Replication

Replication Origin and Replication Initiation

Unlike ^29 phage and adenoviruses, replication of the linear plasmids (and linear chromosomes) of Streptomyces is initiated from an internal origin. Such an internal replication origin was first identified by Shiffman and Cohen (1992) in pSCLl of S. clavuligerus by its ability to support replication of a circular DNA construct in Streptomyces. This autonomously replicating sequence (ARS) contains two rep genes essential for replication. One is a DnaB (replicative helicase) homolog and the other resembles another rep gene of pSLA2 of S. rochei (Chang et al. 1996).

Subsequently, ARSs were identified in several other linear plasmids of Streptomyces. Without exception, they contain at least one gene encoding a protein necessary for replication (presumably its initiation) and probably the replication origin in the form of a series of direct repeats (iterons) or AT-rich sequences or both. The ARS of pSLA2 contains an operon consisting of rep1pSLA2 encoding a DNA-binding protein and rep2pSLA2 encoding a helicase (Chang et al. 1996). The ARS of SCP1 is central and contains an open reading frame (ORF) (SCP1.196) encoding a 506-aa putative primase/helicase, an overlapping ORF (SCP1.197) encoding a 133-aa hypothetical protein, and a putative iteron nearby (Redenbach et al. 1999). The same gene pair encoding a primase/helicase and a ca. 130-aa protein is also found in two ARSs (ori2 and ori3) on the 210-kb linear plasmid pSLA2-L in S. rochei (Mochizuki et al. 2001). The ca. 330-aa proteins are of limited occurrence, but the pri-mase/helicase proteins are widespread, particularly among plasmids and bac-teriophages.

pSLA2-L contains a third ARS (ori1) adjacent to ori2. ori1 includes an essential gene, repL1, a subsidiary gene, repL2, and a putative replication origin lying about 800 bp from repL1 (Hiratsu et al. 2000). Search of the database found only a homologous pair of repL1 and repL2 on pSCL1 in a syntenous arrangement (including a 4-bp overlap). It is not known which of the three ARSs (all of which can support replication of plasmids in a circular form) is functional in vivo for pSLA2-L replication. In view of the great difference between pSLA2-L and pSCL1 in size and genetic content, it is likely that ori1 was acquired during evolution. However, the joining of ori1 and ori2 next to each other is puzzling.

The SLP2 ARS represents yet a different type. It contains an iteron and a dnaB-type helicase gene (repSLP2) (Xu et al. 2006), which is found in a wide variety of bacterial plasmids, but this is the only example among linear Streptomyces plasmids characterized so far.

The diversity of the genetic constituents (oriP and rep enzymes) involved in initiation of replication of Streptomyces linear plasmids is in contrast to the greater homogeneity of those (telomeres and Tpg/Tap proteins) involved in end patching (at least of the archetypal telomeres). This indicates that there have been diverse origins of the rep-iteron system of the linear Streptomyces plasmids. A recent study shows that new rep-iteron combinations produced by artificial exchanges of rep genes or iterons among three plasmids, SLP2, SCP1, and pSLA2, are competent to support plasmid replication, although the plasmid transformation efficiencies were decreased by two to three orders of magnitude (Xu et al. 2006). This indicates a relatively loose Rep-iteron specificity for these replication initiation systems.

Elements Required for Replication in Linear Form

Qin et al. (2003) discovered a novel gene rlrApSLA2 on pSLA2, which is required for replication of the plasmid in linear but not circular form. RlrApSLA2 is a 308-aa protein with two LuxR family regulatory domains, which on overexpression interferes with the replication of pSLA2 in circular form. On pSLA2, rlrA appears to be regulated by a divergently transcribed gene rorR encoding a KorA-like regulator.

No rlrA homolog has been found in the database except for a weak ho-mology (27% identity) with a hypothetical protein encoded by pSCL1 (ORF pSCL_p5), suggesting that other linear plasmids may encode their own functional counterparts of rlrApSLA2. Indeed, replication of SLP2 in linear form does require an additional sequence containing an ORF (SLP2.13, z'lrASLP2, with no homology to rlrApSLA2) adjacent to a KorA-like regulatory gene (SLP2.14) on SLP2 (Xu et al. 2006). Interestingly, this requirement for linear replication may be substituted by either rlrApSLA2 (Qin et al. 2003) or by a truncated tap homolog on SLP2 (mtapSLP2) that encodes an 88-aa polypeptide corresponding to the N terminus of Tap proteins (Xu et al. 2006). In a yeast two-hybrid assay, IlrASLP2 interacts with TapSli. Therefore, it has been suggested that IlrASLP2 and RlrApSLA2 are involved in coordinating initiation from the origin with end patching during replication of linear plasmids. However, in these studies, no trans-acting function of these genetic elements has been tested, and therefore the possibility that rlrApSLA2, ilrASLP2, and mtapSLP2 contain an essential cis-acting element, such as an origin of replication, which is essential specifically for replication in a linear form, cannot be ruled out.

Like their circular counterparts, the linear plasmids of Streptomyces generally do not encode a DNA polymerase, and presumably rely on one of the chromosomally encoded polymerases (such as Pol III; see below) for replication. An exception so far has been SCP1, which encodes a (catalytic) and P (clamp) subunits of Pol III (Bentley et al. 2004), the significance of which is not clear.

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