Characterization of a Novel Origin Recognition Complex-Like Complex: Implications for DNA Recognition, Cell Cycle Control, and Locus-Specific Gene Amplification

Abstract

The origin recognition complex (ORC) plays a central role in eukaryotic DNA replication. Here we describe a unique ORC-like complex in Tetrahymena thermophila, TIF4, which bound in an ATP-dependent manner to sequences required for cell cycle-controlled replication and gene amplification (ribosomal DNA [rDNA] type I elements). TIF4's mode of DNA recognition was distinct from that of other characterized ORCs, as it bound exclusively to single-stranded DNA. In contrast to yeast ORCs, TIF4 DNA binding activity was cell cycle regulated and peaked during S phase, coincident with the redistribution of the Orc2-related subunit, p69, from the cytoplasm to the macronucleus. Origin-binding activity and nuclear p69 immunoreactivity were further regulated during development, where they distinguished replicating from nonreplicating nuclei. Both activities were lost from germ line micronuclei following the programmed arrest of micronuclear replication. Replicating macronuclei stained with Orc2 antibodies throughout development in wild-type cells but failed to do so in the amplification-defective rmm11 mutant. Collectively, these findings indicate that the regulation of TIF4 is intimately tied to the cell cycle and developmentally programmed replication cycles. They further implicate TIF4 in rDNA gene amplification. As type I elements interact with other sequence-specific single-strand breaks (in vitro and in vivo), the dynamic interplay of Orc-like (TIF4) and non-ORC-like proteins with this replication determinant may provide a novel mechanism for regulation.

TIF1 Activates the Intra-S-Phase Checkpoint Response in the Diploid Micronucleus and Amitotic Polyploid Macronucleus ofTetrahymena

“…It is unclear how Tif1p is differentially localized to distinct nuclei that inhabit the same cytoplasm. By analogy, the subcellular localization of the TIF4 Orc2p cross-reactive subunit Tt-p69 is similarly regulated (Mohammad et al, 2003). We speculate that these proteins are imported into micro-and macronuclei by nucleus-specific "licensing factors."…”

“…For cell division analysis, DAPI-stained cells were examined microscopically at defined intervals after drug treatment and removal. The input cells for the latter analysis were synchronized by starvation and refeeding before the addition of HU and/or caffeine (Mohammad et al, 2003). The cell division index corresponds to the percentage of cells exhibiting a cytokinetic furrow, as determined by light microscopy.…”

“…Fixed cells were incubated with antibodies before mounting onto slides (Morrison et al, 2005) or mounted before antibody incubation (Loidl and Scherthan, 2004). For cell cycle studies, vegetative cultures were starved, refed, and harvested at 30-min intervals as described previously (Mohammad et al, 2003).…”

Yeast Rmi1/Nce4 Controls Genome Stability as a Subunit of the Sgs1-Top3 Complex

“…Loading dye was added to a final concentration of 8% glycerol and 0.25% bromophenol blue. Gel electrophoresis was modified from a previously described method (34). After the gel was loaded, the sample was electrophoresed at 10 V/cm through a 7.5% polyacrylamide gel (29:1 acrylamide:bis) in 12.5 mM Tris-HCl (pH 8.7), 55 mM glycine, and 1.5 mM EDTA at 4°C.…”

TIF1 Represses rDNA Replication Initiation, but Promotes Normal S Phase Progression and Chromosome Transmission inTetrahymena

“…Promoter-proximal type I elements are also required for rRNA transcription (Gallagher and Blackburn, 1998 Umthun et al, 1994;Mohammad et al, 2000). In contrast to ORC and non-ORC replicator proteins in other systems, all four TIFs associate exclusively with single-stranded DNA in vitro (Mohammad et al, 2000(Mohammad et al, , 2003Saha and Kapler, 2000). Consistent with an in vivo role for these sequence-specific single-stranded DNA binding proteins (ss-SSBs), in vivo footprinting studies indicate that the Tetrahymena rDNA origin and promoter regions are naturally unwound in native chromosomes (Saha et al, 2001).…”