The terminal 5′ phosphate and proximate phosphorothioate promote ligation‐independent cloning

Liu, Xipeng; Liu, Jianhua

doi:10.1002/pro.374

Cited by 23 publications

(20 citation statements)

References 18 publications

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“…Phosphorothioate (PT) bonds have been reported to block Lambda Exo from degrading dsDNA cassettes [30], an observation that we have confirmed in an in vitro experiment using recombinant Lambda Exo (Figure 1A–B). Despite this, placing PT bonds on both 5′ ends of a dsDNA cassette does not decrease (and actually enhances) the recombination frequency of that cassette in vivo [21], [22].…”

Section: Resultssupporting

confidence: 78%

Improving Lambda Red Genome Engineering in Escherichia coli via Rational Removal of Endogenous Nucleases

et al. 2012

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Lambda Red recombineering is a powerful technique for making targeted genetic changes in bacteria. However, many applications are limited by the frequency of recombination. Previous studies have suggested that endogenous nucleases may hinder recombination by degrading the exogenous DNA used for recombineering. In this work, we identify ExoVII as a nuclease which degrades the ends of single-stranded DNA (ssDNA) oligonucleotides and double-stranded DNA (dsDNA) cassettes. Removing this nuclease improves both recombination frequency and the inheritance of mutations at the 3′ ends of ssDNA and dsDNA. Extending this approach, we show that removing a set of five exonucleases (RecJ, ExoI, ExoVII, ExoX, and Lambda Exo) substantially improves the performance of co-selection multiplex automatable genome engineering (CoS-MAGE). In a given round of CoS-MAGE with ten ssDNA oligonucleotides, the five nuclease knockout strain has on average 46% more alleles converted per clone, 200% more clones with five or more allele conversions, and 35% fewer clones without any allele conversions. Finally, we use these nuclease knockout strains to investigate and clarify the effects of oligonucleotide phosphorothioation on recombination frequency. The results described in this work provide further mechanistic insight into recombineering, and substantially improve recombineering performance.

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Section: Resultssupporting

confidence: 78%

Improving Lambda Red Genome Engineering in Escherichia coli via Rational Removal of Endogenous Nucleases

et al. 2012

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“…Genes encoding the RecJ-like protein (PF2055), primase (PF0110 and PF0111), GINS (PF0483 and PF0982), Proliferating cell nuclear antigen (PCNA, PF0983), RPA (PF2018-2020) and PolB (PF0212) were amplified from P. furiosus genomic DNA by PCR using their respective primers (Supplementary Table S1) and then inserted into pDEST17 as described previously (32). Amino acid substitutions were introduced into RecJ and PolB with a QuikChange® Site-Directed Mutagenesis Kit using KOD-plus DNA polymerase and the appropriate primers (Supplementary Table S1).…”

Section: Methodsmentioning

confidence: 99%

RecJ-like protein from Pyrococcus furiosus has 3′–5′ exonuclease activity on RNA: implications for proofreading of 3′-mismatched RNA primers in DNA replication

Yuan

Liu

Han

et al. 2013

Nucleic Acids Research

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Replicative DNA polymerases require an RNA primer for leading and lagging strand DNA synthesis, and primase is responsible for the de novo synthesis of this RNA primer. However, the archaeal primase from Pyrococcus furiosus (Pfu) frequently incorporates mismatched nucleoside monophosphate, which stops RNA synthesis. Pfu DNA polymerase (PolB) cannot elongate the resulting 3′-mismatched RNA primer because it cannot remove the 3′-mismatched ribonucleotide. This study demonstrates the potential role of a RecJ-like protein from P. furiosus (PfRecJ) in proofreading 3′-mismatched ribonucleotides. PfRecJ hydrolyzes single-stranded RNA and the RNA strand of RNA/DNA hybrids in the 3′–5′ direction, and the kinetic parameters (Km and Kcat) of PfRecJ during RNA strand digestion are consistent with a role in proofreading 3′-mismatched RNA primers. Replication protein A, the single-stranded DNA–binding protein, stimulates the removal of 3′-mismatched ribonucleotides of the RNA strand in RNA/DNA hybrids, and Pfu DNA polymerase can extend the 3′-mismatched RNA primer after the 3′-mismatched ribonucleotide is removed by PfRecJ. Finally, we reconstituted the primer-proofreading reaction of a 3′-mismatched ribonucleotide RNA/DNA hybrid using PfRecJ, replication protein A, Proliferating cell nuclear antigen (PCNA) and PolB. Given that PfRecJ is associated with the GINS complex, a central nexus in archaeal DNA replication fork, we speculate that PfRecJ proofreads the RNA primer in vivo.

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“…Double-stranded DNA with phosphorothioate linkages are resistant to digestion by λ Exonuclease (380). Therefore, a PCR substrate containing phosphorothioates at the 5′ ends should be resistant to λ Exo, and be a poor substrate for recombineering.…”

Section: Red Recombineering Via a Ssdna Intermediatementioning

confidence: 99%

λ Recombination and Recombineering

Murphy

2016

EcoSal Plus

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The bacteriophage λ Red homologous recombination system has been studied over the past 50 years as a model system to define the mechanistic details of how organisms exchange DNA segments that share extended regions of homology. The λ Red system proved useful as a system to study because recombinants could be easily generated by co-infection of genetically marked phages. What emerged from these studies was the recognition that replication of phage DNA was required for substantial Red-promoted recombination in vivo, and the critical role that double-stranded DNA ends play in allowing the Red proteins access to the phage DNA chromosomes. In the past 16 years, however, the λ Red recombination system has gained a new notoriety. When expressed independently of other λ functions, the Red system is able to promote recombination of linear DNA containing limited regions of homology (∼50 bp) with the Escherichia coli chromosome, a process known as recombineering. This review explains how the Red system works during a phage infection, and how it is utilized to make chromosomal modifications of E. coli with such efficiency that it changed the nature and number of genetic manipulations possible, leading to advances in bacterial genomics, metabolic engineering, and eukaryotic genetics.

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The terminal 5′ phosphate and proximate phosphorothioate promote ligation‐independent cloning

Cited by 23 publications

References 18 publications

Improving Lambda Red Genome Engineering in Escherichia coli via Rational Removal of Endogenous Nucleases

Improving Lambda Red Genome Engineering in Escherichia coli via Rational Removal of Endogenous Nucleases

RecJ-like protein from Pyrococcus furiosus has 3′–5′ exonuclease activity on RNA: implications for proofreading of 3′-mismatched RNA primers in DNA replication

λ Recombination and Recombineering

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