Discovery of a Major D-Loop Replication Origin Reveals Two Modes of Human mtDNA Synthesis Jennifer Fish,* Nicola Raule,* Giuseppe Attardi. Mammalian mitochondrial DNA (mtDNA) replication has long been consid- ered to occur by asymmetric synthesis of the two strands, starting at the multiple origins of the strand-displacement loop (D-loop). We report the discovery of a major replication origin at position 57 in the D-loop of several human cell lines (HeLa, A549, and 143B.TK – ) and immortalized lymphocytes. The nascent chains starting at this origin, in contrast to those initiated at the previously described origins, do not terminate prematurely at the 3¶ end of the D-loop but proceed well beyond this control point, behaving as ‘‘true’’ replicating strands. This origin is mainly responsible for mtDNA maintenance under steady-state conditions, whereas mtDNA synthesis from the formerly identified D-loop origins may be more important for recovery after mtDNA depletion and for accelerating mtDNA replication in response to physiological demands. Traditionally, mammalian mtDNA replica- tion has been thought to occur through an asymmetric mechanism. This was proposed in a model introduced more than 25 years ago, which held that the two mtDNA strands replicate in partial asynchrony (1–3). Re- cently, however, evidence favoring the exis- tence of a bidirectional, strand-coupled mechanism has been presented (4–6), raising a lively debate (7–9). The proponents of this alternative model suggest that the multiple origins within the D-loop represent points of fork arrest of bidirectional replication start- ing downstream. Here, we examine the role of the mtDNA heavy strand (H-strand) replication origins in three human cell lines—HeLa, A549 lung carcinoma, and 143B.TK – osteosarcoma cells—and in sev- eral immortalized lymphocyte cell lines. To measure the rate of mtDNA replication, we used primer extension to make a comple- mentary copy of each mtDNA nascent H- strand chain present in a preparation of total cell DNA (10, 11). This technique uses VENT DNA polymerase and an appropriate E5¶- 32 P^-labeled light strand (L-strand) oligo- deoxynucleotide primer, chosen within the D- loop sequence (internal) or 3¶ to this sequence (external) (Fig. 1A). The extended primers are then separated by polyacrylamide gel electro- phoresis (PAGE). From the radioactivity associated with extended primers synthesized in vitro on nascent chains, one can estimate the relative amounts of these chains, which should reflect their relative rates of synthesis and, consequently, the relative activities of the corresponding origins. Figure 2A shows the PAGE patterns of the extended L-strand primers obtained from total cell DNA samples of the three human cell lines with the use of the D-loop–internal primer Ip1 (Fig. 1A). These patterns exhibited species of nascent chains, absent in mtDNA-less 143B.r-206 cells (12), which corresponded to origins at or very near positions 191, 167, 151, 146, and 110 in the mtDNA sequence ( 13). These origins matched the major initiation sites of H- strand synthesis determined earlier in the mtDNA D-loop of human cells (14–16). The slowly migrating, highly labeled band ob- served in Fig. 2 derived mainly from primer extension on the parental H-strand and was thus labeled PSex (parental strand extended) (Fig. 2C) (11). It was previously shown that most of the nascent H-strands originating in the D-loop terminate prematurely at the 3¶-end of the loop (17), forming the 7S DNA (1) (Fig. 1A). To identify the nascent H-strand chains that extended beyond the D-loop, we used the external primer Ep1 (Fig. 1A). The patterns for all three cell lines (Fig. 2B) exhibited a set of extended primers corresponding in size to those expected for nascent chains starting from the various origins previously detected with the internal primer (Fig. 2A); these bands varied in intensity in each cell line and between different cell lines, in a manner similar to what was observed with internal primers. Unexpectedly, the three patterns also showed a strong band representing a new species of extended primers, absent in the pattern from 143B.r-206 cell DNA (Fig. 2D), which corresponded to an origin at an mtDNA position near 60 in the D-loop. The same results were obtained with the external primers Ep2 (Fig. 2D), Ep3 (fig. S1), and Ep4 (Fig. 2E). Premature termination H5' L3' H3' L5' LSP D-Loop Cytochrome b Ip1 Ep1 Ep3 Ep4 191 57 RNA primers A 7S DNA True Replicating DNA 151 PT B 57 Ip2 Ip3 Ep2 T A549: 20 clones of 24 HeLa: 13 clones of 14 143B: 9 clones of 14 16,100 191 14,747 Fig. 1. (A) Schematic representation of H-strand replication initiation in human mtDNA D-loop region. P, tRNA Pro gene; T, tRNA Thr gene; H, heavy strand; L, light strand; LSP, L- strand transcription promoter; Ip1 to Ip3, L-strand internal primers 1, 2, and 3; Ep1 to Ep4, L-strand external primers 1, 2, 3, and 4 (11). Numbers indicate nucleotide positions accord- ing to the human mtDNA sequence (13). (B) Identification by sequencing of the position-57 origin. The sequence shows a representative 5¶-end–3¶-end junction in a cloned PCR product of a circularized extended primer. Numbers of clones from mtDNA of different cell lines that exhibited the 5¶-end–3¶-end junction sequence shown are indicated. Division of Biology, California Institute of Technolo- gy, Pasadena, CA 91125, USA. *These authors contributed equally to this work. .To whom correspondence should be addressed. E-mail: attardi@caltech.edu R EPORTS 17 DECEMBER 2004 VOL 306 SCIENCE www.sciencemag.org 2098