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Heteroduplex formation, mismatch resolution, and genetic sectoring during homologous recombination in the hyperthermophilic archaeon sulfolobus acidocaldarius.

Mao D, Grogan DW - Front Microbiol (2012)

Bottom Line: From 20 to 40% of the resulting colonies were found to contain two Pyr(+) clones with distinct sets of the non-selected markers.The conversion events that occur during transformation by single-stranded DNA do not show the strand bias necessary for a system that corrects replication errors effectively; similar events also occur in pre-formed heteroduplex electroporated into the cells.Although numerous mechanistic details remain obscure, the results demonstrate that the HR system of S. acidocaldarius can generate remarkable genetic diversity from short intervals of moderately diverged DNAs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Cincinnati Cincinnati, OH, USA.

ABSTRACT
Hyperthermophilic archaea exhibit certain molecular-genetic features not seen in bacteria or eukaryotes, and their systems of homologous recombination (HR) remain largely unexplored in vivo. We transformed a Sulfolobus acidocaldariuspyrE mutant with short DNAs that contained multiple non-selected genetic markers within the pyrE gene. From 20 to 40% of the resulting colonies were found to contain two Pyr(+) clones with distinct sets of the non-selected markers. The dual-genotype colonies could not be attributed to multiple DNAs entering the cells, or to conjugation between transformed and non-transformed cells. These colonies thus appear to represent genetic sectoring in which regions of heteroduplex DNA formed and then segregated after partial resolution of inter-strand differences. Surprisingly, sectoring was also frequent in cells transformed with single-stranded DNAs. Oligonucleotides produced more sectored transformants when electroporated as single strands than as a duplex, although all forms of donor DNA (positive-strand, negative-strand, and duplex) produced a diversity of genotypes, despite the limited number of markers. The marker patterns in the recombinants indicate that S. acidocaldarius resolves individual mismatches through un-coordinated short-patch excision followed by re-filling of the resulting gap. The conversion events that occur during transformation by single-stranded DNA do not show the strand bias necessary for a system that corrects replication errors effectively; similar events also occur in pre-formed heteroduplex electroporated into the cells. Although numerous mechanistic details remain obscure, the results demonstrate that the HR system of S. acidocaldarius can generate remarkable genetic diversity from short intervals of moderately diverged DNAs.

No MeSH data available.


Related in: MedlinePlus

Transformation by differentially marked oligonucleotides. Two synthetic DNAs (Table 2), representing the top strand (T) and bottom strand (B) of base pairs 43–227 of pyrE, were electroporated individually (A,B) or as a duplex (C,D). Markers are given in Table 2; the transformants were selected and analyzed as for Figure 2. (C) Shows pairs of genotypes from sectored colonies, whereas (D) shows the single genotypes of non-sectored colonies recovered in the same experiment. Symbols: top-strand donor allele, black; bottom-strand donor allele, gray; recipient allele, white.
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Figure 4: Transformation by differentially marked oligonucleotides. Two synthetic DNAs (Table 2), representing the top strand (T) and bottom strand (B) of base pairs 43–227 of pyrE, were electroporated individually (A,B) or as a duplex (C,D). Markers are given in Table 2; the transformants were selected and analyzed as for Figure 2. (C) Shows pairs of genotypes from sectored colonies, whereas (D) shows the single genotypes of non-sectored colonies recovered in the same experiment. Symbols: top-strand donor allele, black; bottom-strand donor allele, gray; recipient allele, white.

Mentions: Based on this result from enzymatically produced ssDNA, we designed a new assay that used long synthetic oligonucleotides as multiply marked but chemically defined donor. In this scheme, two oligonucleotides represent the top and bottom strands, respectively, of the same 185-bp interval of the functional S. acidocaldariuspyrE gene and incorporated silent BPSs within the selected region and at regular intervals on both sides (Table 2). When electroporated separately into MR31 cells, the two oligonucleotides showed no obvious strand bias (averages of 2.6 vs. 1.8 Pyr+ transformants per pmol for top and bottom strand, respectively). In addition, both top- (T-) and bottom- (B-) strand transformants included sectored colonies, indicating that these short ssDNAs form extensive heteroduplex structures with the S. acidocaldarius chromosome which can segregate two distinct genotypes (Figure 4). The six non-selected, phenotypically silent, markers in each oligonucleotide (Table 2), define 26 = 64 possible genotypes for the transformants in each case. We found 13 of these genotypes among 48 T-strand transformants, and 15 among 48 B-strand transformants. Figure 5 compares the two or three most common of the genotypes recovered under these and other conditions.


Heteroduplex formation, mismatch resolution, and genetic sectoring during homologous recombination in the hyperthermophilic archaeon sulfolobus acidocaldarius.

Mao D, Grogan DW - Front Microbiol (2012)

Transformation by differentially marked oligonucleotides. Two synthetic DNAs (Table 2), representing the top strand (T) and bottom strand (B) of base pairs 43–227 of pyrE, were electroporated individually (A,B) or as a duplex (C,D). Markers are given in Table 2; the transformants were selected and analyzed as for Figure 2. (C) Shows pairs of genotypes from sectored colonies, whereas (D) shows the single genotypes of non-sectored colonies recovered in the same experiment. Symbols: top-strand donor allele, black; bottom-strand donor allele, gray; recipient allele, white.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3367456&req=5

Figure 4: Transformation by differentially marked oligonucleotides. Two synthetic DNAs (Table 2), representing the top strand (T) and bottom strand (B) of base pairs 43–227 of pyrE, were electroporated individually (A,B) or as a duplex (C,D). Markers are given in Table 2; the transformants were selected and analyzed as for Figure 2. (C) Shows pairs of genotypes from sectored colonies, whereas (D) shows the single genotypes of non-sectored colonies recovered in the same experiment. Symbols: top-strand donor allele, black; bottom-strand donor allele, gray; recipient allele, white.
Mentions: Based on this result from enzymatically produced ssDNA, we designed a new assay that used long synthetic oligonucleotides as multiply marked but chemically defined donor. In this scheme, two oligonucleotides represent the top and bottom strands, respectively, of the same 185-bp interval of the functional S. acidocaldariuspyrE gene and incorporated silent BPSs within the selected region and at regular intervals on both sides (Table 2). When electroporated separately into MR31 cells, the two oligonucleotides showed no obvious strand bias (averages of 2.6 vs. 1.8 Pyr+ transformants per pmol for top and bottom strand, respectively). In addition, both top- (T-) and bottom- (B-) strand transformants included sectored colonies, indicating that these short ssDNAs form extensive heteroduplex structures with the S. acidocaldarius chromosome which can segregate two distinct genotypes (Figure 4). The six non-selected, phenotypically silent, markers in each oligonucleotide (Table 2), define 26 = 64 possible genotypes for the transformants in each case. We found 13 of these genotypes among 48 T-strand transformants, and 15 among 48 B-strand transformants. Figure 5 compares the two or three most common of the genotypes recovered under these and other conditions.

Bottom Line: From 20 to 40% of the resulting colonies were found to contain two Pyr(+) clones with distinct sets of the non-selected markers.The conversion events that occur during transformation by single-stranded DNA do not show the strand bias necessary for a system that corrects replication errors effectively; similar events also occur in pre-formed heteroduplex electroporated into the cells.Although numerous mechanistic details remain obscure, the results demonstrate that the HR system of S. acidocaldarius can generate remarkable genetic diversity from short intervals of moderately diverged DNAs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Cincinnati Cincinnati, OH, USA.

ABSTRACT
Hyperthermophilic archaea exhibit certain molecular-genetic features not seen in bacteria or eukaryotes, and their systems of homologous recombination (HR) remain largely unexplored in vivo. We transformed a Sulfolobus acidocaldariuspyrE mutant with short DNAs that contained multiple non-selected genetic markers within the pyrE gene. From 20 to 40% of the resulting colonies were found to contain two Pyr(+) clones with distinct sets of the non-selected markers. The dual-genotype colonies could not be attributed to multiple DNAs entering the cells, or to conjugation between transformed and non-transformed cells. These colonies thus appear to represent genetic sectoring in which regions of heteroduplex DNA formed and then segregated after partial resolution of inter-strand differences. Surprisingly, sectoring was also frequent in cells transformed with single-stranded DNAs. Oligonucleotides produced more sectored transformants when electroporated as single strands than as a duplex, although all forms of donor DNA (positive-strand, negative-strand, and duplex) produced a diversity of genotypes, despite the limited number of markers. The marker patterns in the recombinants indicate that S. acidocaldarius resolves individual mismatches through un-coordinated short-patch excision followed by re-filling of the resulting gap. The conversion events that occur during transformation by single-stranded DNA do not show the strand bias necessary for a system that corrects replication errors effectively; similar events also occur in pre-formed heteroduplex electroporated into the cells. Although numerous mechanistic details remain obscure, the results demonstrate that the HR system of S. acidocaldarius can generate remarkable genetic diversity from short intervals of moderately diverged DNAs.

No MeSH data available.


Related in: MedlinePlus