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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

Effects of blocking conjugation or transforming with single-stranded DNA. The genotypes of dual-genotype transformants are shown as for Figure 2. (A) Strain SA1 (conjugation-deficient) transformed with the double-stranded pyrEv3 cassette. (B) Strain MR31 transformed with single-stranded pyrEv3 DNA (see Materials and Methods).
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Figure 3: Effects of blocking conjugation or transforming with single-stranded DNA. The genotypes of dual-genotype transformants are shown as for Figure 2. (A) Strain SA1 (conjugation-deficient) transformed with the double-stranded pyrEv3 cassette. (B) Strain MR31 transformed with single-stranded pyrEv3 DNA (see Materials and Methods).

Mentions: We also considered whether two-genotype colonies could have resulted from diversification of an initial recombinant cell through a second round of recombination by conjugation with a non-transformed cell on the selective plate. The efficiency of the DNA transfer step in S. acidocaldarius has not been measured experimentally and may not be especially high, since recombinants rarely form at frequencies above 10−4 (Grogan, 1996). However, in contrast to the conjugation system of S. solfataricus, the S. acidocaldarius system functions constitutively (Ajon et al., 2011) and thus cannot be discounted on the basis that the recipient cells were not treated with DNA-damaging agents under our conditions. We therefore tested the dependence of genetic sectoring on conjugation, by performing the same experiments with the conjugation-defective Pil− strain SA1 (Ajon et al., 2011). Dual-genotype colonies represented three of 19 colonies examined, and their patterns of markers resembled those found in the isogenic Pil+ recipient MR31, i.e., uninterrupted tracts of donor markers differing at one end but not the other (Figure 3A). This result demonstrated that the process leading to the dual-genotype colonies was fully functional in the ΔupsE strain, and, therefore, that conjugation is not necessary to produce this class of transformants.


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

Mao D, Grogan DW - Front Microbiol (2012)

Effects of blocking conjugation or transforming with single-stranded DNA. The genotypes of dual-genotype transformants are shown as for Figure 2. (A) Strain SA1 (conjugation-deficient) transformed with the double-stranded pyrEv3 cassette. (B) Strain MR31 transformed with single-stranded pyrEv3 DNA (see Materials and Methods).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Effects of blocking conjugation or transforming with single-stranded DNA. The genotypes of dual-genotype transformants are shown as for Figure 2. (A) Strain SA1 (conjugation-deficient) transformed with the double-stranded pyrEv3 cassette. (B) Strain MR31 transformed with single-stranded pyrEv3 DNA (see Materials and Methods).
Mentions: We also considered whether two-genotype colonies could have resulted from diversification of an initial recombinant cell through a second round of recombination by conjugation with a non-transformed cell on the selective plate. The efficiency of the DNA transfer step in S. acidocaldarius has not been measured experimentally and may not be especially high, since recombinants rarely form at frequencies above 10−4 (Grogan, 1996). However, in contrast to the conjugation system of S. solfataricus, the S. acidocaldarius system functions constitutively (Ajon et al., 2011) and thus cannot be discounted on the basis that the recipient cells were not treated with DNA-damaging agents under our conditions. We therefore tested the dependence of genetic sectoring on conjugation, by performing the same experiments with the conjugation-defective Pil− strain SA1 (Ajon et al., 2011). Dual-genotype colonies represented three of 19 colonies examined, and their patterns of markers resembled those found in the isogenic Pil+ recipient MR31, i.e., uninterrupted tracts of donor markers differing at one end but not the other (Figure 3A). This result demonstrated that the process leading to the dual-genotype colonies was fully functional in the ΔupsE strain, and, therefore, that conjugation is not necessary to produce this class of transformants.

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