Limits...
Phage annealing proteins promote oligonucleotide-directed mutagenesis in Escherichia coli and mouse ES cells.

Zhang Y, Muyrers JP, Rientjes J, Stewart AF - BMC Mol. Biol. (2003)

Bottom Line: The ssOR activity, unlike DSBR, does not require a phage exonuclease (RecE or Redalpha) but only requires a phage annealing protein (RecT or Redbeta).Notably, the P22 phage annealing protein Erf, which does not mediate the same DSBR reactions, also delivers ssOR activity.This expands the repertoire of useful DNA engineering strategies, shows promise for applications in eukaryotic cells, and has implications for the unanswered questions regarding DSBR mediated by RecE/RecT and Redalpha/Redbeta.

View Article: PubMed Central - HTML - PubMed

Affiliation: Gene Expression Program, EMBL, Meyerhofstr, 1, 69117, Heidelberg. youming.zhang@genebridges.com <youming.zhang@genebridges.com>

ABSTRACT

Background: The phage protein pairs, RecE/RecT from Rac or Redalpha/Redbeta from lambda, initiate efficient double strand break repair (DSBR) in Escherichia coli that has proven very useful for DNA engineering. These phage pairs initiate DSBR either by annealing or by another mechanism that is not defined.

Results: Here we report that these proteins also mediate single strand oligonucleotide repair (ssOR) at high efficiencies. The ssOR activity, unlike DSBR, does not require a phage exonuclease (RecE or Redalpha) but only requires a phage annealing protein (RecT or Redbeta). Notably, the P22 phage annealing protein Erf, which does not mediate the same DSBR reactions, also delivers ssOR activity. By altering aspects of the oligonucleotides, we document length and design parameters that affect ssOR efficiency to show a simple relationship to homologies either side of the repair site. Notably, ssOR shows strand bias. Oligonucleotides that can prime lagging strand replication deliver more ssOR than their leading complements. This suggests a model in which the annealing proteins hybridize the oligonucleotides to single stranded regions near the replication fork. We also show that ssOR is a highly efficient way to engineer BACs and can be detected in a eukaryotic cell upon expression of a phage annealing protein.

Conclusion: Phage annealing proteins can initiate the recombination of single stranded oligonucleotides into endogenous targets in Escherichia coli at very high efficiencies. This expands the repertoire of useful DNA engineering strategies, shows promise for applications in eukaryotic cells, and has implications for the unanswered questions regarding DSBR mediated by RecE/RecT and Redalpha/Redbeta.

Show MeSH

Related in: MedlinePlus

Exploration of ssOR by alterations in oligo design. The contribution of the oligo to ssOR was explored by altering the oligos in three different ways. A. The position of the repair site was shifted from a central position in 50 mers (23/23) to more 3' (35/11) or 5' (11/35), as well as at the 5' (0/20; 0/100) or 3' (20/0; 100/0) terminae, for both lagging and leading oligos. Results are from a single experiment performed with competent cell batches made in parallel after induction of RecT or Redβ expression. B. The ability of ssOR to repair larger deletions in the template was evaluated with three further derivatives of pGKneoΔ that lacked 15, 33 or 60 bps around the NcoI site of the kanamycin resistance gene. Oligos that retained 22 nucleotides of homology either side of the the 4, 15, 33 or 60 nucleotides required to restore kanamycin resistance were co-electroporated with pGKneoΔ. Results are from a single experiment performed with lagging oligos and competent cell batches made in parallel after induction of RecT or Redβ expression. C. The impact of a second mismatch in the oligo was evaluated using 92 mers with the NcoI site off center leaving a short side of 29 nts from either the 5' or 3' end. The longer side of 59 nts was interrupted either by a point mutation 14 or 29 nts from the NcoI site. Results shown are from a single experiment using a lagging oligo. Qualitatively similar results were obtained with the corresponding leading strand oligos (data not shown). The two, rare, events in which the point mutation was also incorporated were found with the 44/14/29 oligo illustrated.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC149363&req=5

Figure 3: Exploration of ssOR by alterations in oligo design. The contribution of the oligo to ssOR was explored by altering the oligos in three different ways. A. The position of the repair site was shifted from a central position in 50 mers (23/23) to more 3' (35/11) or 5' (11/35), as well as at the 5' (0/20; 0/100) or 3' (20/0; 100/0) terminae, for both lagging and leading oligos. Results are from a single experiment performed with competent cell batches made in parallel after induction of RecT or Redβ expression. B. The ability of ssOR to repair larger deletions in the template was evaluated with three further derivatives of pGKneoΔ that lacked 15, 33 or 60 bps around the NcoI site of the kanamycin resistance gene. Oligos that retained 22 nucleotides of homology either side of the the 4, 15, 33 or 60 nucleotides required to restore kanamycin resistance were co-electroporated with pGKneoΔ. Results are from a single experiment performed with lagging oligos and competent cell batches made in parallel after induction of RecT or Redβ expression. C. The impact of a second mismatch in the oligo was evaluated using 92 mers with the NcoI site off center leaving a short side of 29 nts from either the 5' or 3' end. The longer side of 59 nts was interrupted either by a point mutation 14 or 29 nts from the NcoI site. Results shown are from a single experiment using a lagging oligo. Qualitatively similar results were obtained with the corresponding leading strand oligos (data not shown). The two, rare, events in which the point mutation was also incorporated were found with the 44/14/29 oligo illustrated.

Mentions: Second, the relative contributions of homologies on the 5' and 3' sides of the repair site were evaluated. No repair was observed when the repair site was located at either the 5' or 3' ends, regardless of the length of the single-sided homology region, up to 100 nt (Fig. 3A and not shown). Hence homology regions on both sides of the repair site are required. Next, a series of six 50 mers were synthesized, (3 pairs for lagging and leading) differing in the relative position of the repair site within the oligo (Fig. 3B). For both lagging and leading oligos, the centrally positioned repair site (23/4/23) was most efficient. Again the lagging oligo delivered better efficiency than the leading. For both lagging and leading oligos, more homology 5' to the repair site (35/4/11) worked better than oligos with more 3' homology (11/4/35). Notably, the 11/4/35 and 35/4/11 oligos functioned with RecT whereas the 11/4/11 26 mer oligo did not (Fig. 2). This indicates that the stability of hybrid formation on one side of the repair enhances the efficacy of the hybrid formed on the other side.


Phage annealing proteins promote oligonucleotide-directed mutagenesis in Escherichia coli and mouse ES cells.

Zhang Y, Muyrers JP, Rientjes J, Stewart AF - BMC Mol. Biol. (2003)

Exploration of ssOR by alterations in oligo design. The contribution of the oligo to ssOR was explored by altering the oligos in three different ways. A. The position of the repair site was shifted from a central position in 50 mers (23/23) to more 3' (35/11) or 5' (11/35), as well as at the 5' (0/20; 0/100) or 3' (20/0; 100/0) terminae, for both lagging and leading oligos. Results are from a single experiment performed with competent cell batches made in parallel after induction of RecT or Redβ expression. B. The ability of ssOR to repair larger deletions in the template was evaluated with three further derivatives of pGKneoΔ that lacked 15, 33 or 60 bps around the NcoI site of the kanamycin resistance gene. Oligos that retained 22 nucleotides of homology either side of the the 4, 15, 33 or 60 nucleotides required to restore kanamycin resistance were co-electroporated with pGKneoΔ. Results are from a single experiment performed with lagging oligos and competent cell batches made in parallel after induction of RecT or Redβ expression. C. The impact of a second mismatch in the oligo was evaluated using 92 mers with the NcoI site off center leaving a short side of 29 nts from either the 5' or 3' end. The longer side of 59 nts was interrupted either by a point mutation 14 or 29 nts from the NcoI site. Results shown are from a single experiment using a lagging oligo. Qualitatively similar results were obtained with the corresponding leading strand oligos (data not shown). The two, rare, events in which the point mutation was also incorporated were found with the 44/14/29 oligo illustrated.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC149363&req=5

Figure 3: Exploration of ssOR by alterations in oligo design. The contribution of the oligo to ssOR was explored by altering the oligos in three different ways. A. The position of the repair site was shifted from a central position in 50 mers (23/23) to more 3' (35/11) or 5' (11/35), as well as at the 5' (0/20; 0/100) or 3' (20/0; 100/0) terminae, for both lagging and leading oligos. Results are from a single experiment performed with competent cell batches made in parallel after induction of RecT or Redβ expression. B. The ability of ssOR to repair larger deletions in the template was evaluated with three further derivatives of pGKneoΔ that lacked 15, 33 or 60 bps around the NcoI site of the kanamycin resistance gene. Oligos that retained 22 nucleotides of homology either side of the the 4, 15, 33 or 60 nucleotides required to restore kanamycin resistance were co-electroporated with pGKneoΔ. Results are from a single experiment performed with lagging oligos and competent cell batches made in parallel after induction of RecT or Redβ expression. C. The impact of a second mismatch in the oligo was evaluated using 92 mers with the NcoI site off center leaving a short side of 29 nts from either the 5' or 3' end. The longer side of 59 nts was interrupted either by a point mutation 14 or 29 nts from the NcoI site. Results shown are from a single experiment using a lagging oligo. Qualitatively similar results were obtained with the corresponding leading strand oligos (data not shown). The two, rare, events in which the point mutation was also incorporated were found with the 44/14/29 oligo illustrated.
Mentions: Second, the relative contributions of homologies on the 5' and 3' sides of the repair site were evaluated. No repair was observed when the repair site was located at either the 5' or 3' ends, regardless of the length of the single-sided homology region, up to 100 nt (Fig. 3A and not shown). Hence homology regions on both sides of the repair site are required. Next, a series of six 50 mers were synthesized, (3 pairs for lagging and leading) differing in the relative position of the repair site within the oligo (Fig. 3B). For both lagging and leading oligos, the centrally positioned repair site (23/4/23) was most efficient. Again the lagging oligo delivered better efficiency than the leading. For both lagging and leading oligos, more homology 5' to the repair site (35/4/11) worked better than oligos with more 3' homology (11/4/35). Notably, the 11/4/35 and 35/4/11 oligos functioned with RecT whereas the 11/4/11 26 mer oligo did not (Fig. 2). This indicates that the stability of hybrid formation on one side of the repair enhances the efficacy of the hybrid formed on the other side.

Bottom Line: The ssOR activity, unlike DSBR, does not require a phage exonuclease (RecE or Redalpha) but only requires a phage annealing protein (RecT or Redbeta).Notably, the P22 phage annealing protein Erf, which does not mediate the same DSBR reactions, also delivers ssOR activity.This expands the repertoire of useful DNA engineering strategies, shows promise for applications in eukaryotic cells, and has implications for the unanswered questions regarding DSBR mediated by RecE/RecT and Redalpha/Redbeta.

View Article: PubMed Central - HTML - PubMed

Affiliation: Gene Expression Program, EMBL, Meyerhofstr, 1, 69117, Heidelberg. youming.zhang@genebridges.com <youming.zhang@genebridges.com>

ABSTRACT

Background: The phage protein pairs, RecE/RecT from Rac or Redalpha/Redbeta from lambda, initiate efficient double strand break repair (DSBR) in Escherichia coli that has proven very useful for DNA engineering. These phage pairs initiate DSBR either by annealing or by another mechanism that is not defined.

Results: Here we report that these proteins also mediate single strand oligonucleotide repair (ssOR) at high efficiencies. The ssOR activity, unlike DSBR, does not require a phage exonuclease (RecE or Redalpha) but only requires a phage annealing protein (RecT or Redbeta). Notably, the P22 phage annealing protein Erf, which does not mediate the same DSBR reactions, also delivers ssOR activity. By altering aspects of the oligonucleotides, we document length and design parameters that affect ssOR efficiency to show a simple relationship to homologies either side of the repair site. Notably, ssOR shows strand bias. Oligonucleotides that can prime lagging strand replication deliver more ssOR than their leading complements. This suggests a model in which the annealing proteins hybridize the oligonucleotides to single stranded regions near the replication fork. We also show that ssOR is a highly efficient way to engineer BACs and can be detected in a eukaryotic cell upon expression of a phage annealing protein.

Conclusion: Phage annealing proteins can initiate the recombination of single stranded oligonucleotides into endogenous targets in Escherichia coli at very high efficiencies. This expands the repertoire of useful DNA engineering strategies, shows promise for applications in eukaryotic cells, and has implications for the unanswered questions regarding DSBR mediated by RecE/RecT and Redalpha/Redbeta.

Show MeSH
Related in: MedlinePlus