Strand invasion structures in the inverted repeat of Candida albicans mitochondrial DNA reveal a role for homologous recombination in replication.

Molecular recombination and transcription are proposed mechanisms to initiate mitochondrial DNA (mtDNA) replication in yeast. We conducted a comprehensive analysis of mtDNA from the yeast Candida albicans. Two-dimensional agarose gel electrophoresis of mtDNA intermediates reveals no bubble structures diagnostic of specific replication origins, but rather supports recombination-driven replication initiation of mtDNA in yeast. Specific species of Y structures together with DNA copy number analyses of a C. albicans mutant strain provide evidence that a region in a mainly noncoding inverted repeat is predominantly involved in replication initiation via homologous recombination. Our further findings show that the C. albicans mtDNA forms a complex branched network that does not contain detectable amounts of circular molecules. We provide topological evidence for recombination-driven mtDNA replication initiation and introduce C. albicans as a suitable model organism to study wild-type mtDNA maintenance in yeast.

Double-stranded DNA-dependent ATPase Irc3p is directly involved in mitochondrial genome maintenance.

Nucleic acid-dependent ATPases are involved in nearly all aspects of DNA and RNA metabolism. Previous studies have described a number of mitochondrial helicases. However, double-stranded DNA-dependent ATPases, including translocases or enzymes remodeling DNA-protein complexes, have not been identified in mitochondria of the yeast Saccharomyces cerevisae. Here, we demonstrate that Irc3p is a mitochondrial double-stranded DNA-dependent ATPase of the Superfamily II. In contrast to the other mitochondrial Superfamily II enzymes Mss116p, Suv3p and Mrh4p, which are RNA helicases, Irc3p has a direct role in mitochondrial DNA (mtDNA) maintenance. Specific Irc3p-dependent mtDNA metabolic intermediates can be detected, including high levels of double-stranded DNA breaks that accumulate in irc3Δ mutants. irc3Δ-related topology changes in rho- mtDNA can be reversed by the deletion of mitochondrial RNA polymerase RPO41, suggesting that Irc3p counterbalances adverse effects of transcription on mitochondrial genome stability.

Replication intermediates of the linear mitochondrial DNA of Candida parapsilosis suggest a common recombination based mechanism for yeast mitochondria.

Variation in the topology of mitochondrial DNA (mtDNA) in eukaryotes evokes the question if differently structured DNAs are replicated by a common mechanism. RNA-primed DNA synthesis has been established as a mechanism for replicating the circular animal/mammalian mtDNA. In yeasts, circular mtDNA molecules were assumed to be templates for rolling circle DNA-replication. We recently showed that in Candida albicans, which has circular mapping mtDNA, recombination driven replication is a major mechanism for replicating a complex branched mtDNA network. Careful analyses of C. albicans-mtDNA did not reveal detectable amounts of circular DNA molecules. In the present study we addressed the question of how the unit sized linear mtDNA of Candida parapsilosis terminating at both ends with arrays of tandem repeats (mitochondrial telomeres) is replicated. Originally, we expected to find replication intermediates diagnostic of canonical bi-directional replication initiation at the centrally located bi-directional promoter region. However, we found that the linear mtDNA of Candida parapsilosis also employs recombination for replication initiation. The most striking findings were that the mitochondrial telomeres appear to be hot spots for recombination driven replication, and that stable RNA:DNA hybrids, with a potential role in mtDNA replication, are also present in the mtDNA preparations.

UvrD-like helicase Hmi1 Has an ATP independent role in yeast mitochondrial DNA maintenance.

Hmi1 is a UvrD-like DNA helicase required for the maintenance of the yeast Saccharomyces cerevisiae mitochondrial DNA (mtDNA). Deletion of the HMI1 ORF leads to the formation of respiration-deficient petite mutants, which either contain a short fragment of mtDNA arranged in tandem repeats or lack mtDNA completely. Here we characterize point mutants of the helicase designed to target the ATPase or ssDNA binding activity and show that these mutations do not separately lead to complete loss of the Hmi1 function. The mutant strains support ATP production via oxidative phosphorylation and enable us to directly analyze the impact of both activities on the stability of wild-type mtDNA in this petite-positive yeast. Our data reveal that Hmi1 mutants affecting ssDNA binding display a stronger defect in the maintenance of mtDNA compared to the mutants of ATP binding/hydrolysis. Hmi1 mutants impaired in ssDNA binding demonstrate sensitivity to UV irradiation and lower levels of Cox2 encoded by the mitochondrial genome. This suggests a complex and multifarious role for Hmi1 in mtDNA maintenance-linked transactions, some of which do not require the ATP-dependent helicase activity.

Mitochondrial Irc3 helicase of the thermotolerant yeast Ogataea polymorpha displays dual DNA- and RNA-stimulated ATPase activity.

Irc3 is one of the six mitochondrial helicases described in Saccharomyces cerevisiae. Physiological functions of Irc3 are not completely understood as both DNA metabolic processes and mRNA translation have been suggested to be direct targets of the helicase. In vitro analysis of Irc3 has been hampered by the modest thermostability of the S. cerevisiae protein. Here, we purified a homologous helicase (Irc3op) of the thermotolerant yeast Ogataea polymorpha that retains structural integrity and catalytic activity at temperatures above 40 °C. Irc3op can complement the respiratory deficiency phenotype of a S. cerevisiae irc3Δ mutant, indicating conservation of biochemical functions. The ATPase activity of Irc3op is best stimulated by branched and double- stranded DNA cofactors. Single-stranded DNA binds Irc3op tightly but is a weak activator of the ATPase activity. We could also detect a lower level stimulation with RNA, especially with molecules possessing a compact three-dimensional structure. These results support the idea that that Irc3 might have dual specificity and remodel both DNA and RNA molecules in vivo. Furthermore, our analysis of kinetic parameters predicts that Irc3 could have a regulatory function via sensing changes of the mitochondrial ATP pool or respond to the accumulation of single-stranded DNA.

Relicensing of transcriptionally inactivated replication origins in budding yeast.

DNA replication origins are licensed in early G(1) phase of the cell cycle where the origin recognition complex (ORC) recruits the minichromosome maintenance (MCM) helicase to origins. These pre-replicative complexes (pre-RCs) remain inactive until replication is initiated in the S phase. However, transcriptional activity in the regions of origins can eliminate their functionality by displacing the components of pre-RC from DNA. We analyzed genome-wide data of mRNA and cryptic unstable transcripts in the context of locations of replication origins in yeast genome and found that at least one-third of the origins are transcribed and therefore might be inactivated by transcription. When investigating the fate of transcriptionally inactivated origins, we found that replication origins were repetitively licensed in G(1) to reestablish their functionality after transcription. We propose that reloading of pre-RC components in G(1) might be utilized for the maintenance of sufficient number of competent origins for efficient initiation of DNA replication in S phase.

Irc3 is a monomeric DNA branch point-binding helicase in mitochondria of the yeast Saccharomyces cerevisiae.

Irc3 is a superfamily II DNA helicase required for the maintenance of mitochondrial DNA stability in Saccharomyces cerevisiae. Here, we show that recombinant Irc3 is a monomeric protein and that it can form a binary complex with forked DNA. The catalytically active enzyme is a monomer as no positive cooperativity of ATP hydrolysis or DNA unwinding can be detected. Interestingly, we find that Irc3 prefers to unwind the nascent lagging strand at a replication fork. Using DNase I footprinting, we demonstrate that Irc3 captures DNA substrates by establishing a strong contact at the DNA branching point. Additional protections on the lagging strand template suggest a 3'-to-5' polarity for Irc3 movement.

Mitochondrial helicase Irc3 translocates along double-stranded DNA.

Irc3 is a superfamily II helicase required for mitochondrial DNA stability in Saccharomyces cerevisiae. Irc3 remodels branched DNA structures, including substrates without extensive single-stranded regions. Therefore, it is unlikely that Irc3 uses the conventional single-stranded DNA translocase mechanism utilized by most helicases. Here, we demonstrate that Irc3 disrupts partially triple-stranded DNA structures in an ATP-dependent manner. Our kinetic experiments indicate that the rate of ATP hydrolysis by Irc3 is dependent on the length of the double-stranded DNA cosubstrate. Furthermore, the previously uncharacterized C-terminal region of Irc3 is essential for these two characteristic features and forms a high affinity complex with branched DNA. Together, our experiments demonstrate that Irc3 has double-stranded DNA translocase activity.

Irc3 is a mitochondrial DNA branch migration enzyme.

Integrity of mitochondrial DNA (mtDNA) is essential for cellular energy metabolism. In the budding yeast Saccharomyces cerevisiae, a large number of nuclear genes influence the stability of mitochondrial genome; however, most corresponding gene products act indirectly and the actual molecular mechanisms of mtDNA inheritance remain poorly characterized. Recently, we found that a Superfamily II helicase Irc3 is required for the maintenance of mitochondrial genome integrity. Here we show that Irc3 is a mitochondrial DNA branch migration enzyme. Irc3 modulates mtDNA metabolic intermediates by preferential binding and unwinding Holliday junctions and replication fork structures. Furthermore, we demonstrate that the loss of Irc3 can be complemented with mitochondrially targeted RecG of Escherichia coli. We suggest that Irc3 could support the stability of mtDNA by stimulating fork regression and branch migration or by inhibiting the formation of irregular branched molecules.

The helicase CaHmi1p is required for wild-type mitochondrial DNA organization in Candida albicans.

The mechanistic details of mtDNA maintenance in petite-negative yeasts have remained largely unexplored. We report here that the DNA helicase Hmi1p plays a crucial role in mtDNA stability in Candida albicans. Like its counterpart in Saccharomyces cerevisiae, Hmi1p in C. albicans (CaHmi1p) contains a C-terminal mitochondrial targeting signal that is functional in both organisms. Biochemical analysis demonstrates that CaHmi1p is a protein possessing ATP-dependent 3'-5' DNA-unwinding activity. Deletion of both HMI1 alleles does not lead to complete loss of mtDNA in C. albicans; however, substantial fragmentation of the wild-type mitochondrial genome, reduction of mtDNA mass and loss of wild-type nucleoid distribution occur. Specific regions of the mitochondrial genome give rise to mtDNA molecule populations with altered characteristics upon CaHMI1 deletion. Fragmentation of the mitochondrial genome can be reversed by reintroduction of CaHmi1p. This is the first time that a gene required for wild-type mtDNA maintenance in S. cerevisiae has been demonstrated to be nonessential in a petite-negative yeast.

Hmi1p from Saccharomyces cerevisiae mitochondria is a structure-specific DNA helicase.

Hmi1p is a Saccharomyces cerevisiae mitochondrial DNA helicase that is essential for the maintenance of functional mitochondrial DNA. Hmi1p belongs to the superfamily 1 of helicases and is a close homologue of bacterial PcrA and Rep helicases. We have overexpressed and purified recombinant Hmi1p from Escherichia coli and describe here the biochemical characteristics of its DNA helicase activities. Among nucleotide cofactors, the DNA unwinding by Hmi1p was found to occur efficiently only in the presence of ATP and dATP. Hmi1p could unwind only the DNA substrates with a 3'-single-stranded overhang. The length of the 3'-overhang needed for efficient targeting of the helicase to the substrate depended on the substrate structure. For substrates consisting of duplex DNA with a 3'-single-stranded DNA overhang, at least a 19-nt 3'-overhang was needed. In the case of forked substrates with both 3'- and 5'-overhangs, a 9-nt 3'-overhang was sufficient provided that the 5'-overhang was also 9 nt in length. In flap-structured substrates mimicking the chain displacement structures in DNA recombination process, only a 5-nt 3'-single-stranded DNA tail was required for efficient unwinding by Hmi1p. These data indicate that Hmi1p may be targeted to a specific 3'-flap structure, suggesting its possible role in DNA recombination.

Helicase Hmi1 stimulates the synthesis of concatemeric mitochondrial DNA molecules in yeast Saccharomyces cerevisiae.

Hmi1p is a helicase in the yeast Saccharomyces cerevisiae required for maintenance of the wild-type mitochondrial genome. Disruption of the HMI1 ORF generates rho(-) and rho(0) cells. Here we demonstrate that, in rho(-) yeast strains, Hmi1p stimulates the synthesis of long concatemeric mitochondrial DNA molecules associated with a reduction in the number of nucleoids used for mitochondrial DNA packaging. Surprisingly, the ATPase negative mutants of Hmi1p can also stimulate the synthesis of long concatemeric rho(-) mitochondrial DNA molecules and support the maintenance of the wild-type mitochondrial genome, albeit with reduced efficiency. We show that, in the mutant hmi1-5 background, the wild-type mitochondrial DNA is fragmented; and we propose that, in hmi1Delta yeast cells, the loss of the wild-type mitochondrial genome is caused by this fragmentation of the mitochondrial DNA.