DNA replication is a dynamic process that occurs in a temporal order along each of the chromosomes. A consequence of the temporally coordinated activation of replication origins is the establishment of broad domains (>100 kb) that replicate either early or late in S phase. This partitioning of the genome into early and late replication domains is important for maintaining genome stability, gene dosage, and epigenetic inheritance; however, the molecular mechanisms that define and establish these domains are poorly understood. The modENCODE Project provided an opportunity to investigate the chromatin features that define the Drosophila replication timing program in multiple cell lines. The majority of early and late replicating domains in the Drosophila genome were static across all cell lines; however, a small subset of domains was dynamic and exhibited differences in replication timing between the cell lines. Both origin selection and activation contribute to defining the DNA replication program. Our results suggest that static early and late replicating domains were defined at the level of origin selection (ORC binding) and likely mediated by chromatin accessibility. In contrast, dynamic domains exhibited low ORC densities in both cell types, suggesting that origin activation and not origin selection governs the plasticity of the DNA replication program. Finally, we show that the male-specific early replication of the X chromosome is dependent on the dosage compensation complex (DCC), suggesting that the transcription and replication programs respond to the same chromatin cues. Specifically, MOF-mediated hyperacetylation of H4K16 on the X chromosome promotes both the up-regulation of male-specific transcription and origin activation.
The T box transcription antitermination mechanism regulates the expression of unique genes in many Gram-positive bacteria by responding, in a magnesium-dependent manner, to uncharged cognate tRNA base pairing with an antiterminator RNA element and other regions of the 5′-untranslated region. Model T box antiterminator RNA are known to bind aminoglycosides, ligands that typically bind RNA in divalent metal ion binding sites. In this study, enzymatic footprinting and spectroscopic assays were used to identify and characterize the binding site of neomycin B to an antiterminator model RNA. Neomycin B binds the antiterminator bulge nucleotides in an electrostatic-dependent manner and displaces 3-4 monovalent cations, indicating that the antiterminator likely contains a divalent metal ion-binding site. Neomycin B facilitates rather than inhibits tRNA binding indicating that bulge-targeted inhibitors that bind the antiterminator via non-electrostatic interactions may be the more optimal candidates for antiterminator-targeted ligand design.
In order to duplicate the genome within S‐phase, DNA replication must initiate at multiple start sites. We are using genomic approaches in the model organism Drosophila to elucidate how origins of replication are selected and regulated to maintain genomic stability. As part of the model organism ENCODE consortium we have characterized the average time of replication for all unique sequences, mapped early origins of replication and identified sites of preRC localization in multiple cell lines. We have found that ORC preferentially localizes to open chromatin near the transcription start site of active genes. ORC binding sites are enriched for the histone variant H3.3 which marks open and dynamic chromatin. The large number of ORC binding sites has provided us the opportunity to use machine learning algorithms to identify complex sequence elements that direct ORC localization. We have also found that the single X chromosome of male cell lines replicates significantly earlier than the autosomes, suggesting a link between DNA replication and dosage compensation. Funding from NIH grant HG004279.
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