SUMMARY Replication forks face multiple obstacles that slow their progression. By two-dimensional gel analysis, yeast forks pause at stable DNA protein complexes, and this pausing is greatly increased in the absence of the Rrm3 helicase. We used a genome wide approach to identify 96 sites of very high DNA polymerase binding in wild type cells. Most of these binding sites were not previously identified pause sites. Rather, the most highly represented genomic category among high DNA polymerase binding sites was the open reading frames (ORFs) of highly transcribed RNA polymerase II genes. Twice as many pause sites were identified in rrm3 compared to wild type cells as pausing in this strain occurred at both highly transcribed RNA polymerase II genes and the previously identified protein DNA complexes. ORFs of highly transcribed RNA polymerase II genes are the first class of natural pause sites that are not exacerbated in rrm3 cells.

Summary Chromosome replication initiates at multiple replicons and terminates when forks converge. In E. coli, the Tus-TER complex mediates polar fork converging at the terminator region and aberrant termination events challenge chromosome integrity and segregation. Since in eukaryotes termination is less characterized, we used budding yeast to identify the factors assisting fork fusion at replicating chromosomes. Using genomic and mechanistic studies we have identified and characterized 71 chromosomal termination regions (TERs). TERs contain fork pausing elements that influence fork progression and merging. The Rrm3 DNA helicase assists fork progression across TERs counteracting the accumulation of X-shaped structures. The Top2 DNA topoisomerase associates at TERs in S-phase and G2/M facilitates fork fusion and prevents DNA breaks and genome rearrangements at TERs. We propose that in eukaryotes replication fork barriers, Rrm3 and Top2 coordinate replication fork progression and fusion at termination regions thus counteracting abnormal genomic transitions.

The Saccharomyces cerevisiae DNA helicase Rrm3p is needed for normal fork progression through >1000 discrete sites scattered throughout the genome. Here we show that replication of all yeast chromosomes was markedly delayed in rrm3 cells. Delayed replication was seen even in a region that lacks any predicted Rrm3p-dependent sites. Based on the pattern of replication intermediates in two-dimensional gels, the rate of fork movement in rrm3 cells appeared similar to wild-type except at known Rrm3p-dependent sites. These data suggest that although Rrm3p has a global role in DNA replication, its activity is needed only or primarily at specific, difficult-to-replicate sites. By the criterion of chromatin immunoprecipitation, Rrm3p was associated with both Rrm3p-dependent and -independent sites, and moved with the replication fork through both. In addition, Rrm3p interacted with Pol2p, the catalytic subunit of DNA polymerase , in vivo. Thus, rather than being recruited to its sites of action when replication forks stall at these sites, Rrm3p is likely a component of the replication fork apparatus.[Keywords: Rrm3p; Mrc1p; DNA replication; helicase; yeast; chromatin] Supplemental material is available at http://www.genesdev.org.

Populations of terrestrial or freshwater taxa that are separated by oceans can be explained by either oceanic dispersal or fragmentation of a previously contiguous land mass. Amphisbaenians, the worm lizards (approx. 165 species), are small squamate reptiles that are uniquely adapted to a burrowing lifestyle and inhabit Africa, South America, Caribbean Islands, North America, Europe and the Middle East. All but a few species are limbless and they rarely leave their subterranean burrows. Given their peculiar habits, the distribution of amphisbaenians has been assumed to be primarily the result of two land-mass fragmentation events: the split of the supercontinent Pangaea starting 200 Myr ago, separating species on the northern land mass (Laurasia) from those on the southern land mass (Gondwana), and the split of South America from Africa 100 Myr ago. Here we show with molecular evidence that oceanic dispersal—on floating islands—played a more prominent role, and that amphisbaenians crossed the Atlantic Ocean in the Eocene (40 Myr ago) resulting in a tropical American radiation representing one-half of all known amphisbaenian species. Until now, only four or five transatlantic dispersal events were known in terrestrial vertebrates. Significantly, this is the first such dispersal event to involve a group that burrows, an unexpected lifestyle for an oceanic disperser.

Assembly of hepatitis delta virus (HDV) in infected human hepatocytes involves association of the 1,679-nucleotide single-stranded genomic RNA (␦RNA) with multiple copies of both small and large forms of the delta protein (␦Ag) to form a ribonucleoprotein particle which in turn interacts with envelope proteins of the natural helper virus, hepatitis B virus. Subsequently, for initiation of a new round of replication, the amount of small ␦Ag within the assembled HDV particle is both necessary and sufficient. Quantitative assays were used in order to better understand just how much ␦Ag is needed. The molar ratio of ␦Ag species to genomic ␦RNA in assembled HDV particles was approximately 200. Next, this ratio was determined for cells under several different experimental situations in which HDV genome replication was occurring. These included replication in woodchuck liver and also in mouse liver and skeletal muscle, as well as replication in stably and transiently transfected cultured human hepatoblastoma cells. Surprisingly, in almost all these situations the molar ratios were comparable to that observed for HDV particles. This was true for different times after the initiation of replication and was independent of whether or not virus assembly was occurring. Cell fractionation combined with quantitative assays was used to test whether the majority of ␦Ag and ␦RNA were colocalized during HDV replication in transfected cells. The cytoplasmic fraction contained the majority of ␦Ag and genomic ␦RNA. Finally, the quality of ␦Ag and ␦RNA, especially at relatively late times after the initiation of replication, was examined by using reverse transcription-PCR, cloning, and sequencing through the entire ␦Ag open reading frame. When virus assembly and spread were not possible, 20% or less of the predicted ␦Ag would have been able to support HDV replication. In summary, an examination of the quantity, quality and intracellular distribution of ␦Ag and ␦RNA in several different experimental systems has provided a better understanding of the parameters associated with the initiation, maintenance, and ultimate decline of HDV genome replication.Human hepatitis delta virus (HDV) was discovered in 1977 (34) and was soon recognized as a satellite of hepatitis B virus (HBV) (35). The helper role of HBV is limited to providing the envelope proteins needed for HDV assembly and subsequent cycles of infection (20). In other words, all other aspects of the intracellular replication of the HDV genome can be studied independently of assembly and infection.

IntroductionBoth normal hematopoiesis and lymphomagenesis are driven, to a large extent, by expression of lineage-specific transcription factors. Many such factors are overtly oncogenic. For example, the hallmark of Burkitt lymphoma and some diffuse large B cell lymphomas (DLBCLs) is the t(8;14) translocation that places the MYC protooncogene under control of the Ig heavy chain gene enhancer (1, 2); a similar translocation has been identified in murine plasmacytomas (3). The causative role of Myc in B-lymphomagenesis has been validated through the generation of various transgenic (4-7) and nontransgenic (8) murine models of non-Hodgkin lymphoma.Other transcription factors are not known to initiate B-lymphomagenesis, but are consistently mutated in full-fledged neoplasms. One such intriguing protein is paired box gene 5 (Pax5), a B cell activator protein (9) with a characteristic paired box DNA-binding motif (10). Inactivation of Pax5 via homologous recombination precludes normal B cell development (11,12), and several lines of evidence implicate Pax5 as an important factor in B-lymphomagenesis as well.

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