DNA Polymerase SSB and Terminal Recognition Factor

Each autonomous linear plasmid harbors a gene encoding a TP-DNA polymerase (Tommasino et al. 1988; Hishinuma and Hirai 1991; Hishinuma et al.

1984; Stark et al. 1984; Sor and Fukuhara 1985; Klassen et al. 2001; Klassen and Meinhardt 2003; Jeske and Meinhardt 2006). Hence, any cytoplasmic plasmid system possesses at least one viral B-type DNA polymerase; however, as for the pGKL system of K. lactis, another TP-DNA polymerase locus may be present. Since loss or knockout of either of the two polymerase encoding genes cannot be functionally complemented by the remaining locus, each plasmid evidently replicates via its own DNA polymerase (Kitada and Gunge 1988; Schaffrath et al. 1995). Such specificity is supported by the fact that TPs of pGKL1 and pGKL2 differ in size. Indeed, it was experimentally proven that TPs of pGKL2 are encoded as the N-terminal region of pGKL2 Orf2 (Takeda et al. 1986), agreeing with DNA polymerases specifically replicating the plas-mid that encodes them.

In multiple plasmid systems possessing a single TP-DNA polymerase gene only (on the autonomous element), as for pPac1-1, pPac1-2 and pPE1A, pPE1B of P. acaciae and D. etchellsii, respectively, replication of nonau-tonomous elements is probably mediated by the same enzyme. Though it is to be expected that autonomous and nonautonomous elements should have identical TPs in these instances, there is no discernible sequence homology with respect to TIR sequences (Klassen et al. 2002, 2004; Jeske and Meinhardt 2006), which are considered to harbor replication origins. Thus, the TPs rather than the TIRs provide the basis for the specificity seen in systems with more than one DNA polymerase.

The viral B-type DNA polymerase-TP fusion protein is apparently sufficient for replication of linear plasmids in mitochondria. However, cyto-plasmic replication needs additional plasmid encoded functions, such as the single-strand DNA binding protein (SSB), for stabilization of replication intermediates and the (rather basic) terminal recognition factor (TRF) which specifically binds to TIRs and is presumably involved in replication initiation (McNeel and Tamanoi 1991; Tommasino 1991; Schaffrath and Meacock 2001). Indeed, pGKL2 encoded SSB were shown to interact with ssDNA without sequence preference (Schaffrath and Meacock 2001), whereas binding sites for TRF comprise nucleotides 107-183 in the pGKL1 TIR and 126-179 in the pGKL2 TIR. Though there is no obvious sequence similarity, both TIR regions are rich in long dA-dT stretches presumably folding into similar secondary structures (McNeel and Tamanoi 1991).

Genes encoding TP-DNA polymerase, SSB, and TRF were proven to be essential for cytoplasmic inheritance in the pGKL1,2 system (Schaffrath and Meacock 1995; Schaffrath et al. 1995; Tiggemann and Meinhardt, unpublished results). The enzymatic machinery involved in replication of cytoplasmic linear plasmids clearly resembles the scenario seen in phi29-like bacteriophages, since virus encoded ss- and dsDNA binding proteins are involved in replication initiation in addition to the B-type DNA polymerase and TPs (Meijer et al. 2001).

0 0

Post a comment