A revisionist replicon model for higher eukaryotic genomes.

The replicon model devised to explain replication control in bacteria has served as the guiding paradigm in the search for origins of replication in the more complex genomes of eukaryotes. In Saccharomyces cerevisiae, this model has proved to be extremely useful, leading to the identification of specific genetic elements (replicators) and the interacting initiator proteins that activate them. However, replication control in organisms ranging from Schizosaccharomyces pombe to mammals is far more fluid: only a small number of origins seem to represent classic replicators, while the majority correspond to zones of inefficient, closely spaced start sites none of which are indispensable for origin activity. In addition, it is apparent that the epigenetic state of a given sequence largely determines its ability to be used as a replication initiation site. These conclusions were arrived at over a period of three decades, and required the development of several novel replicon mapping techniques, as well as new ways of examining the chromatin architecture of any sequence of interest. Recently, methods have been elaborated for isolating all of the active origins in the genomes of higher eukaryotes en masse. Microarray analyses and more recent high-throughput sequencing technology will allow all the origins to be mapped onto the chromosomes of any organism whose genome has been sequenced. With the advent of whole-genome studies on gene expression and chromatin composition, the field is now positioned to define both the genetic and epigenetic rules that govern origin activity.

The matrix attachment region in the Chinese hamster dihydrofolate reductase origin of replication may be required for local chromatid separation.

Centered in the Chinese hamster dihydrofolate reductase origin of replication is a prominent nuclear matrix attachment region (MAR). Indirect lines of evidence suggested that this MAR might be required for origin activation in early S phase. To test this possibility, we have deleted the MAR from a Chinese hamster ovary variant harboring a single copy of the dihydrofolate reductase locus. However, 2D gel replicon mapping shows that removal of the MAR has no significant effect either on the frequency or timing of initiation in this locus. Rather, fluorescence in situ hybridization studies on cells swollen under either neutral or alkaline conditions show that deletion of the MAR interferes with local separation of daughter chromatids. This surprising result provides direct genetic evidence that at least a subset of MARs performs an important biological function, possibly related to chromatid cohesion and separation.

The dihydrofolate reductase origin of replication does not contain any nonredundant genetic elements required for origin activity.

The Chinese hamster dihydrofolate reductase (DHFR) origin of replication consists of a broad zone of potential initiation sites scattered throughout a 55-kb intergenic spacer, with at least three sites being preferred (ori-beta, ori-beta', and ori-gamma). We previously showed that deletion of the most active site or region (ori-beta) has no demonstrable effect on initiation in the remainder of the intergenic spacer nor on the time of replication of the DHFR locus as a whole. In the present study, we have now deleted ori-beta', both ori-beta and ori-beta', an 11-kb region just downstream from the DHFR gene, or the central approximately 40-kb core of the spacer. The latter two deletions together encompass >95% of the initiation sites that are normally used in this locus. Two-dimensional gel analysis shows that initiation still occurs in the early S phase in the remainder of the intergenic spacer in each of these deletion variants. Even removal of the 40-kb core fails to elicit a significant effect on the time of replication of the DHFR locus in the S period; indeed, in the truncated spacer that remains, the efficiency of initiation actually appears to increase relative to the corresponding region in the wild-type locus. Thus, if replicators control the positions of nascent strand start sites in this complex origin, either (i) there must be a very large number of redundant elements in the spacer, each of which regulates initiation only in its immediate environment, or (ii) they must lie outside the central core in which the vast majority of nascent strand starts occur.

Amplification of the human dihydrofolate reductase gene via double minutes is initiated by chromosome breaks.

DNA sequence amplification is one of the most frequent manifestations of genomic instability in human tumors. We have shown previously that amplification of the dihydrofolate reductase (DHFR) gene in Chinese hamster cells is initiated by chromosome breaks, followed by bridge-breakage-fusion cycles that generate large intrachromosomal repeats; these are ultimately trimmed by an unknown process to smaller, more homogenous units manifested as homogenously staining chromosome regions (HSRs). However, in most human tumor cells, amplified DNA sequences are borne on unstable, extrachromosomal double minutes (DMs), which suggests the operation of a different amplification mechanism. In this study, we have isolated a large number of independent methotrexate-resistant human cell lines, all of which contained DHFR-bearing DMs. Surprisingly, all but one of these also had suffered partial or complete loss of one of the parental DHFR-bearing chromosomes. Cells in a few populations displayed what could be transient intermediates in the amplification process, including an initial HSR, its subsequent breakage, the appearance of DHFR-containing fragments, and, finally, DMs. Our studies suggest that HSRs and DMs both are initiated by chromosome breaks, but that cell types differ in how the extra sequences ultimately are processed and/or maintained.

Phosphoaminoglycosides inhibit SWI2/SNF2 family DNA-dependent molecular motor domains.

Members of the SWI2/SNF2 family of proteins participate in an array of nucleic acid metabolic functions, including chromatin remodeling and transcription. The present studies identify a novel strategy to specifically inhibit the functional DNA-dependent adenosinetriphosphatase (ATPase) motor domain common to SWI2/SNF2 family members. We have identified preparations of phosphoaminoglycosides, which are natural products of aminoglycoside-resistant bacteria, as inhibitors of the in vitro activities of three SWI2/SNF2 family members. These compounds inhibit the ATPase activity of the active DNA-dependent ATPase A domain (ADAAD) by competing with respect to DNA and thus have no effect on DNA-independent ATPases or on RNA-dependent ATPases. Within the superfamily of DNA-dependent ATPases, these compounds are most potent toward SWI2/SNF2 family members and less potent toward other DNA-dependent ATPases. We demonstrate that it is feasible to target DNA-dependent ATPases of a particular type without affecting the function of other ATPases. As the SWI2/SNF2 proteins have been proposed to function in all aspects of DNA metabolism, this paper provides an archetype for development of DNA metabolic inhibitors.

Dispersive initiation of replication in the Chinese hamster rhodopsin locus.

Several higher eukaryotic replication origins appear to be composed of broad zones of potential nascent strand start sites, while others are more circumscribed, resembling those of yeast, bacteria, and viruses. The most delocalized origin identified so far is approximately 55 kb in length and lies between the convergently transcribed dihydrofolate reductase (DHFR) and the 2BE2121 genes on chromosome 2 in the Chinese hamster genome. In some of our studies, we have utilized the rhodopsin origin as an early replicating internal standard for assessing the effects of deleting various parts of the DHFR locus on DHFR origin activity. However, it had not been previously established that the rhodopsin locus was located at a site far enough away to be immune to such deletions, nor had the mechanism of initiation at this origin been characterized. In the present study, we have localized the rhodopsin domain to a pair of small metacentric chromosomes and have used neutral/neutral 2-D gel replicon mapping to show that initiation in this origin is also highly delocalized, encompassing a region more than 50 kb in length that includes the nontranscribed rhodopsin gene itself. The initiation zone is flanked at least on one end by an actively transcribed gene that does not support initiation. Thus, the DHFR and rhodopsin origins belong to a class of complex, polydisperse origins that appears to be unique to higher eukaryotic cells.

A eukaryotic SWI2/SNF2 domain, an exquisite detector of double-stranded to single-stranded DNA transition elements.

Many members of the SWI2/SNF2 family of adenosine triphosphatases participate in the assembly/disassembly of multiprotein complexes involved in the DNA metabolic processes of transcription, recombination, repair, and chromatin remodeling. The DNA molecule serves as an essential effector or catalyst for most of the members of this particular class of proteins, and the structure of the DNA may be more important than the nucleotide sequence. Inspection of the DNA structure at sites where multiprotein complexes are assembled/disassembled for these various DNA metabolic processes reveals the presence of a common element: a double-stranded to single-stranded transition region. We now show that this DNA element is crucial for the ATP hydrolytic function of an SWI2/SNF2 family member: DNA-dependent ATPase A. We further demonstrate that a domain containing the seven helicase-related motifs that are common to the SWI2/SNF2 family of proteins mediates the interaction with the DNA, yielding specific DNA structural recognition. This study forms a primary step toward understanding the physico-biochemical nature of the interaction between a particular class of DNA-dependent ATPase and their DNA effectors. Furthermore, this study provides a foundation for development of mechanisms to specifically target this class of DNA-dependent ATPases.

Distal sequences, but not ori-beta/OBR-1, are essential for initiation of DNA replication in the Chinese hamster DHFR origin.

In the Chinese hamster dihydrofolate reductase replication initiation zone, the ori-beta locus is preferred over other start sites. To test the hypothesis that ori-beta contains a genetic replicator, we restored a deletion in the 3' end of the DHFR gene with a cosmid that provides the missing sequence and simultaneously knocks out the downstream ori-beta locus. Replication initiates normally in ori-beta knockout cell lines, and the DHFR domain is still synthesized in early S phase. However, initiation is completely suppressed in the starting deletion variant lacking the 3' end of the gene. We conclude that ori-beta does not contain an essential replicator, but that distant sequence elements have profound effects on origin activity in this locus.

DNA-dependent adenosinetriphosphatase A: immunoaffinity purification and characterization of immunological reagents.

We describe an immunoaffinity purification of DNA-dependent ATPase A from fetal calf thymus. The rapid purification increases the yield of enzymatically active enzyme approximately 4-fold, with up to a 7-fold increase in specific activity, and significantly improves the yield of a higher molecular weight species of ATPase A. In the presence of a denatured calf thymus DNA effector, the immunoaffinity-purified enzyme has a specific activity that is more than 10-fold higher than reported for any other eukaryotic DNA-dependent ATPase and 100-fold higher than most others. The improvement in yield has allowed several polypeptides to be identified using monoclonal antibodies, and these polypeptides are demonstrated to be structurally related by partial peptide mapping with N-chlorosuccinimide. The preferred DNA effector for ATP hydrolysis continues to be a DNA primer-template junction with an adjacent stretch of single-stranded DNA. We have used the immunoaffinity-purified enzyme to develop additional stable murine hybridoma monoclones, resulting in a bank of antibodies that recognize a number of different epitopes. All of the monoclonal antibodies react with both calf thymus DNA-dependent ATPase A and bacteriophage T4 gene 44 protein, a DNA-dependent ATPase essential for DNA replication in the bacteriophage T4 system. These monoclonal antibodies should facilitate the development of our understanding with respect to the role and regulation of DNA-dependent ATPases in eukaryotic DNA replication.

Laser cross-linking of proteins to nucleic acids. II. Interactions of the bacteriophage T4 DNA replication polymerase accessory proteins complex with DNA.

In this paper we examine the interactions of the polymerase accessory proteins subassembly of the bacteriophage T4 DNA replication complex, using single-pulse ultraviolet laser excitation to induce protein-nucleic acid cross-links. The laser-induced cross-linking permits effective "freezing" of the instantaneous equilibrium state of the complex and thus provides a mechanism to dissect the individual protein-nucleic acid interactions involved in complex assembly. We find that the binding of the gene 44, 62, and 45 proteins is dependent not only on the presence of each of the other proteins, but also on the presence of adenine nucleotide cofactors. We find that the nonhydrolyzable analogs of ATP often behave more like ADP than ATP in these experiments. Gene 45 protein is able to induce an increase in cross-linking of the gp44/62 complex to nucleic acids, and this increased cross-linking correlates with changes in the apparent Km of the gp44/62 complex for polynucleotides and with changes in Vmax during ATP hydrolysis. Our results suggest that the enhanced DNA binding is predominately through the gene 62 protein and not the ATPase catalytic subunit (gene 44 protein). Thus the gene 62 protein seems to play an integral role in gp45-mediated enhancement of the ATP hydrolytic activity of gp44. These results are summarized and integrated in the form of a model for the multiple interactions of the accessory proteins with DNA and one another in the presence of mononucleotide cofactors and substrates.

Probing the energetics of oligo(dT).poly(dA) by laser cross-linking.

Experimentally determined changes in free energy (delta G(o)) for thymine-thymine interactions occurring in oligo(dT).poly(dA) are dependent on the method used for preparation of the double-stranded template. A rapid laser cross-linking technique was used to examine the equilibrium between oligomers of (dT) bound to either poly(dA) or poly(rA). The single-pulse (4-6 nsec) ultraviolet laser excitation of these polynucleotides causes pyrimidine dimer formation between contiguous oligo(dT) molecules, resulting in a "ligation" of the oligomers. Analysis of the resulting data using standard binding isotherms allowed determination of the degree of cooperativity existing between oligomers. Using the cooperativity, delta G(o), delta H(o), and delta S(o) are calculated, thereby providing thermodynamic parameters for this interaction. The measured cooperativity of oligo(dT) molecule interactions allows direct calculation of the number of 3' ends available as nicked structures or the number of 3' ends associated with gaps for oligo(dT).poly(dA) when used as a substrate for DNA synthesis.

DNA-dependent adenosinetriphosphatase A is the eukaryotic analogue of the bacteriophage T4 gene 44 protein: immunological identity of DNA replication-associated ATPases.

We report the construction of three stable murine hybridomas that secrete monoclonal antibodies which recognize calf thymus DNA-dependent adenosinetriphosphatase A. All three of the antibodies react specifically with calf thymus ATPase A and the gene 44 protein from the bacteriophage T4 DNA-dependent ATPase. Each of the three anti-ATPase A antibodies appears to recognize a different epitope and none of the antibodies inhibit DNA-dependent ATP hydrolysis by ATPase A. Furthermore, one of the antibodies has been shown to react with two different preparations of HeLa cell DNA-dependent ATPases and a yeast DNA-dependent ATPase, all of which have been implicated in the enzymology of DNA replication. These findings provide strong evidence for the role of ATPase A in DNA replication. These observations lead us to conclude that, apart from the nucleotide binding sites, there are at least three epitopes common to both the bacteriophage and eukaryotic DNA-dependent ATPases that we have examined and that the different preparations of the eukaryotic ATPases contain the same DNA-dependent ATPase.