Preferential Localization of Human Origins of DNA Replication at the 5′-Ends of Expressed Genes and at Evolutionarily Conserved DNA Sequences

Replication of mammalian genomes requires the activation of thousands of origins which are both spatially and temporally regulated by as yet unknown mechanisms. At the most fundamental level, our knowledge about the distribution pattern of origins in each of the chromosomes, among different cell types, and whether the physiological state of the cells alters this distribution is at present very limited.We have used standard λ-exonuclease resistant nascent DNA preparations in the size range of 0.7–1.5 kb obtained from the breast cancer cell line MCF–7 hybridized to a custom tiling array containing 50–60 nt probes evenly distributed among genic and non-genic regions covering about 1% of the human genome. A similar DNA preparation was used for high-throughput DNA sequencing. Array experiments were also performed with DNA obtained from BT-474 and H520 cell lines. By determining the sites showing nascent DNA enrichment, we have localized several thousand origins of DNA replication. Our major findings are: (a) both array and DNA sequencing assay methods produced essentially the same origin distribution profile; (b) origin distribution is largely conserved (>70%) in all cell lines tested; (c) origins are enriched at the 5′ends of expressed genes and at evolutionarily conserved intergenic sequences; and (d) ChIP on chip experiments in MCF-7 showed an enrichment of H3K4Me3 and RNA Polymerase II chromatin binding sites at origins of DNA replication.Our results suggest that the program for origin activation is largely conserved among different cell types. Also, our work supports recent studies connecting transcription initiation with replication, and in addition suggests that evolutionarily conserved intergenic sequences have the potential to participate in origin selection. Overall, our observations suggest that replication origin selection is a stochastic process significantly dependent upon local accessibility to replication factors

Modular sequence elements associated with origin regions in eukaryotic chromosomal DNA

We have postulated that chromosomal replication origin regions in eukaryotes have in common clusters of certain modular sequence elements (Benbow, Zhao, and Larson, BioEssays 14, 661–670, 1992). In this study, computer analyses of DNA sequences from six origin regions showed that each contained one or more potential initiation regions consisting of a putative DUE (DNA unwinding element) aligned with clusters of SAR (scaffold associated region), and ARS (autonomously replicating sequence) consensus sequences, and pyrimidine tracts. The replication origins analyzed were from the following loci: Tetrahymena thermophila macronuclear rDNA gene, Chinese hamster ovary dihydrofolate reductase amplicon, human c- myc protooncogene, chicken histone H5 gene, Drosophila melanogaster chorion gene cluster on the third chromosome, and Chinese hamster ovary rhodopsin gene. The locations of putative initiation regions identified by the computer analyses were compared with published data obtained using diverse methods to map initiation sites. For at least four loci, the potential initiation regions identified by sequence analysis aligned with previously mapped initiation events. A consensus DNA sequence, WAWTTDDWWWDHWGWHMAWTT, was found within the potential initiation regions in every case. An additional 35 kb of combined flanking sequences from the six loci were also analyzed, but no additional copies of this consensus sequence were found.This article is from Nucleic Acids Research 22 (1994): 2479, doi: 10.1093/nar/22.13.2479. Posted with permission.</p

Modular sequence elements associated with origin regions in eukaryotic chromosomal DNA.

We have postulated that chromosomal replication origin regions in eukaryotes have in common clusters of certain modular sequence elements (Benbow, Zhao, and Larson, BioEssays 14, 661-670, 1992). In this study, computer analyses of DNA sequences from six origin regions showed that each contained one or more potential initiation regions consisting of a putative DUE (DNA unwinding element) aligned with clusters of SAR (scaffold associated region), and ARS (autonomously replicating sequence) consensus sequences, and pyrimidine tracts. The replication origins analyzed were from the following loci: Tetrahymena thermophila macronuclear rDNA gene, Chinese hamster ovary dihydrofolate reductase amplicon, human c-myc proto-oncogene, chicken histone H5 gene, Drosophila melanogaster chorion gene cluster on the third chromosome, and Chinese hamster ovary rhodopsin gene. The locations of putative initiation regions identified by the computer analyses were compared with published data obtained using diverse methods to map initiation sites. For at least four loci, the potential initiation regions identified by sequence analysis aligned with previously mapped initiation events. A consensus DNA sequence, WAWTTDDWWWDHWGWHMAWTT, was found within the potential initiation regions in every case. An additional 35 kb of combined flanking sequences from the six loci were also analyzed, but no additional copies of this consensus sequence were found

Studies of cis- and trans-acting elements in Tetrahymena rDNA replication

Eukaryotic cells must precisely duplicate their genomes before they divide. The mechanisms of eukaryotic chromosomal DNA replication are far from clear, however, because very few cis- and trans-acting factors that function in eukaryotic DNA replication have been identified. To gain further insights into this problem, this work identified and characterized potential cis- and trans-acting replication factors for the replication initiation of Tetrahymena ribosomal RNA gene (rDNA);A search far cis-acting elements identified a cluster of predicted modular sequences and structural elements in the origin region of rDNA. The presence of these elements were verified experimentally; (1) two mung bean nuclease-hypersensitive sites were localized within the 5\u27NTS; (2) three fragments in the 5\u27NTS were found to contain bent DNA structures; (3) nuclear matrices were found to be present in the 5\u27NTS. These structural elements were also identified in five other eukaryotic origin regions, suggesting that clusters of modular structural elements may be a conserved feature in eukaryotic chromosomal origins of replication;Biochemical purification of potential trans-acting factors has led to the identification of the first DNA helicase in protozoan, Tetrahymena thermophila DNA helicase I. It co-fractionated through several steps with ssA-TIBF, an rDNA origin binding protein, indicating that they may functionally associate in rDNA replication in vivo. The ATP-binding subunit was determined to be ~70 kDa, distinct from the 24 kDa DNA binding subunit of ssA-TIBF. The directionality of this helicase was 3\u27 to 5\u27, indicating a possible functional interaction with DNA polymerase moving in the same direction during DNA replication. This helicase preferentially unwound helicase substrates containing a fork-like structure which resembles replication intermediates. Taken together, the properties of T. thermophila DNA helicase I suggest that it could function as a replicative helicase in leading strand DNA synthesis. A model is proposed to describe possible events in the replication initiation of Tetrahymena rDNA

Isolation and genetic dissection of an eukaryotic replicon that supports autonomous DNA replication

Maintenance of genome integrity requires that chromosomes be accurately and faithfully replicated. We are using Tetrahymena thermophila as a model system for studying the initiation and regulation of eukaryotic DNA replication. This organism contains a diploid micronucleus and polyploid macronucleus. During macronuclear development, the five diploid chromosomes of the micronucleus are fragmented into 280 macronuclear minichromosomes that are subsequently replicated to ~45 copies. In stark contrast, the 21 kb ribosomal DNA minichromosome (rDNA) is amplified from 2 to 10,000 copies in the same nucleus. Previous characterization of the rDNA replicon has led to the localization of its origin and the cis-acting regulatory determinants to the 1.9 kb 5'non-transcribed spacer region. The objective of this study was to identify and characterize non-rDNA origins of replication in Tetrahymena. This will help determine the underlying basis for differential regulation of rDNA and non-rDNA origins during development, as well as provide a better understanding of the organization of eukaryotic replicons. To this effect, I developed a DNA transformation assay that I used to isolate new Tetrahymena replication origins. A 6.7 kb non-rDNA fragment, designated TtARS1, was shown to support stable autonomous replication of circular plasmids in Tetrahymena. Genetic dissection revealed that TtARS1 contains two independent replicons, TtARS1-A and TtARS1-B. Full TtARS1-A function requires a minimal sequence of 700 bp, and two small regions in this fragment have been shown to be essential for origin function. TtARS1-B replicon function was localized to a 1.2 kb intergenic segment that contains little sequence similarity to TtARS1-A. Both non-rDNA replicons lack sequence similarity to the rDNA 5' NTS, suggesting that each replicon interact with a different set of regulatory proteins. This study indicates that the rDNA and the non-rDNA replicons have a modular organization, containing discrete, cis-acting replication determinants

Conserved and Unconventional Responses to DNA Damage in Tetrahymena

Here the ciliate protozoa Tetrahymena thermophila was used as a model system to study the DNA damage response. Tetrahymena enclose nuclear dimorphism, a polyploid somatic macronucleus (MAC), which is transcriptionally active and maintains vegetative growth, and a diploid germline micronucleus (MIC) responsible for the transmition of genetic information during conjugation. Previous studies have identified Tif1p, a novel protein involved in the regulation of rDNA replication in Tetrahymena. TIF1 hypomorphic strains acquire spontaneous DNA damage during vegetative cell cycle and are hypersensitive to DNA damaging agents. TIF1-deficient strains acquire DNA damage in both nuclear compartments, suggesting a global role of Tif1p in the maintenance of genomic stability. In my dissertation research, I studied the role of Tif1p during the cell cycle progression. To this end, I generated tagged-Tif1p strains, which revealed that the subcellular localization of Tif1p is dynamic throughout the cell cycle. However, the addition of epitope tag to this protein generated phenotypes analogous to ones observed in a TIF1-deficient strain. This suggested that the addition of epitope tag to Tif1p severely affects the properties of Tif1p and hence the overall integrity of the cell. To overcome these limitations, a peptide antibody specific to Tif1p was generated to study the endogenous protein. This work revealed that the abundance of Tif1p protein is not cell cycle regulated and that Tif1p is absent in starved cells. Furthermore, the specific binding of TIf1p to rDNA minichromosome was studied during vegetative cell cycles. Chromatin immunoprecipitation studies revealed that the specific binding of Tif1p extends beyond the cis-acting determinant of replication present at the rDNA origin and promoter. This suggests that coding regions may be targeted for the binding of Tif1p to previously uncharacterized sequences, and that Tif1p preferentially localizes on the rDNA minichromosome. I also studied the induction of DNA damage response, demonstrating that Tetrahymena activates a checkpoint response mediated by an ATR-like pathway. Studies with a hypomorphic TIF1 strain revealed that Tif1p mediates proper activation of the DNA damage response. Further characterization of the response to genotoxic agents showed that Tetrahymena is able to activate a G1/S and intra-S phase DNA damage response. The results presented here suggest that a caffeine-dependent checkpoint activator protein modulates the response to DNA damage. In addition, a subunit of the replicative helicase, Mcm6p, is directly affected by the induction of DNA damage. This suggests that Tetrahymena uses a novel mechanism to halt the progression of DNA replication forks during genotoxic stress through degradation of Mcm6p