leads to extensive gene amplification, and the organization of the cis-acting DNA elements that regulate this process may provide a model for how such regulation is achieved. Two elements important for amplification of the third chromosome chorion gene cluster, ACE3 and Ori-beta, are directly bound by Orc (origin recognition complex), and two-dimensional gel analysis has revealed that the primary origin used is Ori-beta (refs 7-9). Here we show that the Drosophila homologue of the Myb (Myeloblastosis) oncoprotein family is tightly associated with four additional proteins, and that the complex binds site-specifically to these regulatory DNA elements. Drosophila Myb is required in trans for gene amplification, showing that a Myb protein is directly involved in DNA replication. A Drosophila Myb binding site, as well as the binding site for another Myb complex member (p120), is necessary in cis for replication of reporter transgenes. Chromatin immunoprecipitation experiments localize both proteins to the chorion loci in vivo. These data provide evidence that specific protein complexes bound to replication enhancer elements work together with the general replication machinery for site-specific origin utilization during replication.

Animals↗

Suppression of tumorigenicity in T-cell lymphoma hybrids is correlated with changes in myc expression and DNA replication of the myc chromosomal domain.

Intraspecific somatic cell hybrids between T-lymphoma cells and lymphocytes are highly tumorigenic whereas fusion of T-lymphoma cells with normal fibroblasts leads to reduced or even completely suppressed tumorigenicity of the hybrid cells. A particular cytogenetic phenomenon defines these two classes of hybrids. DNA replication analysis via bromodeoxyuridine pulse labelling reveals an aberrant banding pattern in the c-myc chromosomal domain in tumour cells and highly tumorigenic hybrids. In hybrids with suppressed tumorigenicity the tumor parent derived chromosomes have reverted to normal DNA replication banding. Aberrant DNA replication in tumour cells and highly tumorigenic hybrids coincides with enhanced c-myc expression. In hybrids with suppressed tumorigenicity and with normal DNA replication banding c-myc expression is also reduced. Thus, a correlation between aberrant DNA replication and enhanced expression of a gene located in the same chromosomal domain is observed. Reversion of aberrant DNA replication and reduction of c-myc expression to normal in hybrid cells may be due to a site-specific trans effect which overrides the control brought about in cis by retroviral insertion near the c-myc gene.

Animals↗

Detection of template strand switching during initiation and termination of DNA replication of porcine circovirus.

Nucleotide substitution mutagenesis was conducted to investigate the importance of the inverted repeats (palindrome) at the origin of DNA replication (Ori) of porcine circovirus type 1 (PCV1). Viral genomes with engineered mutations on either arm or both arms of the palindrome were not impaired in protein synthesis and yielded infectious progeny viruses with restored or new palindromes. Thus, a flanking palindrome at the Ori was not essential for initiation of DNA replication, but one was generated inevitably at termination. Among the 26 viruses recovered, 16 showed evidence of template strand switching, from minus-strand genome DNA to palindromic strand DNA, during biosynthesis of the Ori. Here I propose a novel rolling-circle "melting-pot" model for PCV1 DNA replication. In this model, the replicator Rep protein complex binds, destabilizes, and nicks the Ori sequence to initiate leading-strand DNA synthesis. All four strands of the destabilized inverted repeats exist in a "melted" configuration, and the minus-strand viral genome and a palindromic strand are available as templates, simultaneously, during initiation or termination of DNA replication. Inherent in this model is a "gene correction" or "terminal repeat correction" mechanism that can restore mutilated inverted-repeat sequences to a palindrome at the Ori of circular DNAs or at the termini of circularized linear DNAs. Potentially, the melted state of the inverted repeats increases the rate of noncomplementary or illegitimate nucleotide incorporation into the palindrome. Thus, this melting-pot model provides insight into the mechanisms of DNA replication, gene correction, and illegitimate recombination at the Ori of PCV1, and it may be applicable to the replication of other circular DNA molecules.

Animals↗