Mode of Replication and Origins of Replication

The study of DNA replication in B. burgdorferi has just recently commenced. What has been established is the position of the replication origin for the linear chromosome and the minimal complement of open reading frames (ORFs) required for autonomous replication of several examples of the circular and linear replicons.

A key study physically mapped the origin of replication of the linear chromosome to a small 2-kb region almost exactly in the centre of the chromosome (Picardeau et al. 1999). In combination with examination of AT- and CG-skew to identify a candidate origin region, a technique called nascent DNA strand analysis was employed. By further analysis of the switch point of the polarity of CG-skew and sequence analysis for origin-like characteristics, the authors conclude that the origin most likely lies in the intergenic region between dnaA and dnaN. Replication from this origin proceeds bidirection-ally. A further important point is that the hp telomeres show no origin activity in the nascent DNA strand assay, eliminating the possibility of their use as replication origins (Picardeau et al. 1999).

The discovery of a specific binding site for the Hbb protein in the dnaA-dnaN intergenic region lends further support to the designation of this area as the origin (Kobryn et al. 2000). Hbb is the B. burgdorferi homologue of the nucleoid proteins HU and IHF (Tilly et al. 1996). These accessory DNA bending proteins often act in conjunction with DnaA in the open complex formation step of origin firing in other bacterial systems (Hwang and Kornberg 1992; Kornberg 1992; Roth et al. 1994). Hbb binding to its specific site introduces a sharp U-bend between two flanking direct repeats and induces KMnO4 sensitivity of several T nucleotides outwith the Hbb binding site (Ko-bryn et al. 2000). These are all biochemical features consistent with a role of Hbb binding to this site in replication initiation.

The bidirectional internal origin and the demonstration of the lack of origin activity of the hp telomeres strongly argued against replication models for the chromosome that do not invoke a DNA breakage and reunion event (telomere resolution) at the replicated telomeres (rTels; see Casjens 1999 for a review of replication models for the linear replicons). The lack of hp telom-ere origin activity also argues against models similar to those proposed for poxvirus replication, which invoke site-specific nicking at one telomere followed by strand displacement synthesis and telomere resolution to process the resulting linear head-to-head, tail-to-tail concatamers into unit length viral genomes terminated by hp telomeres (Fig. 1; see Kobryn and Chaconas 2001, and references therein).

I Pathway

I Pathway T|

I strand displacement replication

■ bidirectional I replication from

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