Architecture of the yeast origin recognition complex bound to origins of DNA replication.

In many organisms, the replication of DNA requires the binding of a protein called the initiator to DNA sites referred to as origins of replication. Analyses of multiple initiator proteins bound to their cognate origins have provided important insights into the mechanism by which DNA replication is initiated. To extend this level of analysis to the study of eukaryotic chromosomal replication, we have investigated the architecture of the Saccharomyces cerevisiae origin recognition complex (ORC) bound to yeast origins of replication. Determination of DNA residues important for ORC-origin association indicated that ORC interacts preferentially with one strand of the ARS1 origin of replication. DNA binding assays using ORC complexes lacking one of the six subunits demonstrated that the DNA binding domain of ORC requires the coordinate action of five of the six ORC subunits. Protein-DNA cross-linking studies suggested that recognition of origin sequences is mediated primarily by two different groups of ORC subunits that make sequence-specific contacts with two distinct regions of the DNA. Implications of these findings for ORC function and the mechanism of initiation of eukaryotic DNA replication are discussed.     

Human origins of DNA replication selected from a library of nascent DNA

The identification of metazoan origins of DNA replication has so far been hampered by the lack of a suitable genetic screening and by the cumbersomeness of the currently available mapping procedures. Here we describe the construction of a library of nascent DNA, representative of all cellular origin sequences, and its utilization as a screening probe for origin identification in large genomic regions. The procedure developed was successfully applied to the human 5q31.1 locus, encoding for the IL-3 and GM-CSF genes. Two novel origins were identified and subsequently characterized by competitive PCR mapping, located approximately 3.5 kb downstream of the GM-CSF gene. The two origins (GM-CSF Ori1 and Ori2) were shown to interact with different members of the DNA prereplication complex. This observation reinforces the universal paradigm that initiation of DNA replication takes place at, or in close proximity to, the binding sites of the trans-acting initiator proteins.     

AAA+ ATPases in the initiation of DNA replication

All cellular organisms and many viruses rely on large, multi-subunit molecular machines, termed replisomes, to ensure that genetic material is accurately duplicated for transmission from one generation to the next. Replisome assembly is facilitated by dedicated initiator proteins, which serve to both recognize replication origins and recruit requisite replisomal components to the DNA in a cell-cycle coordinated manner. Exactly how imitators accomplish this task, and the extent to which initiator mechanisms are conserved among different organisms have remained outstanding issues. Recent structural and biochemical findings have revealed that all cellular initiators, as well as the initiators of certain classes of double-stranded DNA viruses, possess a common adenine nucleotide-binding fold belonging to the ATPases Associated with various cellular Activities (AAA+) family. This review focuses on how the AAA+ domain has been recruited and adapted to control the initiation of DNA replication, and how the use of this ATPase module underlies a common set of initiator assembly states and functions. How biochemical and structural properties correlate with initiator activity, and how species-specific modifications give rise to unique initiator functions, are also discussed.     

AAA+ ATPases in the Initiation of DNA Replication

All cellular organisms and many viruses rely on large, multi-subunit molecular machines, termed replisomes, to ensure that genetic material is accurately duplicated for transmission from one generation to the next. Replisome assembly is facilitated by dedicated initiator proteins, which serve to both recognize replication origins and recruit requisite replisomal components to the DNA in a cell-cycle coordinated manner. Exactly how imitators accomplish this task, and the extent to which initiator mechanisms are conserved among different organisms have remained outstanding issues. Recent structural and biochemical findings have revealed that all cellular initiators, as well as the initiators of certain classes of double-stranded DNA viruses, possess a common adenine nucleotide-binding fold belonging to the ATPases Associated with various cellular Activities (AAA+) family. This review focuses on how the AAA+ domain has been recruited and adapted to control the initiation of DNA replication, and how the use of this ATPase module underlies a common set of initiator assembly states and functions. How biochemical and structural properties correlate with initiator activity, and how species-specific modifications give rise to unique initiator functions, are also discussed.     

Two compound replication origins in Saccharomyces cerevisiae contain redundant origin recognition complex binding sites

While many of the proteins involved in the initiation of DNA replication are conserved between yeasts and metazoans, the structure of the replication origins themselves has appeared to be different. As typified by ARS1, replication origins in Saccharomyces cerevisiae are <150 bp long and have a simple modular structure, consisting of a single binding site for the origin recognition complex, the replication initiator protein, and one or more accessory sequences. DNA replication initiates from a discrete site. While the important sequences are currently less well defined, metazoan origins appear to be different. These origins are large and appear to be composed of multiple, redundant elements, and replication initiates throughout zones as large as 55 kb. In this report, we characterize two S. cerevisiae replication origins, ARS101 and ARS310, which differ from the paradigm. These origins contain multiple, redundant binding sites for the origin recognition complex. Each binding site must be altered to abolish origin function, while the alteration of a single binding site is sufficient to inactivate ARS1. This redundant structure may be similar to that seen in metazoan origins.     

Negative control of DNA replication in composite SV40-bovine papilloma virus plasmids

To identify DNA sequences that function in the control of DNA replication, we designed a hybrid replicon consisting of linked SV40 and BPV DNA sequences. In the composite SV40-BPV plasmid negative control encoded by BPV is dominant over the uncontrolled replication encoded by the positive factor, SV40 T antigen. Using a transient replication assay, we show that replication control requires three BPV elements. Two cis-acting sequences are closely linked to BPV replication origins. A third trans-acting element is encoded within the 5' part of the BPV E1 open reading frame (ORF) and is separable from the positive replication factor encoded within the 3' part of the same ORF. The controlled replication of SV-BPV composite replicons has enabled us to create permanent COS cell lines that stably maintain these plasmids as episomes.     

Relaxation of replication control in chaperone-independent initiator mutants of plasmid P1.

Escherichia coli chaperones DnaJ, DnaK and GrpE increase P1 plasmid initiator binding to the origin by promoting initiator folding. The binding allows initiation and also promotes pairing of origins which is believed to control initiation frequency. Chaperone-independent DNA binding mutants are often defective in replication control. We show here that these mutants have increased rates of association for DNA binding and defects in origin pairing. The increases in association rates were found to be due either to increased protein folding into active forms or to increases in the association rate constant, kon. Since the dissociation rate constants for DNA release with these mutants are not changed, it is unlikely that the DNA binding domain is affected. The pairing domain may thus control replication and modulate DNA binding. The role of the pairing domain in DNA binding can be significant in vivo as the selection for chaperone-independent binding favors pairing-defective mutants.     

Identification of herpes simplex virus type 1 genes required for origin-dependent DNA synthesis.

The herpes simplex virus (HSV) genome contains both cis- and trans-acting elements which are important in viral DNA replication. The cis-acting elements consist of three origins of replication: two copies of oriS and one copy of oriL. It has previously been shown that five cloned restriction fragments of HSV-1 DNA together can supply all of the trans-acting functions required for the replication of plasmids containing oriS or oriL when cotransfected into Vero cells (M. D. Challberg, Proc. Natl. Acad. Sci. USA, 83:9094-9098, 1986). These observations provide the basis for a complementation assay with which to locate all of the HSV sequences which encode trans-acting functions necessary for origin-dependent DNA replication. Using this assay in combination with the data from large-scale sequence analysis of the HSV-1 genome, we have now identified seven HSV genes which are necessary for transient replication of plasmids containing either oriS or oriL. As shown previously, two of these genes encode the viral DNA polymerase and single-stranded DNA-binding protein, which are known from conventional genetic analysis to be essential for viral DNA replication in infected cells. The functions of the products of the remaining five genes are unknown. We propose that the seven genes essential for plasmid replication comprise a set of genes whose products are directly involved in viral DNA synthesis.     

Rolling-circle replication of bacterial plasmids.

Many bacterial plasmids replicate by a rolling-circle (RC) mechanism. Their replication properties have many similarities to as well as significant differences from those of single-stranded DNA (ssDNA) coliphages, which also replicate by an RC mechanism. Studies on a large number of RC plasmids have revealed that they fall into several families based on homology in their initiator proteins and leading-strand origins. The leading-strand origins contain distinct sequences that are required for binding and nicking by the Rep proteins. Leading-strand origins also contain domains that are required for the initiation and termination of replication. RC plasmids generate ssDNA intermediates during replication, since their lagging-strand synthesis does not usually initiate until the leading strand has been almost fully synthesized. The leading- and lagging-strand origins are distinct, and the displaced leading-strand DNA is converted to the double-stranded form by using solely the host proteins. The Rep proteins encoded by RC plasmids contain specific domains that are involved in their origin binding and nicking activities. The replication and copy number of RC plasmids, in general, are regulated at the level of synthesis of their Rep proteins, which are usually rate limiting for replication. Some RC Rep proteins are known to be inactivated after supporting one round of replication. A number of in vitro replication systems have been developed for RC plasmids and have provided insight into the mechanism of plasmid RC replication.     

Relationship of eukaryotic DNA replication to committed gene expression: general theory for gene control.

The historic arguments for the participation of eukaryotic DNA replication in the control of gene expression are reconsidered along with more recent evidence. An earlier view in which gene commitment was achieved with stable chromatin structures which required DNA replication to reset expression potential (D. D. Brown, Cell 37:359-365, 1984) is further considered. The participation of nonspecific stable repressor of gene activity (histones and other chromatin proteins), as previously proposed, is reexamined. The possible function of positive trans-acting factors is now further developed by considering evidence from DNA virus models. It is proposed that these positive factors act to control the initiation of replicon-specific DNA synthesis in the S phase (early or late replication timing). Stable chromatin assembles during replication into potentially active (early S) or inactive (late S) states with prevailing trans-acting factors (early) or repressing factors (late) and may asymmetrically commit daughter templates. This suggests logical schemes for programming differentiation based on replicons and trans-acting initiators. This proposal requires that DNA replication precede major changes in gene commitment. Prior evidence against a role for DNA replication during terminal differentiation is reexamined along with other results from terminal differentiation of lower eukaryotes. This leads to a proposal that DNA replication may yet underlie terminal gene commitment, but that for it to do so there must exist two distinct modes of replication control. In one mode (mitotic replication) replicon initiation is tightly linked to the cell cycle, whereas the other mode (terminal replication) initiation is not cell cycle restricted, is replicon specific, and can lead to a terminally differentiated state. Aberrant control of mitotic and terminal modes of DNA replication may underlie the transformed state. Implications of a replicon basis for chromatin structure-function and the evolution of metazoan organisms are considered.     

Attachment of DNA loops to an artificial matrix does not affect the origin of replication initiation in early development of African clawed frog

Replication initiation proceeds in a random fashion in early development of Xenopus laevis. The replication origins become fixed only at later stages of development after the mid-blastula transition. Specification of replication origins occurs at the same time with the specification of the DNA attachment to the nuclear matrix. Replication origins of many species coincide or are located in the vicinity of sites of DNA attachment to the nuclear matrix. The present work was dedicated to development of an experimental system where DNA loops were specifically attached to an artificial matrix and a study of an effect of this attachment on specificity of DNA replication initiation in extracts of Xenopus laevis oocytes. We have found that DNA attachment to the artificial matrix increases the efficacy of DNA replication as compared to the control, but does not affect the replication specificity. It is likely that the transition from non-specific to specific replication is determined by a combination of several factors, and specificity of DNA attachment to a matrix alone is not sufficient for specification of a replication origin.     

Role of the Orc6 protein in origin recognition complex-dependent DNA binding and replication in Drosophila melanogaster

The six-subunit origin recognition complex (ORC) is a DNA replication initiator protein in eukaryotes that defines the localization of the origins of replication. We report here that the smallest Drosophila ORC subunit, Orc6, is a DNA binding protein that is necessary for the DNA binding and DNA replication functions of ORC. Orc6 binds DNA fragments containing Drosophila origins of DNA replication and prefers poly(dA) sequences. We have defined the core replication domain of the Orc6 protein which does not include the C-terminal domain. Further analysis of the core replication domain identified amino acids that are important for DNA binding by Orc6. Alterations of these amino acids render reconstituted Drosophila ORC inactive in DNA binding and DNA replication. We show that mutant Orc6 proteins do not associate with chromosomes in vivo and have dominant negative effects in Drosophila tissue culture cells. Our studies provide a molecular analysis for the functional requirement of Orc6 in replicative functions of ORC in Drosophila and suggest that Orc6 may contribute to the sequence preferences of ORC in targeting to the origins.     

Origin inactivation in bacterial DNA replication control

Initiation of DNA replication is a highly regulated process in all organisms. Proteins that are required to recruit DNA polymerase - initiator proteins - are often used to regulate the timing or frequency of initiation in the cell cycle by limiting either their own synthesis or availability. Studies of the Escherichia coli chromosome and of bacterial plasmids with iterated initiator binding sites (iterons) have revealed that, in addition to initiator limitation, replication origin inactivation is used to prevent replication that is untimely or excessive. Our recent studies of plasmid P1 revealed that this additional mode of control becomes a requirement when initiator availability is limited only by autoregulation. Thus, although initiator limitation appears to be a well-conserved and central mode of replication control, optimal replication might require additional control mechanisms. This review gives examples of how the multiple mechanisms can act synergistically, antagonistically or be partially redundant to guarantee low frequency events. The lessons learned are likely to help understand many other regulatory systems in the bacterial cell.     

Attachment of DNA Loops to an Artificial Matrix Does Not Affect the Replication Origin Specificity in Early Development of Xenopus laevis

Replication initiation proceeds in a random fashion in early development of Xenopus laevis. The replication origins become fixed only at later stages of development after the mid-blastula transition. Specification of replication origins occurs at the same time with the specification of the DNA attachment to the nuclear matrix. Replication origins of many species coincide or are located in the vicinity of sites of DNA attachment to the nuclear matrix. The present work was dedicated to development of an experimental system where DNA loops were specifically attached to an artificial matrix and a study of an effect of this attachment on specificity of DNA replication initiation in extracts of Xenopus laevis oocytes. We have found that DNA attachment to the artificial matrix increases the efficacy of DNA replication as compared to the control, but does not affect the replication specificity. It is likely that the transition from non-specific to specific replication is determined by a combination of several factors, and specificity of DNA attachment to a matrix alone is not sufficient for specification of a replication origin.     

Genetic and physical mapping of DNA replication origins in Haloferax volcanii

The halophilic archaeon Haloferax volcanii has a multireplicon genome, consisting of a main chromosome, three secondary chromosomes, and a plasmid. Genes for the initiator protein Cdc6/Orc1, which are commonly located adjacent to archaeal origins of DNA replication, are found on all replicons except plasmid pHV2. However, prediction of DNA replication origins in H. volcanii is complicated by the fact that this species has no less than 14 cdc6/orc1 genes. We have used a combination of genetic, biochemical, and bioinformatic approaches to map DNA replication origins in H. volcanii. Five autonomously replicating sequences were found adjacent to cdc6/orc1 genes and replication initiation point mapping was used to confirm that these sequences function as bidirectional DNA replication origins in vivo. Pulsed field gel analyses revealed that cdc6/orc1-associated replication origins are distributed not only on the main chromosome (2.9 Mb) but also on pHV1 (86 kb), pHV3 (442 kb), and pHV4 (690 kb) replicons. Gene inactivation studies indicate that linkage of the initiator gene to the origin is not required for replication initiation, and genetic tests with autonomously replicating plasmids suggest that the origin located on pHV1 and pHV4 may be dominant to the principal chromosomal origin. The replication origins we have identified appear to show a functional hierarchy or differential usage, which might reflect the different replication requirements of their respective chromosomes. We propose that duplication of H. volcanii replication origins was a prerequisite for the multireplicon structure of this genome, and that this might provide a means for chromosome-specific replication control under certain growth conditions. Our observations also suggest that H. volcanii is an ideal organism for studying how replication of four replicons is regulated in the context of the archaeal cell cycle.     

Replication origins, factors and attachment sites

The initiation of eukaryotic DNA synthesis occurs at specific sites determined by both cis- and trans-acting elements. Here I review advances in the characterization of yeast origins, origin-binding proteins and the relationship of DNA replication to nuclear substructure in yeast.