1. Introduction
DNA replication in prokaryotes, in budding yeast and in mammalian DNA viruses initiates from fixed origins (
Using an
Usually, the mutants are confirmed by co-immunoprecipitation of cell lysates with the anti-Y antibody (Ab) retained on agarose beads, stripping of the wild type (WT) X (or mutant X that should be in the wash), separation by gel electrophoresis and visualization with anti-Y Ab. Naturally, the authentic non-interaction mutant forms of X should no longer bind to Y or bind poorly. These “pull down” assays are used to confirm the reverse 2-hybrid results. If the interaction of X and Y is necessary for a biological function (
The yeast forward and reverse 2-hybrid analyses followed by biochemical analysis of Tus, showed that it contacted DnaB probably at the L1 loop because the only mutations that impaired helicase arrest and fork arrest without abolishing or significantly reducing Tus-
If there is protein-protein interaction between Tus and DnaB and if this is necessary for fork arrest, how does Tus also promote polar arrest of RNA polymerase, an enzyme apparently different in structure from DnaB? One possible explanation is that Tus might make an equivalent contact with RNA polymerase to inhibit its progression, or else a different mechanism could be operating here. It should, however, be clearly stated that this line of reasoning does not necessarily disprove the first explanation. Based on the data discussed above, we have suggested a model of fork arrest that involves not only stable Tus-
The Fob1 protein is multifunctional and loads histone deacetylase to silence intra-chromatid recombination in the tandem array of ~200 rDNA repeats that might otherwise lead to unscheduled loss or gain of rDNA repeats (Bairwa et al., 2010; Huang et al., 2006; Huang and Moazed, 2003). Fob1 protein is also a transcriptional activator and controls exit from mitosis (Bastia and Mohanty, 2006; Stegmeier et al., 2004).
One of the facile techniques to study Fob1 function is to perform segment-directed mutagenesis, which is shown schematically (Fig.6). A segment of an ORF flanked by regions of homology (also from the ORF) is amplified by PCR under conditions of low fidelity synthesis in which one of the dNTPs is present at a suboptimal concentration. This leads to misincorporation of the base into DNA causing random mutations. A plasmid containing a gap corresponding to the segment being mutagenized and the PCR products are used to transform yeast. The mutagenized DNA segment gets incorporated into the plasmid by gap repair caused by the homologous recombination machinery of yeast with high efficiency, thus generating a pool of potential mutants contained in the plasmid. The plasmid contains a marker expressed in yeast (
We have recently reported that the Reb1 terminator protein binding to 2 Ter sites of fission yeast act in a cooperative fashion. The dimeric Reb1 protein, for example, brings into contact a Ter site located on chromosome 2 with two Ter sites located on chromosome 1. Interestingly there was no interaction observed between sites on chromosome 1 and 2 with the Ter sites located in the two rDNA clusters present on chromosome 3. It seems that the Ter-Ter interactions are not random. We further reported that the interactions called "chromosome kissing' modulated the activities of the Ter sites (Singh et al., 2010).
In summary, replication termination at site-specific termini is an important part of DNA replication that invites further investigation, especially in eukaryotes, because of its role in various DNA transactions including maintenance of genome stability.
Acknowledgement
: We thank Dr. G. Krings and other members of our group for their valuable contributions to the investigations of replication termination. Our work was supported by a grant from the NIGMS.
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