The high mobility group protein Abf2p influences the level of yeast mitochondrial DNA recombination intermediates in vivo

Abstract

Abf2p is a high mobility group (HMG) protein found in yeast mitochondria that is required for the maintenance of wild-type (rho+) mtDNA in cells grown on fermentable carbon sources, and for efficient recombination of mtDNA markers in crosses. Here, we show by two-dimensional gel electrophoresis that Abf2p promotes or stabilizes Holliday recombination junction intermediates in rho+ mtDNA in vivo but does not influence the high levels of recombination intermediates readily detected in the mtDNA of petite mutants (rho-). mtDNA recombination junctions are not observed in rho+ mtDNA of wild-type cells but are elevated to detectable levels in cells with a null allele of the MGT1 gene (Deltamgt1), which codes for a mitochondrial cruciform-cutting endonuclease. The level of recombination intermediates in rho+ mtDNA of Deltamgt1 cells is decreased about 10-fold if those cells contain a null allele of the ABF2 gene. Overproduction of Abf2p by >/= 10-fold in wild-type rho+ cells, which leads to mtDNA instability, results in a dramatic increase in mtDNA recombination intermediates. Specific mutations in the two Abf2p HMG boxes required for DNA binding diminishes these responses. We conclude that Abf2p functions in the recombination of rho+ mtDNA.

Figure 1

(A) Schematic of 2D gel electrophoresis (22). Restriction fragments of total DNA enriched for single-stranded character (21) are separated by agarose gel electrophoresis in the first dimension according to their mass. The second-dimension agarose gel resolves the restriction fragments by both mass and shape. The DNA then is transferred to a membrane and hybridized with a specific probe. The majority of the DNA will be present as linear molecules at the 1N spot. Replication intermediates will have a characteristic pattern depending on whether an origin fires inside (giving rise to a bubble arc) or outside (giving rise to a Y-arc) of the fragment of interest. Recombination intermediates are detected as a vertical spike (X-arc) rising from the 2N position. (B) Abf2p influences the levels of Holliday junctions in ρ⁺ mtDNAs. Replication and recombination intermediates in ρ⁺ mtDNAs of 14WW strains ABF2 MGT1 (a), Δabf2 MGT1 (b), ABF2 Δmgt1 (c), and Δabf2 Δmgt1 (d) were resolved by 2D gel electrophoresis and hybridized with a probe specific for a 5-kb EcoRI fragment from the COX1 gene. Replication and recombination intermediates are indicated in a and c, respectively. Positions of linear 5-kb (1N) and 10-kb (2N) fragments are indicated in a. DNA was prepared from log-phase cells cultured in YPG medium.

Figure 2

The levels of Holliday junctions in ρ⁻ mtDNAs are independent of Abf2p. (A) 2D gel electrophoresis of replication and recombination intermediates from the 2-kb VAR1 petite, 2–33, in ABF2 (Upper) and Δabf2 (Lower) 14WW cells. The blots were hybridized with a probe specific for petite mtDNA. The arrows indicate high molecular weight species, which may be representative of single-strand tailing resulting from rolling circle replication. (B) One-dimensional gel electrophoresis of ρ⁻ 2–33 mtDNA linearized with increasing concentrations of HincII (0.25, 0.5, and 1 unit/μl) from ABF2 and Δabf2 14WW cells. Molecular size markers and the expected migration positions of linear double-stranded and Holliday junction DNAs are indicated.

Figure 3

Overproduction of Abf2p results in an increase in Holliday junctions in ρ⁺ mtDNA. (A) Schematic of Abf2p with the HMG-box domains represented by the gray boxes. Mutations were made in conserved basic residues, as indicated, from both HMG-box domains resulting in mutant protein Abf2p1⁻2⁻. (B) Overproduction of Abf2p and Abf2p1⁻2⁻ in 14WW ρ⁺ cells after the transition from YNBG to YNBGal medium for the indicated time points. Abf2p and Abf21⁻2⁻ were overexpressed from transformants containing the CEN plasmid GAL1–10 promoter constructs pGal-Abf2 and pGal-Abf21⁻2⁻, respectively. Protein levels were detected by Western blotting with polyclonal antiserum raised against Abf2p (18). (C) 2D gel electrophoresis of ρ⁺ mtDNA replication and recombination intermediates from 14WW cells containing either pGal-Abf2 or pGal-Abf21⁻2⁻ after the transition from YNBG to YNBGal medium. The blots were hybridized with a probe specific for a 5-kb EcoRI fragment from the COX1 gene.

Figure 4

Effect of ABF2 and MGT1 alleles on the stability of ρ⁺ mtDNA. The kinetics of petite induction were determined for ABF2 MGT1 (○), Δabf2 MGT1 (□), ABF2 Δmgt1 (▴), and Δabf2 Δmgt1 (⧫) 14WW cells. Cells were shifted from YPG to YPD medium, and aliquots were plated at various intervals and on YPD medium. The fraction of ρ⁺ colonies was scored by overlaying with 2,3,5-triphenyl tetrazolium chloride soft agar as described in Materials and Methods.