Extended Data Figure 3: Characterization of oriC3.
From: Accelerated growth in the absence of DNA replication origins
a, Sequence features of oriC3. Double-headed arrow indicates the autonomously replicating fragment recovered from a genomic library of H1023 (pTA1100; see Methods for details); solid arrows, open reading frames; triangles, repeats. The intergenic region upstream of orc2 is typical of archaeal origins. It is enlarged to show the sequence features of oriC3 duplex unwinding element (DUE). HVO_0635 encodes a conserved hypothetical protein. b, Sequence of intergenic repeats upstream of orc2 (numbered in a, triangles show repeat orientation). Dark grey shading indicates match to consensus origin recognition box (ORB); bases conserved between repeats are indicated by light grey shading. c, Plasmid-based assays for the three chromosomal origins. Recombination-deficient strain H112 was transformed with 1 µg of pTA441 (oriC1), pTA612 (oriC2) or pTA1100 (oriC3). Transformants were plated with 100-fold dilution on Hv-Ca and incubated at 45 °C for 14 days. Numbers indicate transformation efficiency in colony-forming units (c.f.u.) per microgram of DNA. d, GC-disparity of main chromosome in wild isolate DS2 (adapted from ref. 4); positions of orc genes and replication origins are shown. The lack of a nucleotide disparity inflection point at oriC3 suggests that this origin has been acquired recently or is used infrequently, consistent with the replication profile (Fig. 1a) and plasmid-based assay (Extended Data Fig. 3c).
