Abstract
The Saccharomyces cerevisiae MGM101 gene encodes a DNA-binding protein targeted to mitochondrial nucleoids. MGM101 is essential for maintenance of a functional rho(+) genome because meiotic segregants, with a disrupted mgm101 allele, cannot undergo more than 10 divisions on glycerol medium. Quantitative analysis of mtDNA copy number in a rho(+) strain carrying a temperature-sensitive allele, mgm101-1, revealed that the amount of mtDNA is halved each cell division upon a shift to the restrictive temperature. These data suggest that mtDNA replication is rapidly blocked in cells lacking MGM101. However, a small proportion of meiotic segregants, disrupted in MGM101, have rho(-) genomes that are stably maintained. Interestingly, all surviving rho(-) mtDNAs contain an ori/rep sequence. Disruption of MGM101 in hypersuppressive (HS) strains does not have a significant effect on the propagation of HS rho(-) mtDNA. However, in petites lacking an ori/rep, disruption of MGM101 leads to either a complete loss or a dramatically decreased stability of mtDNA. This discriminatory effect of MGM101 suggests that replication of rho(+) and ori/rep-devoid rho(-) mtDNAs is carried out by the same process. By contrast, the persistence of ori/rep-containing mtDNA in HS petites lacking MGM101 identifies a distinct replication pathway. The alternative mtDNA replication mechanism provided by ori/rep is independent of mitochondrial RNA polymerase encoded by RPO41 as a HS rho(-) genome is stably maintained in a mgm101, rpo41 double mutant.
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- Asai T., Bates D. B., Kogoma T. DNA replication triggered by double-stranded breaks in E. coli: dependence on homologous recombination functions. Cell. 1994 Sep 23;78(6):1051–1061. doi: 10.1016/0092-8674(94)90279-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Baldacci G., Chérif-Zahar B., Bernardi G. The initiation of DNA replication in the mitochondrial genome of yeast. EMBO J. 1984 Sep;3(9):2115–2120. doi: 10.1002/j.1460-2075.1984.tb02099.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Blanc H., Dujon B. Replicator regions of the yeast mitochondrial DNA responsible for suppressiveness. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3942–3946. doi: 10.1073/pnas.77.7.3942. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chen X. J., Guan M. X., Clark-Walker G. D. MGM101, a nuclear gene involved in maintenance of the mitochondrial genome in Saccharomyces cerevisiae. Nucleic Acids Res. 1993 Jul 25;21(15):3473–3477. doi: 10.1093/nar/21.15.3473. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chi N. W., Kolodner R. D. Purification and characterization of MSH1, a yeast mitochondrial protein that binds to DNA mismatches. J Biol Chem. 1994 Nov 25;269(47):29984–29992. [PubMed] [Google Scholar]
- Clark-Walker G. D., Chen X. J. A vital function for mitochondrial DNA in the petite-negative yeast Kluyveromyces lactis. Mol Gen Genet. 1996 Oct 28;252(6):746–750. doi: 10.1007/BF02173982. [DOI] [PubMed] [Google Scholar]
- Contamine V., Picard M. Maintenance and integrity of the mitochondrial genome: a plethora of nuclear genes in the budding yeast. Microbiol Mol Biol Rev. 2000 Jun;64(2):281–315. doi: 10.1128/mmbr.64.2.281-315.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Desai S. D., Pasupathy K., Chetty K. G., Pradhan D. S. Evidence for the presence of DNA primase in mitochondria of Saccharomyces cerevisiae. Biochem Biophys Res Commun. 1989 Apr 28;160(2):525–534. doi: 10.1016/0006-291x(89)92464-9. [DOI] [PubMed] [Google Scholar]
- Fangman W. L., Henly J. W., Churchill G., Brewer B. J. Stable maintenance of a 35-base-pair yeast mitochondrial genome. Mol Cell Biol. 1989 May;9(5):1917–1921. doi: 10.1128/mcb.9.5.1917. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Foury F. Cloning and sequencing of the nuclear gene MIP1 encoding the catalytic subunit of the yeast mitochondrial DNA polymerase. J Biol Chem. 1989 Dec 5;264(34):20552–20560. [PubMed] [Google Scholar]
- Foury F., Kolodynski J. pif mutation blocks recombination between mitochondrial rho+ and rho- genomes having tandemly arrayed repeat units in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1983 Sep;80(17):5345–5349. doi: 10.1073/pnas.80.17.5345. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Foury F., Roganti T., Lecrenier N., Purnelle B. The complete sequence of the mitochondrial genome of Saccharomyces cerevisiae. FEBS Lett. 1998 Dec 4;440(3):325–331. doi: 10.1016/s0014-5793(98)01467-7. [DOI] [PubMed] [Google Scholar]
- Gietz R. D., Schiestl R. H., Willems A. R., Woods R. A. Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast. 1995 Apr 15;11(4):355–360. doi: 10.1002/yea.320110408. [DOI] [PubMed] [Google Scholar]
- Graves T., Dante M., Eisenhour L., Christianson T. W. Precise mapping and characterization of the RNA primers of DNA replication for a yeast hypersuppressive petite by in vitro capping with guanylyltransferase. Nucleic Acids Res. 1998 Mar 1;26(5):1309–1316. doi: 10.1093/nar/26.5.1309. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Greenleaf A. L., Kelly J. L., Lehman I. R. Yeast RPO41 gene product is required for transcription and maintenance of the mitochondrial genome. Proc Natl Acad Sci U S A. 1986 May;83(10):3391–3394. doi: 10.1073/pnas.83.10.3391. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Güldener U., Heck S., Fielder T., Beinhauer J., Hegemann J. H. A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res. 1996 Jul 1;24(13):2519–2524. doi: 10.1093/nar/24.13.2519. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hermann G. J., Shaw J. M. Mitochondrial dynamics in yeast. Annu Rev Cell Dev Biol. 1998;14:265–303. doi: 10.1146/annurev.cellbio.14.1.265. [DOI] [PubMed] [Google Scholar]
- Kao L. R., Megraw T. L., Chae C. B. Essential role of the HMG domain in the function of yeast mitochondrial histone HM: functional complementation of HM by the nuclear nonhistone protein NHP6A. Proc Natl Acad Sci U S A. 1993 Jun 15;90(12):5598–5602. doi: 10.1073/pnas.90.12.5598. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kaufman B. A., Newman S. M., Hallberg R. L., Slaughter C. A., Perlman P. S., Butow R. A. In organello formaldehyde crosslinking of proteins to mtDNA: identification of bifunctional proteins. Proc Natl Acad Sci U S A. 2000 Jul 5;97(14):7772–7777. doi: 10.1073/pnas.140063197. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kleff S., Kemper B., Sternglanz R. Identification and characterization of yeast mutants and the gene for a cruciform cutting endonuclease. EMBO J. 1992 Feb;11(2):699–704. doi: 10.1002/j.1460-2075.1992.tb05102.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lecrenier N., Foury F. New features of mitochondrial DNA replication system in yeast and man. Gene. 2000 Apr 4;246(1-2):37–48. doi: 10.1016/s0378-1119(00)00107-4. [DOI] [PubMed] [Google Scholar]
- Ling F., Makishima F., Morishima N., Shibata T. A nuclear mutation defective in mitochondrial recombination in yeast. EMBO J. 1995 Aug 15;14(16):4090–4101. doi: 10.1002/j.1460-2075.1995.tb00081.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ling F., Morioka H., Ohtsuka E., Shibata T. A role for MHR1, a gene required for mitochondrial genetic recombination, in the repair of damage spontaneously introduced in yeast mtDNA. Nucleic Acids Res. 2000 Dec 15;28(24):4956–4963. doi: 10.1093/nar/28.24.4956. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lockshon D., Zweifel S. G., Freeman-Cook L. L., Lorimer H. E., Brewer B. J., Fangman W. L. A role for recombination junctions in the segregation of mitochondrial DNA in yeast. Cell. 1995 Jun 16;81(6):947–955. doi: 10.1016/0092-8674(95)90014-4. [DOI] [PubMed] [Google Scholar]
- Lorimer H. E., Brewer B. J., Fangman W. L. A test of the transcription model for biased inheritance of yeast mitochondrial DNA. Mol Cell Biol. 1995 Sep;15(9):4803–4809. doi: 10.1128/mcb.15.9.4803. [DOI] [PMC free article] [PubMed] [Google Scholar]
- MacAlpine D. M., Perlman P. S., Butow R. A. The high mobility group protein Abf2p influences the level of yeast mitochondrial DNA recombination intermediates in vivo. Proc Natl Acad Sci U S A. 1998 Jun 9;95(12):6739–6743. doi: 10.1073/pnas.95.12.6739. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Macino G., Tzagoloff A. Assembly of the mitochondrial membrane system: partial sequence of a mitochondrial ATPase gene in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1979 Jan;76(1):131–135. doi: 10.1073/pnas.76.1.131. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Maleszka R., Skelly P. J., Clark-Walker G. D. Rolling circle replication of DNA in yeast mitochondria. EMBO J. 1991 Dec;10(12):3923–3929. doi: 10.1002/j.1460-2075.1991.tb04962.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Meeusen S., Tieu Q., Wong E., Weiss E., Schieltz D., Yates J. R., Nunnari J. Mgm101p is a novel component of the mitochondrial nucleoid that binds DNA and is required for the repair of oxidatively damaged mitochondrial DNA. J Cell Biol. 1999 Apr 19;145(2):291–304. doi: 10.1083/jcb.145.2.291. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Megraw T. L., Chae C. B. Functional complementarity between the HMG1-like yeast mitochondrial histone HM and the bacterial histone-like protein HU. J Biol Chem. 1993 Jun 15;268(17):12758–12763. [PubMed] [Google Scholar]
- Miyakawa I., Fumoto S., Kuroiwa T., Sando N. Characterization of DNA-binding proteins involved in the assembly of mitochondrial nucleoids in the yeast Saccharomyces cerevisiae. Plant Cell Physiol. 1995 Oct;36(7):1179–1188. [PubMed] [Google Scholar]
- Miyakawa I., Sando N., Kawano S., Nakamura S., Kuroiwa T. Isolation of morphologically intact mitochondrial nucleoids from the yeast, Saccharomyces cerevisiae. J Cell Sci. 1987 Nov;88(Pt 4):431–439. doi: 10.1242/jcs.88.4.431. [DOI] [PubMed] [Google Scholar]
- Myers A. M., Pape L. K., Tzagoloff A. Mitochondrial protein synthesis is required for maintenance of intact mitochondrial genomes in Saccharomyces cerevisiae. EMBO J. 1985 Aug;4(8):2087–2092. doi: 10.1002/j.1460-2075.1985.tb03896.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Newman S. M., Zelenaya-Troitskaya O., Perlman P. S., Butow R. A. Analysis of mitochondrial DNA nucleoids in wild-type and a mutant strain of Saccharomyces cerevisiae that lacks the mitochondrial HMG box protein Abf2p. Nucleic Acids Res. 1996 Jan 15;24(2):386–393. doi: 10.1093/nar/24.2.386. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nobrega F. G., Tzagoloff A. Assembly of the mitochondrial membrane system. DNA sequence and organization of the cytochrome b gene in Saccharomyces cerevisiae D273-10B. J Biol Chem. 1980 Oct 25;255(20):9828–9837. [PubMed] [Google Scholar]
- Preiser P. R., Wilson R. J., Moore P. W., McCready S., Hajibagheri M. A., Blight K. J., Strath M., Williamson D. H. Recombination associated with replication of malarial mitochondrial DNA. EMBO J. 1996 Feb 1;15(3):684–693. [PMC free article] [PubMed] [Google Scholar]
- Reenan R. A., Kolodner R. D. Isolation and characterization of two Saccharomyces cerevisiae genes encoding homologs of the bacterial HexA and MutS mismatch repair proteins. Genetics. 1992 Dec;132(4):963–973. doi: 10.1093/genetics/132.4.963. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schmitt M. E., Clayton D. A. Conserved features of yeast and mammalian mitochondrial DNA replication. Curr Opin Genet Dev. 1993 Oct;3(5):769–774. doi: 10.1016/s0959-437x(05)80097-8. [DOI] [PubMed] [Google Scholar]
- Sedman T., Kuusk S., Kivi S., Sedman J. A DNA helicase required for maintenance of the functional mitochondrial genome in Saccharomyces cerevisiae. Mol Cell Biol. 2000 Mar;20(5):1816–1824. doi: 10.1128/mcb.20.5.1816-1824.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sor F., Fukuhara H. Identification of two erythromycin resistance mutations in the mitochondrial gene coding for the large ribosomal RNA in yeast. Nucleic Acids Res. 1982 Nov 11;10(21):6571–6577. doi: 10.1093/nar/10.21.6571. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Van Dyck E., Clayton D. A. Transcription-dependent DNA transactions in the mitochondrial genome of a yeast hypersuppressive petite mutant. Mol Cell Biol. 1998 May;18(5):2976–2985. doi: 10.1128/mcb.18.5.2976. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Van Dyck E., Foury F., Stillman B., Brill S. J. A single-stranded DNA binding protein required for mitochondrial DNA replication in S. cerevisiae is homologous to E. coli SSB. EMBO J. 1992 Sep;11(9):3421–3430. doi: 10.1002/j.1460-2075.1992.tb05421.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wach A., Brachat A., Pöhlmann R., Philippsen P. New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast. 1994 Dec;10(13):1793–1808. doi: 10.1002/yea.320101310. [DOI] [PubMed] [Google Scholar]
- Weislogel P. O., Butow R. A. Low temperature and chloramphenicol induction of respiratory deficiency in a cold-sensitive mutant of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1970 Sep;67(1):52–58. doi: 10.1073/pnas.67.1.52. [DOI] [PMC free article] [PubMed] [Google Scholar]
- White M. F., Lilley D. M. The structure-selectivity and sequence-preference of the junction-resolving enzyme CCE1 of Saccharomyces cerevisiae. J Mol Biol. 1996 Mar 29;257(2):330–341. doi: 10.1006/jmbi.1996.0166. [DOI] [PubMed] [Google Scholar]
- Willer M., Rainey M., Pullen T., Stirling C. J. The yeast CDC9 gene encodes both a nuclear and a mitochondrial form of DNA ligase I. Curr Biol. 1999 Oct 7;9(19):1085–1094. doi: 10.1016/s0960-9822(99)80477-1. [DOI] [PubMed] [Google Scholar]
- Williamson D. H., Fennell D. J. Visualization of yeast mitochondrial DNA with the fluorescent stain "DAPI". Methods Enzymol. 1979;56:728–733. doi: 10.1016/0076-6879(79)56065-0. [DOI] [PubMed] [Google Scholar]
- Xu B., Clayton D. A. A persistent RNA-DNA hybrid is formed during transcription at a phylogenetically conserved mitochondrial DNA sequence. Mol Cell Biol. 1995 Jan;15(1):580–589. doi: 10.1128/mcb.15.1.580. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zelenaya-Troitskaya O., Newman S. M., Okamoto K., Perlman P. S., Butow R. A. Functions of the high mobility group protein, Abf2p, in mitochondrial DNA segregation, recombination and copy number in Saccharomyces cerevisiae. Genetics. 1998 Apr;148(4):1763–1776. doi: 10.1093/genetics/148.4.1763. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zelenaya-Troitskaya O., Perlman P. S., Butow R. A. An enzyme in yeast mitochondria that catalyzes a step in branched-chain amino acid biosynthesis also functions in mitochondrial DNA stability. EMBO J. 1995 Jul 3;14(13):3268–3276. doi: 10.1002/j.1460-2075.1995.tb07330.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zinn A. R., Pohlman J. K., Perlman P. S., Butow R. A. In vivo double-strand breaks occur at recombinogenic G + C-rich sequences in the yeast mitochondrial genome. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2686–2690. doi: 10.1073/pnas.85.8.2686. [DOI] [PMC free article] [PubMed] [Google Scholar]