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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

Mechanistic alternatives for “ends-out” HR in Sulfolobus. (A) ssDNA annealing at a transient gap in the chromosome; (B) ssDNA annealing to a transiently regressed replication fork; (C) dsDNA incorporated at a gap in the chromosome, or alternatively, at a regressed replication fork (dotted arrow). Arrowheads indicate 3′ ends, black bars correspond to the top (sense) strand of the pyrE gene, gray bars indicate the bottom (anti-sense) strand.
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Figure 8: Mechanistic alternatives for “ends-out” HR in Sulfolobus. (A) ssDNA annealing at a transient gap in the chromosome; (B) ssDNA annealing to a transiently regressed replication fork; (C) dsDNA incorporated at a gap in the chromosome, or alternatively, at a regressed replication fork (dotted arrow). Arrowheads indicate 3′ ends, black bars correspond to the top (sense) strand of the pyrE gene, gray bars indicate the bottom (anti-sense) strand.

Mentions: Although the four features proposed above leave a number of important steps undefined, including those that form and stabilize heteroduplex DNA, they provide a context for formulating and testing models of HR involving linear DNA in S. acidocaldarius and other hyperthermophilic archaea. The schemes of Figure 8 illustrate a few of the possibilities, but various alternatives also cannot be excluded. Short-patch excision events play central roles in these schemes, and the molecular details of those events also remain unclear, although we favor the idea of a process analogous to NER, in which each mispair promotes formation of a small bubble, one strand of which is excised by structure-specific endonucleases cutting at the boundaries.


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

Mao D, Grogan DW - Front Microbiol (2012)

Mechanistic alternatives for “ends-out” HR in Sulfolobus. (A) ssDNA annealing at a transient gap in the chromosome; (B) ssDNA annealing to a transiently regressed replication fork; (C) dsDNA incorporated at a gap in the chromosome, or alternatively, at a regressed replication fork (dotted arrow). Arrowheads indicate 3′ ends, black bars correspond to the top (sense) strand of the pyrE gene, gray bars indicate the bottom (anti-sense) strand.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: Mechanistic alternatives for “ends-out” HR in Sulfolobus. (A) ssDNA annealing at a transient gap in the chromosome; (B) ssDNA annealing to a transiently regressed replication fork; (C) dsDNA incorporated at a gap in the chromosome, or alternatively, at a regressed replication fork (dotted arrow). Arrowheads indicate 3′ ends, black bars correspond to the top (sense) strand of the pyrE gene, gray bars indicate the bottom (anti-sense) strand.
Mentions: Although the four features proposed above leave a number of important steps undefined, including those that form and stabilize heteroduplex DNA, they provide a context for formulating and testing models of HR involving linear DNA in S. acidocaldarius and other hyperthermophilic archaea. The schemes of Figure 8 illustrate a few of the possibilities, but various alternatives also cannot be excluded. Short-patch excision events play central roles in these schemes, and the molecular details of those events also remain unclear, although we favor the idea of a process analogous to NER, in which each mispair promotes formation of a small bubble, one strand of which is excised by structure-specific endonucleases cutting at the boundaries.

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