Ori Sequences Research Articles

Surrogate origins of replication in the mitochondrial genomes of ori-zero petite mutants of yeast.

We have investigated the mitochondrial genome of eight ori-zero spontaneous petite mutants of Saccharomyces cerevisiae. The tandem repeat units of these genomes do not contain any of the seven canonical ori sequences of the wild-type genome. Instead, they contain one, or more, ori-S sequences. These 44-nucleotide long surrogate origins of replication are a subset of GC clusters characterized by a potential secondary fold with two sequences ATAG and GGAG , inserted in AT spacers, two AT base pairs just following them, a GC stem (broken in the middle, and, in most cases also near the base, by non-paired nucleotides), and a terminal loop. This structure is reminiscent of that of GC clusters A and B from canonical ori sequences and supports the view (Bernardi, 1982a ) that the GC clusters of the mitochondrial genome arose, by an expansion process, from the canonical ori sequences. Like the latter, ori-S sequences are present in both orientations, are located in intergenic regions, and can be used as excision sequences when tandemly oriented. Again as in the case of canonical ori sequences, the density of ori-S sequences on the repeat units of petite genomes are correlated with the replication efficiency of the latter, as assessed by the outcome of crosses with wild-type or petite tester strains.

A small subclass of SV40 T antigen binds to the viral origin of replication.

We examined the affinities of SV40 large T antigen for unique viral DNA sequences by binding SV40 Bst NI DNA fragments in extracts of infected or transformed cells, and then immunoprecipitating the T antigen-DNA complex. The G fragment, which spans the viral origin of replication (ori) was quantitatively bound to T antigen. A T-antigen-specific monoclonal antibody (McI 7), which recognized only 5%-10% of the T antigen from infected or transformed cells, immunoprecipitated the majority of the ori-binding activity. This suggests that only a minor subclass of wild-type T antigen is active in binding to the origin. C6 cells contain a replication-defective mutant T antigen that when tested in the DNA-binding immunoassay, showed no affinity for the ori fragment. McI 7 not only failed to immunoprecipitate ori binding in C6 cells, but also did not detect any labeled C6 T antigen whatever. Thus McI 7 recognizes an immunologically distinct subset of wild-type 7 antigen that comprises the origin-binding form of the viral protein, which is absent in the C6 T antigen population. McI 122, which recognizes a 53 kilodalton host protein that complexes with T antigen, immunoprecipitated ori-binding activity from extracts of infected or transformed cells, but not from C6 cells. Thus wild-type T antigen can bind ori sequences even when complexed to the host protein. These data suggest that T antigen consists of different subpopulations with different functions.

A region of extreme instability in the mitochondrial genome of yeast.

About half of the spontaneous petite mutants produced by wild-type Saccharomyces cerevisiae strain B (as well as by several other strains) have the same defective mitochondrial genome. Its repeat unit is a segment, 2200 base pairs (bp) long, which derives from an excision between the origins of replication ori 2 and ori 7 of the wild-type genome, and contains a hybrid ori 2-ori 7 sequence. The spontaneous petites carrying this defective ori.h genome are supersuppressive , i.e., they very rapidly compete out the wild-type genome in crosses. The main reasons for the exceptional frequency of ori.h petites are an extremely high excision frequency, due to the extended homology between the two tandemly oriented ori sequences 265 bp long and the short distance separating them. Such an excision frequency is very strongly increased in petite genomes encompassing the ori 2-ori 7 region, because of their higher concentration in these ori sequences.

The origins of replication of the yeast mitochondrial genome and the phenomenon of suppressivity.

The ‘petite colonie’ mutation of Saccharomyces cerevisiae1,2 is characterized by an irreversible loss of respiration and by an extraordinarily high spontaneous mutation rate3,4. Crosses of wild-type cells with petite mutants exhibit a non-mendelian segregation of the mutation, yielding either wild-type progeny only1,2, or both wild-type and petite mutants in proportions essentially dependent on the particular petite used3. In the first case, the petites entering the cross are called neutral, in the second one suppressive3. While the molecular basis of the spontaneous petite mutation is now understood4–12, suppressivity has remained an elusive phenomenon for the past 25 yr. We report here that the mitochondrial genome of most spontaneous petites (which is exclusively made up by the tandem repetition of a DNA segment excised from the genome of parental wild-type cells5–8) carries at least one of the ori sequences of the parental wild-type genome. These are long homologous DNA stretches showing striking similarities with the origins of replication of mitochondrial DNAs from mammalian cells. The properties (intact or altered primary structure, high or low number) of the ori sequences of petite genomes seem to determine suppressivity—the level of transmission of petite genomes to the progeny of crosses with wild-type cells. These results indicate that ori sequences are indeed origins of DNA replication and that suppressivity depends on the relative replication efficiencies of petite and wild-type genomes.

Mutational analysis of the simian virus 40 replicon: pseudorevertants of mutants with a defective replication origin.

The circular genome of simian virus 40 is a model mammalian replicon, containing a unique origin of replication (ori) and coding for a protein (SV40 T antigen) known to be involved in initiation of viral DNA replication and to bind in vitro to the origin region. Mutations within the ori sequence lead to defective viral DNA replication and the formation of small viral plaques after infection of a cell monolayer. Second-site revertants (pseudorevertants) of ori mutants were isolated by random local mutagenesis of mutant DNA followed by transfection of cultured cells and the selection of large plaques. In each case, reversion of the plaque phenotype was associated with an increased rate of viral DNA replication. The second-site mutations that suppressed the replication defects were localized by in vitro recombination or marker rescue experiments to the gene for T antigen. Their map positions differ from those of previously described T antigen mutants, possibly reflecting a specific ori-binding domain of T antigen. From these results we infer that T antigen interacts with the ori signal during virus development as it does in vitro and that this interaction regulates the rate of viral DNA replication.

Inceptor and origin of DNA replication in lambdoid coliphages II. The λ DNA maximal replication system

In pBR313-λ dv hybrid plasmids a second system for initiation of DNA replication has been detected in lambdoid replicator DNAs (in the absence of the p 0 promoter). This “maximal” (or “maxi”) initiation system depends on the origin of replication ( ori) sequence, in conjunction with the “inceptor” ( ice) element located in the lambdoid cII genes. Only leftward, but not bidirectional, primer RNA synthesis seems to be initiated at ori in its newly defined boundaries, and it appears to be catalysed by dnaG-coded primase. Only if transcriptionally activated, will ori effectively initiate λ specific, O and P- dependent “maximal” hybrid-plasmid replication. In addition, it will repress a complete λ “minimal” initiation system in cis, i.e., if present on the same plasmid molecule. This newly discovered repressive activity of the ori system depends on only three factors: an intact left section of ori, the O product, and transcriptional activation of ori (rightward or leftward). A repressed minimal initiation system will regain its activity as soon as a segment carrying either part of the O gene or a promoter for transcriptional activation is deleted from such a plasmid which was combining both the “mini” and “maxi” systems of λ replication.