Limits...
Manipulating replisome dynamics to enhance lambda Red-mediated multiplex genome engineering.

Lajoie MJ, Gregg CJ, Mosberg JA, Washington GC, Church GM - Nucleic Acids Res. (2012)

Bottom Line: We exploited this feature to increase the amount of ssDNA at the lagging strand of the replication fork that is available for λ Red-mediated Multiplex Automatable Genome Engineering (MAGE).Additionally, we demonstrate that both synthetic oligonucleotides and accessible ssDNA targets on the lagging strand of the replication fork are limiting factors for MAGE.These improvements will facilitate ambitious genome engineering projects by minimizing dependence on time-consuming clonal isolation and screening.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT
Disrupting the interaction between primase and helicase in Escherichia coli increases Okazaki fragment (OF) length due to less frequent primer synthesis. We exploited this feature to increase the amount of ssDNA at the lagging strand of the replication fork that is available for λ Red-mediated Multiplex Automatable Genome Engineering (MAGE). Supporting this concept, we demonstrate that MAGE enhancements correlate with OF length. Compared with a standard recombineering strain (EcNR2), the strain with the longest OFs displays on average 62% more alleles converted per clone, 239% more clones with 5 or more allele conversions and 38% fewer clones with 0 allele conversions in 1 cycle of co-selection MAGE (CoS-MAGE) with 10 synthetic oligonucleotides. Additionally, we demonstrate that both synthetic oligonucleotides and accessible ssDNA targets on the lagging strand of the replication fork are limiting factors for MAGE. Given this new insight, we generated a strain with reduced oligonucleotide degradation and increased genomic ssDNA availability, which displayed 111% more alleles converted per clone, 527% more clones with 5 or more allele conversions and 71% fewer clones with 0 allele conversions in 1 cycle of 10-plex CoS-MAGE. These improvements will facilitate ambitious genome engineering projects by minimizing dependence on time-consuming clonal isolation and screening.

Show MeSH

Related in: MedlinePlus

Effect of dnaG attenuation on replication fork dynamics. (A) Schematic showing the replication fork in E. coli, including the leading and lagging strands undergoing DNA synthesis. DnaG synthesizes RNA primers (red) onto the lagging template strand, which in turn initiate OF synthesis (blue) by PolIII. Compared with wt DnaG primase, the variants tested in this study have lower affinities for DnaB helicase (12). Since the DnaG–DnaB interaction is necessary for primase function, primer synthesis occurs less frequently, thereby exposing larger regions of ssDNA on the lagging template strand (13). (B) A schematic representing the E. coli MG1655 genome with the origin (oriC) and terminus (T) of replication indicated, splitting the genome into Replichore 1 and Replichore 2. Each oligo set converts 10 TAG codons to TAA codons within the genomic regions indicated in gray. Co-selection marker positions are denoted by radial lines.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3526312&req=5

gks751-F1: Effect of dnaG attenuation on replication fork dynamics. (A) Schematic showing the replication fork in E. coli, including the leading and lagging strands undergoing DNA synthesis. DnaG synthesizes RNA primers (red) onto the lagging template strand, which in turn initiate OF synthesis (blue) by PolIII. Compared with wt DnaG primase, the variants tested in this study have lower affinities for DnaB helicase (12). Since the DnaG–DnaB interaction is necessary for primase function, primer synthesis occurs less frequently, thereby exposing larger regions of ssDNA on the lagging template strand (13). (B) A schematic representing the E. coli MG1655 genome with the origin (oriC) and terminus (T) of replication indicated, splitting the genome into Replichore 1 and Replichore 2. Each oligo set converts 10 TAG codons to TAA codons within the genomic regions indicated in gray. Co-selection marker positions are denoted by radial lines.

Mentions: The fact that CoS-MAGE is most effective for oligos targeted in close proximity to the selectable marker suggests that replication fork position and accessibility are limiting factors in Redβ-mediated recombination (9). Thus, we reasoned that we could improve AR frequencies by manipulating replication fork dynamics to increase the amount of ssDNA on the lagging strand of the replication fork. Since Okazaki fragment (OF) size can be modulated by the frequency of OF primer synthesis by DnaG primase (11), we hypothesized that attenuating the interaction between DnaG primase and the replisome would increase the amount of accessible ssDNA on the lagging strand of the replication fork and enhance multiplex AR frequencies (Figure 1). Tougu et al. (12) have reported E. coli primase variants with impaired helicase binding, resulting in less-frequent OF initiation, but normal replication fork rate, priming efficiency and primer utilization during in vitro replication. These variants, K580A and Q576A, resulted in in vitro OFs that were ∼1.5- and 8-fold longer, respectively, than those initiated by wild-type DnaG (13). Therefore, these variants were used to explore whether increasing accessible ssDNA on the lagging strand can improve multiplex AR frequency.Figure 1.


Manipulating replisome dynamics to enhance lambda Red-mediated multiplex genome engineering.

Lajoie MJ, Gregg CJ, Mosberg JA, Washington GC, Church GM - Nucleic Acids Res. (2012)

Effect of dnaG attenuation on replication fork dynamics. (A) Schematic showing the replication fork in E. coli, including the leading and lagging strands undergoing DNA synthesis. DnaG synthesizes RNA primers (red) onto the lagging template strand, which in turn initiate OF synthesis (blue) by PolIII. Compared with wt DnaG primase, the variants tested in this study have lower affinities for DnaB helicase (12). Since the DnaG–DnaB interaction is necessary for primase function, primer synthesis occurs less frequently, thereby exposing larger regions of ssDNA on the lagging template strand (13). (B) A schematic representing the E. coli MG1655 genome with the origin (oriC) and terminus (T) of replication indicated, splitting the genome into Replichore 1 and Replichore 2. Each oligo set converts 10 TAG codons to TAA codons within the genomic regions indicated in gray. Co-selection marker positions are denoted by radial lines.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3526312&req=5

gks751-F1: Effect of dnaG attenuation on replication fork dynamics. (A) Schematic showing the replication fork in E. coli, including the leading and lagging strands undergoing DNA synthesis. DnaG synthesizes RNA primers (red) onto the lagging template strand, which in turn initiate OF synthesis (blue) by PolIII. Compared with wt DnaG primase, the variants tested in this study have lower affinities for DnaB helicase (12). Since the DnaG–DnaB interaction is necessary for primase function, primer synthesis occurs less frequently, thereby exposing larger regions of ssDNA on the lagging template strand (13). (B) A schematic representing the E. coli MG1655 genome with the origin (oriC) and terminus (T) of replication indicated, splitting the genome into Replichore 1 and Replichore 2. Each oligo set converts 10 TAG codons to TAA codons within the genomic regions indicated in gray. Co-selection marker positions are denoted by radial lines.
Mentions: The fact that CoS-MAGE is most effective for oligos targeted in close proximity to the selectable marker suggests that replication fork position and accessibility are limiting factors in Redβ-mediated recombination (9). Thus, we reasoned that we could improve AR frequencies by manipulating replication fork dynamics to increase the amount of ssDNA on the lagging strand of the replication fork. Since Okazaki fragment (OF) size can be modulated by the frequency of OF primer synthesis by DnaG primase (11), we hypothesized that attenuating the interaction between DnaG primase and the replisome would increase the amount of accessible ssDNA on the lagging strand of the replication fork and enhance multiplex AR frequencies (Figure 1). Tougu et al. (12) have reported E. coli primase variants with impaired helicase binding, resulting in less-frequent OF initiation, but normal replication fork rate, priming efficiency and primer utilization during in vitro replication. These variants, K580A and Q576A, resulted in in vitro OFs that were ∼1.5- and 8-fold longer, respectively, than those initiated by wild-type DnaG (13). Therefore, these variants were used to explore whether increasing accessible ssDNA on the lagging strand can improve multiplex AR frequency.Figure 1.

Bottom Line: We exploited this feature to increase the amount of ssDNA at the lagging strand of the replication fork that is available for λ Red-mediated Multiplex Automatable Genome Engineering (MAGE).Additionally, we demonstrate that both synthetic oligonucleotides and accessible ssDNA targets on the lagging strand of the replication fork are limiting factors for MAGE.These improvements will facilitate ambitious genome engineering projects by minimizing dependence on time-consuming clonal isolation and screening.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT
Disrupting the interaction between primase and helicase in Escherichia coli increases Okazaki fragment (OF) length due to less frequent primer synthesis. We exploited this feature to increase the amount of ssDNA at the lagging strand of the replication fork that is available for λ Red-mediated Multiplex Automatable Genome Engineering (MAGE). Supporting this concept, we demonstrate that MAGE enhancements correlate with OF length. Compared with a standard recombineering strain (EcNR2), the strain with the longest OFs displays on average 62% more alleles converted per clone, 239% more clones with 5 or more allele conversions and 38% fewer clones with 0 allele conversions in 1 cycle of co-selection MAGE (CoS-MAGE) with 10 synthetic oligonucleotides. Additionally, we demonstrate that both synthetic oligonucleotides and accessible ssDNA targets on the lagging strand of the replication fork are limiting factors for MAGE. Given this new insight, we generated a strain with reduced oligonucleotide degradation and increased genomic ssDNA availability, which displayed 111% more alleles converted per clone, 527% more clones with 5 or more allele conversions and 71% fewer clones with 0 allele conversions in 1 cycle of 10-plex CoS-MAGE. These improvements will facilitate ambitious genome engineering projects by minimizing dependence on time-consuming clonal isolation and screening.

Show MeSH
Related in: MedlinePlus