Research at the DNA replication group aims at understanding how information from the cell cycle machinery leads to the initiation of DNA replication. Proper cell growth depends on an intricate network of signaling molecules that monitor cellular metabolism and environmental signals. This network insures that cells do not start duplicating their genetic material under unfavorable conditions, such as the absence of sufficient nutrients or the presence of potentially damaging agents. When DNA replication escapes cell cycle controls, the results are developmental abnormalities, genomic instability, and cancer. To study cell cycle signaling at the chromatin level, we specify DNA sequences that determine whether, where, and when replication will occur. Recent work used the human beta globin locus as a model to delineate the sequence requirements for dictating the location of starting points for DNA replication. We had also recently reported that the timing of DNA replication during the S-phase of the cell cycle can be altered, and are now elucidating the genetic and epigenetic factors that determine replication timing. We perform these analyses in established cell lines and in embryonic or hematopoietic stem cells. Biochemical studies in mammalian cells revealed that DNA replication initiates from fixed genomic regions called replication origins or initiation regions (IRs). In bacteria, viruses and yeast, these regions carry genetic information essential for the initiation of DNA replication. However, until recently it was not clear whether the initiation of DNA replication in mammalian cells similarly depends on specific DNA sequences. This question was particularly hard to analyze in mammalian cells because, unlike yeast and bacterial replication origins, mammalian DNA sequences that functioned as replication origins within their native loci did not initiate DNA replication extrachromosomally. Our previous studies used the human beta-globin locus as a model system. Our studies show that replication indeed requires specific DNA sequences. In recent studies, we asked which sequences within IR dictate replicator activity. Surprisingly, we found that there are two independent replicators within the globin IR. Each one of these replicators can act as a DNA replication starting site. Within each replicator, we identified two sequences that cooperate to facilitate replication . We now continue the genetic analysis to delineate the requirements for self-contained """"""""minimal"""""""" replicators, and study protein binding patterns of replicator sequences to determine how replicators communicate with cell cycle regulators at various times during the cell cycle. In the human beta-globin locus, we showed that two sequence elements were essential for initiating DNA replication: the initiation region (IR), and the locus control region (LCR) residing 50 kb upstream of the IR. The LCR is also involved in regulating globin gene expression, and in the establishment of tissue and developmental specific chromatin structures. The involvement of LCR in replication implies that initiation of DNA replication may require interactions between distant sequences. These observations suggested that the chromosomal environment may play an important role in determining origin activity. We have also demonstrated that in mouse cells, DNA replication initiates from multiple sites within the beta-globin locus. Nevertheless, the replication timing pattern remaines similar to that observed in human cells - cells that do not express globin replicate the locus late during S-phase whereas cells that express the protein replicate the locus early during S-phase. This study had also shown that murine embryonic stem cells (ES cells) initiated DNA replication at specific sites, unlike some other embryonic systems in which replication initiation appeared random. This was especially interesting because we have previously observed that ES cells do not activate some of the cell cycle controls that regulate the growth of normal somatic cells. We now use the murine system in an attempt to understand the interelations betweem the timing of DNA replication, gene expression and cell cycle control. The human beta-globin locus is a good model to study the regulation of replication timing, since this locus replicates early in S-phase in cells that express globin, but replicates later in cells that do not express globin. We found that insertion of sequences from the human beta-globin locus into a late replicating site on murine chromosome 15 alters replication timing in a sequence- and orientation-dependent manner. These observations suggest that the timing of DNA replication is controlled at the DNA sequence level, and that the sequences that modulate replication timing are distinct from the sequence elements required for replicator activity. We now determine which DNA sequences control of replication timing and ask whether these sequences also modify the condensation state of chromatin or cooperate with replicator sequences.
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