As a graduate student with Julius Adler, I identified the basal body of the bacterial flagellum, develop methods for its purification, and elucidate its fine structure and specific attachments to the bacterial cell envelope. The results of this work, which have been cited widely in text books, remain as accurate today as when it was published in 1971. As a post-doctoral fellow with W. Wallace Cleland, I synthesized the novel compound, Cr(III)ATP, and showed it to be a useful paramagnetic, dead-end inhibitor for exploring the active site and kinetic mechanisms of enzymes that required ATP as one of their substrates. As a post-doctoral fellow with Paul Berg, I developed a subcellular system that allowed simian virus 40 (SV40) DNA to continue replication in vitro. In 1973, I continued these studies on the replication and structure of SV40 chromosomes at Harvard Medical School where they culminated in promotion to Full Professor with tenure in 1985. My laboratory has developed new technologies and applied them towards understanding the molecular biology and enzymology of DNA replication in animal cells and viruses (SV40, polyomavirus, papillomavirus, and herpes simplex virus), and at the beginning of animal development (mouse preimplantation embryos and frog eggs). The results of these studies have been reported in over 150 publications.
Our current research now focuses on two basic, interrelated questions: (1) How do mammalian cells decide where and when to initiate DNA replication? (2) What are the requirements for DNA replication and transcription at the beginning of mammalian development?
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Studies on Viral DNA Replication in Mammalian Cells
DNA replication is the primary event that regulates cellular and viral proliferation. Failure of mammalian cells to regulate their proliferation cycle leads to cancer. Drugs that block DNA replication can arrest the spread of cancer cells and eliminate viral pathogens. In fact, amplification of genes by over replication of certain regions of DNA is one of the primary mechanisms by which cancer cells become resistant to drug therapy. Therefore, the overall goal of our work is to discover how DNA replication is regulated both in the large chromosomes of cells and in the "mini-chromosomes" of viruses and small extrachromosomal DNA molecules. This information will be useful in understanding a basic biological function common to all animals, in designing rational drug therapies, and in engineering transgenic animals by introducing new genes on their own "artificial chromosome".
In the past, our research focused on viral genomes as models for DNA replication in mammalian cell nuclei. We were among the first to develop and exploit sub-cellular systems that allowed SV40 and polyomavirus to complete DNA replication in vitro. We used isolated nuclei from virus infected cells supplemented with cytoplasm, and discovered that viral replicating chromosomes could continue replication in the absence of a nucleus. Results from our lab, as well as from other labs, led to the identification of all of the various DNA replication intermediates in SV40 replication. Noteworthy, was our demonstration that although termination of DNA replication did not require specific DNA sequences, some DNA sequences did promote pausing of DNA replication forks in vivo (and DNA polymerase in vitro), and some sequences, such as thosewithin the termination region for SV40 DNA replication, did promote formation of catenated intertwines during separation of sibling chromosomes. However, catenated molecules are not an obligatory intermediate in replication; the region where replication forks terminate directs the mode of separation for the two sibling molecules.
Of particular importance were our studies on DNA replication forks. We demonstrated that DNA synthesis occurs discontinuously only on one arm of replication forks (the arm where the direction of synthesis is opposite to the direction of fork movement) through the repeated initiation, synthesis and joining of Okazaki fragments (transient nascent DNA chains of 40 to 300 nucleotides). Moreover, we were able to identify and characterize transient oligoribonucleotides consisting of 7 to 10 ribonucleotides in which the 5'-terminus was a triphosphate and the 3'-terminus was covalently attached to the 5'-ends of Okazaki fragments. This was one of the first demonstrations of RNA-primed DNA synthesis in mammalian cells. In contrast to RNA polymerases which initiate synthesis de novo, all DNA polymerases require a primer on which to initiate synthesis. These replication intermediates are common to DNA replication in all eukaryotic chromosomes and in all double-stranded DNA viruses that replicate in mammalian nuclei.
Our lab pioneered the development of methods to identify with nucleotide resolution the sites for initiation of RNA-primed DNA synthesis in SV40 and polyomavirus replicating DNA molecules isolated from virus infected cells. When we applied these methods to the genomic region containing the replication origin in SV40 and polyomavirus, we discovered the nucleotide locations for the transition between discontinuous and continuous DNA synthesis that occurs on each DNA strand where bi-directional replication begins. This site was coincident with sequences genetically required to initiate DNA replication, and was latter shown by others to be the site where DNA unwinding begins. These methods were later adapted to mammalian chromosomes and led to the first identification of an origin of bi-directional replication in mammalian cells.
We also isolated and characterized DNA primase-DNA polymerase-a, the enzyme responsible for RNA primed DNA synthesis. In collaboration with Earl Baril's lab, we identified two accessory proteins ("primer recognition proteins") that specifically and strongly stimulated the ability of this enzyme to initiate synthesis on ssDNA. Our discovery that ddTTP did not inhibit this enzyme, but did inhibit other cellular DNA polymerases complimented the specificity of other DNA synthesis inhibitors and led to the identification of DNA primase-DNA polymerase-a as the enzyme responsible for initiation of DNA synthesis in mammalian cells.
Concurrent with these studies, we applied a variety of nucleases to SV40 chromosomes in order to produce the first detailed analyses of the nucleosome arrangement in and around the actual sites of DNA synthesis. We demonstrated that histone octamers are distributed in an apparently random fashion throughout the genome, and that when replication forks pass through, the "old" octamers are distributed equally to both arms of the fork and new histone octamers are assembled on both arms. This work was originally in sharp contrast to results from another lab that concluded histone octamers were segregated conservatively to the forward arm. However, subsequent experiments in several laboratories has since confirmed the distributive nature of histone segregation. My lab went on to show that the actual sites of DNA synthesis are free of nucleosomes, revealing that the enzymes responsible for DNA synthesis utilize nonnucleosomal DNA templates. Based on exonuclease digestion of nascent DNA, the pre-nucleosomal DNA at replication forks contains an average of 123±20 nucleotides of newly replicated DNA on the forward arm and up to one Okazaki fragment plus an average of 126±20 nucleotides of newly replicated DNA on the retrograde arm. Subsequent electron microscopy analyses confirmed these conclusions. Finally, the initial structure of newly replicated chromatin on both arms of the fork was found to be hypersensitive to nonspecific endonucleases, a result later shown by other labs to result from a step wise assembly of nucleosomes.
All replication origins analyzed so far contain one or more transcription factor binding sites identical to those used in transcription promoters and enhancers. My laboratory was instrumental in identifying the role of transcription elements in SV40 and polyomavirus replication origins. We showed that specific transcription factors stimulate both of these replication origins, but that the factors differ for each origin, suggesting a specific interaction with each unique viral origin recognition protein (T-antigen). We went on to show that transcription factors only weakly facilitate binding of T-antigen to the SV40 replication origin, but strongly facilitate initiation of DNA unwinding. This conclusion was later substantiated by results from other labs that showed transcription factors can interact directly with RP-A, the cellular single-stranded DNA binding protein that is required for DNA unwinding and DNA synthesis at viral and cellular replication forks. There are now four different mechanisms known by which transcription factors stimulate replication origins.
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Studies on Mammalian DNA Replication
In 1989, we initiated studies on the nature of site-specific initiation of DNA replication in mammalian chromosomes. We developed a novel approach to mapping replication origins based on identifying the transition from discontinuous to continuous DNA synthesis that occurs at origins of bi-directional replication. These studies led to the first identification of site-specific DNA replication in mammalian cells, a 1 kb locus located 17 kb downstream from the hamster DHFR gene. In addition, we participated in developing two other mapping methods based on quantitative measurements of the lengths or abundance of nascent DNA strands, and one based on selective inhibition of Okazaki fragment synthesis by emetine. We succeeded in identifying specific initiation sites in hamster chromosomes in vivo, and we were the first to demonstrate that site-specific initiation of mammalian DNA replication can be achieved in a cell free system. We showed that Xenopus egg extract can initiate DNA replication specifically at mammalian replication origins in nuclei from G1-phase cells, but not in damaged nuclei or bare DNA, demonstrating that initiation sites for DNA replication in mammalian cells are established prior to S-phase by components of nuclear structure. We and others then went on to show that specific initiation sites are formed in nuclei during each G1-phase of the cell proliferation cycle, an event referred to as the "origin decision point".
What determines where DNA replication begins? In the budding yeast (S. cerevisiae), a six subunit "origin recognition complex" (ORC) binds to specific DNA sequences, resulting in the assembly of a pre-replication complex (pre-RC) that, when activated, initiates bi-directional DNA replication at these sites. A similar story is emerging in mammals. We and others have found that mammalian replication origins are about 10-times larger (1 ¬ 2 kb) than those in S. cerevisiae, AT-rich and lack a recognizable consensus sequence, despite the fact that they are genetically definable DNA loci. The fission yeast, S. pombe, which contains similar complex origins, may provide a model for metazoan origins. We have recently discovered that the S. pombe Orc4 subunit is solely responsible for origin recognition, and that it binds to specific, genetically required, sequences in S. pombe origins where pre-replication complexes are assembled and bi-directional DNA replication begins. We are now examining the relationship between S. pombe origins and those in metazoan cells. In addition, we have demonstrated that epigenetic factors such as DNA methylation can affect site specificity in mammalian cells.
What determines when DNA replication begins? All eukaryotes limit initiation events to once-per-origin-per-cell cycle. This is accomplished by at least four mechanisms, three of which regulate assembly of pre-RCs at ORC/chromatin sites. However, the premier step in establishing replication origins, the assembly of ORC on chromatin, is also regulated. In mammals, we discovered that Orc1 is selectively released during S-phase, ubiquitinated, sometimes degraded, and then rebound to chromatin during the M to G1-transition. Since Orc1 binding to chromatin immediately precedes the origin decision point, it appears to be the rate limiting step in assembly of pre-RCs at specific genomic sites. In frogs egg extracts, we discovered that the entire ORC is released upon association of Mcm proteins with chromatin to complete the assembly of pre-RCs. These studies reveal that one or more ORC proteins cycles on and off of chromatin during cell division, thereby determining when and where pre-RC assembly can occur. We are now trying to identify the mechanisms involved in regulating ORC assembly and the biological roles this regulation plays during animal development.
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Studies on Mammalian Embryos
In the mid-1980s, our efforts to understand the nature of origins of cellular DNA replication, the potential role that transcription elements may play in regulating their activity, and the feasibility of constructing a mouse with an artificial chromosome led me to investigate the requirements for DNA replication and transcription at the beginning of mammalian development. By injecting plasmid DNA into individual nuclei of mouse oocytes and cleavage stage embryos and by transplanting injected nuclei from one cell to another, we have been able to determine the capacity of oocytes and cleavage stage embryos to utilize specific cis-acting sequences and trans-acting factors that constitute transcription promoters, replication origins or enhancers, and to identify biological parameters that regulate the activation of zygotic gene expression (ZGA).
We have shown that ZGA is a time dependent rather than a cell cycle dependent mechanism that delays both initiation of transcription and translation of nascent transcripts from injected DNA. Thus, zygotic gene transcripts are handled differently than maternal mRNA, a phenomenon also observed in Xenopus. Concurrent with formation of a 2-cell embryo, a general chromatin mediated repression of promoter activities appears. Repression factors are inherited by the maternal pronucleus from the oocyte, but are absent in the paternal pronucleus and not available until sometime during the transition from a late 1-cell to a 2-cell embryo. This means that paternally inherited genes are exposed to a different environment in fertilized eggs than are maternally inherited genes, a situation that could contribute to genomic imprinting. Chromatin mediated repression of promoter activities prior to ZGA is similar to what is observed during Xenopus embryogenesis and insures that genes are not expressed until the appropriate time in development when positive acting factors, such as enhancers, can relieve this repression.
We have been able to show that the ability to use enhancers depends on the acquisition of specific co-activators at the 2-cell stage, concurrent with ZGA. In addition, DNA sequences that bind TEAD(TEF) transcription factors provide a powerful enhancer activity in cleavage stage embryos. The mammalian TEAD transcription factor family consists of four highly conserved proteins that bind to the same DNA sequence, but appear to serve different functions. Since TEAD-2 is the only one expressed during the first 7 days of mouse development, it most likely is responsible for the TEAD transcription factor activity that first appears during ZGA. All four TEAD genes are expressed at later embryonic stages and in adult tissues; virtually every tissue expresses at least one family member, consistent with a critical role for TEAD proteins in either cell proliferation or differentiation. We recently discovered that TEAD transcription factors use the activation domain of YAP65, a SRC/YES-associated protein that is localized in the cytoplasm, suggesting that TEAD-dependent transcription is regulated by association with its cytoplasmic co-activator protein in response to extracellular signals. We are currently trying to identify the biological function of TEAD-2 during early mouse development.
We have also identified a novel gene (Soggy) only 3.8 kb upstream of TEAD-2 that is expressed specifically in developing spermatocytes and lymphocytes. It appears that once cell differentiation begins, cells expresses either Sgy or TEAD-2 from this locus. Sgy expression during mouse development is strongly correlated with DNA methylation in this region, and we are now identifying the sequences that determine which gene is expressed in which cell type.
Finally, we have shown that the mechanism by which enhancers communicate with promoters changes during development. In differentiated cells, we discovered that a TATA box is required for enhancer mediated stimulation of promoters, but in undifferentiated cells (e.g. mouse cleavage stage embryos) a TATA box is dispensable and enhancer stimulation is mediated via an Sp1 site. This provides an opportunity for enhancer mediated stimulation of TATA-less promoters (e.g. housekeeping genes) early during development while reducing stimulation later on.
These events impose a directionality at the beginning of animal development that is evident from the inability of fertilized mouse eggs to reprogram gene expression in nuclei taken from cells at developmentally advanced stages. It should now be possible to identify the roles of specific transcription factors and chromosomal changes in activating specific genes at the beginning of mammalian development.
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