Analysis of the two heavy and light strand origins and the direction of replication of mitochondrial DNA within a detailed physical map of this genome in transformed hamster cells

Abstract

The precise physical map positions of the two origins of replication of mtDNA † † Abbreviations used: mtDNA, mitochondrial DNA; H-strand and L-strand, heavy and light complementary strand; O H and O L, heavy and light strand origins of replication; D-mtDNA, mtDNA containing non-expanded D-loops; Exp-D mtDNA, mtDNA with an expanded D-loop; C-mtDNA, D-mtDNA after removal of the 7 S initiation sequence; E-mtDNA, closed circular mtDNA daughter molecule prior to D-loop formation (for details of terminology for replication intermediates see Nass, 1980; Kasamatsu et al., 1971; Berk & Clayton, 1974); PrI 2, propidium diiodide; bp, base-pairs; kb, base-pairs × 10 −3. have been determined in C 13 B 4 cells, a line of Syrian baby hamster kidney fibroblasts transformed with the Bryan strain of Rous sarcoma virus. A detailed physical map with 27 restriction endonuclease recognition sites was constructed, which aligns eight HpaI and two PstI sites with previously mapped cleavage positions for HpaII, HindIII, EcoRI and BamHI. The origin of heavy strand mtDNA replication, which corresponds to the 5′ end of the 7 S initiation sequence in the D-loop structure, was aligned on restriction fragments HpaI A, PstI A, BamHI A, HindIII B, EcoRI B and HpaII F. The location of the origin within the 891 bp fragment HpaII F is approximately 220 bp from the HpaII F A junction which bisects the D-loop. The remaining about 180 bp sequence extends from this border into a ~ 642 bp subfragment of HpaII A, HpaII + EcoRI 8, and expansion of the D-loop proceeds in this direction. Fragment HpaII F can be further cut into at least two subfragments by HaeIII. The identical map position of the D-loop was found for mtDNA from the non-transformed parent cell line BHK 21 C 13 . The 5′ → 3′ orientation of heavy chain replication was specified by the use of exonuclease III. Replication of C 13 B 4 mtDNA appears exclusively asymmetrical, as determined by electron microscopic analysis of replication intermediates and by hybridization of pulse-labeled isolated initiation and expanded sequences with restriction fragments and with H- and L-strands of mtDNA. Unidirectional H-strand displacement synthesis proceeds to 67± 3% of the genome before any duplex synthesis occurs at this location in the opposite direction, marking L-strand initiation. The majority of clustered L-strand initiation sequences flank the restriction site HpaI D B within fragment HpaII E at map position 67.0. The Syrian hamster mtDNA replication system provides an excellent model for studies of genome substructure and function.