Crystal structure of the Redβ C-terminal domain in complex with λ Exonuclease reveals an unexpected homology with λ Orf and an interaction with<i>Escherichia coli</i>single stranded DNA binding protein

Caldwell, Brian J.; Zakharova, Ekaterina; Filsinger, Gabriel T.; Wannier, Timothy M.; Hempfling, Jordan P; Chun-Der, Lee; Pei, Dehua; Church, George M.; Bell, Charles E.

doi:10.1093/nar/gky1309

Cited by 29 publications

(41 citation statements)

References 60 publications

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“…However, compared to the conserved N-terminus, the residues in the C-terminal region of different SSAP types are irregular. This might be consistent with the fact that the conserved N-terminus of SSAP is responsible for homologous pairing, while the C-terminal interacts with EXO or host factors (20–23).…”

Section: Resultssupporting

confidence: 76%

“…The residues in the N-terminal half of EXOs are conserved across types, while the residues in the C-terminal half vary considerably even in the same type. The residue Leu-91 and residue Phe-94 of λ Exo have been found to be important for forming the λ Exo-Beta complex (23). These residues lie within a helix of the α-α-β-β fold of the λ Exo (Fig.…”

Section: Resultsmentioning

confidence: 99%

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The efficiency for recombineering is dependent on the source of the phage recombinase function unit

Chang¹,

Wang

Su³

et al. 2019

Preprint

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21Phage recombinase function units (PRFUs) such as lambda-Red or Rac RecET have been proven 22 to be powerful genetic tools in the recombineering of Escherichia coli. Studies have focused on 23 developing such systems in other bacteria as it is believed that these PRFUs have limited efficiency 24 in distant species. However, how the species evolution distance relates to the efficiency of 25 recombineering remains unclear. Here, we present a thorough study of PRFUs to find features that 26 might be related to the efficiency of PRFUs for recombineering. We first identified 59 unique sets 27 of PRFUs in the genus Corynebacterium and classified them based on their sequence as well as 28 secondary structure similarities. Then both PRFUs from this genus and other bacteria were chosen 29 for experiment based on sequential and secondary structure similarity as well as species distance. 30These PRFUs were compared for their ability in mediating recombineering with oligo or double-31 stranded DNA substrates in Corynebacterium glutamicum. We demonstrate that the source of the 32 PRFU is more critical than species distance for the efficiency of recombineering. Our work will 33 provide new ideas for efficient recombineering using PRFUs. 34 35 Importance 36 Recombineering using phage recombinase function units (PRFUs) such as lambda-Red or Rac 37RecET has gained success in Escherichia coli, while efforts applying these systems in other 38 bacteria were limited by the efficiency. It is believed that the species distance may be a major 39 reason for the low efficiency. In this study, however, we showed that it is the source of PRFU 40 rather than the species distance that matters for the recombineering in Corynebacterium 41 glutamicum. Besides, we also showed that the lower transformation efficiency in other bacteria 42 compared to that of E. coli could be a major reason for the low performance of heterogeneously 43 expressed RecET. These findings will be helpful for the recombineering using PRFUs. 44 Keywords: phage recombinase function unit, RecET, recombineering, efficiency 45 46 Recombineering using a phage recombinase function unit (PRFU), which usually contains one 48 single-stranded annealing protein (SSAP) and one 5'-3' exonuclease (EXO), is powerful in certain 49 bacteria (1-5). Expression of the SSAP alone can mediate recombineering with single-stranded 50 DNA (ssDNA) substrates, while expression of the whole system can use double-stranded DNA 51 (dsDNA) as substrates for genome fragment editing (1, 2).52 Well-known PRFUs such as lambda-Red or Rac RecET have been used for successful 53 recombineering in Escherichia coli. The Red system is phage lambda derived, while the RecET 54 system is from the defective prophage Rac of E. coli (1). Lambda-Red contains three adjacent 55 genes gam, bet, and exo, which encode a host nuclease inhibiting the protein Gam, an SSAP Beta, 56 and an EXO, respectively (6). In E. coli, it is possible to mediate recombineering using a dsDNA 57 fragment with a homology arm as short as 35 base...

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Section: Resultssupporting

confidence: 76%

Section: Resultsmentioning

confidence: 99%

The efficiency for recombineering is dependent on the source of the phage recombinase function unit

Chang¹,

Wang

Su³

et al. 2019

Preprint

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“…However, the nature and origin of the recombinase that catalyzes invasion of the DNA replication fork by the synthetic oligo makes a considerable difference (Chang et al, 2019). It is possible that such recombinases act in concert with additional endogenous proteins that could be characteristic of each species (Caldwell et al, 2019; Yin et al, 2019). It seems thus desirable that future alternatives to the Rec2 activity encoded in pSEVA2314- rec2 - mutL E36K PP (Fig.…”

Section: Resultsmentioning

confidence: 99%

High-efficiency multi-site genomic editing (HEMSE) ofPseudomonas putidathrough thermoinducible ssDNA recombineering

Aparicio

Nyerges

Martínez‐García

et al. 2019

Preprint

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SUMMARYWhile single-stranded DNA recombineering is a powerful strategy for higher-scale genome editing, its application to species other than enterobacteria is typically limited by the efficiency of the recombinase and the action of native mismatch repair (MMR) systems. By building on [i] availability of the Erf-like single-stranded DNA-annealing protein Rec2, [ii] adoption of tightly-regulated thermoinducible device and [iii] transient expression of a MMR-supressing mutL allele, we have set up a coherent genetic platform for entering multiple changes in the chromosome of Pseudomononas putida with an unprecedented efficacy and reliability. The key genetic construct to this end is a broad host range plasmid encoding co-transcription of rec2 and P. putida’s mutLE36KPP at high levels under the control of the PL/cI857 system. Cycles of short thermal shifts of P. putida cells followed by transformation with a suite of mutagenic oligos delivered different types of high-fidelity genomic changes at frequencies up to 10% per single change—that can be handled without selection. The same approach was instrumental to super-diversify short chromosomal portions for creating libraries of functional genomic segments—as shown in this case with ribosomal binding sites. These results enable the multiplexing of genome engineering of P. putida, as required for metabolic engineering of this important biotechnological chassis.

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“…The C-terminal tail of E. coli SSB is known to be the binding domain for host proteins involved in DNA replication and repair 25 , and a 9-amino-acid EcSSB C-terminal tail peptide was shown to bind to Redβ in vitro 22 . We therefore evaluated the importance of the SSB C-terminal tail by coexpressing Redβ in L. lactis, along with a version of EcSSB that had a 9-amino-acid C-terminal deletion (EcSSB∆9) (Fig 2a).…”

Section: Compatibility Between Rects and Ssbs Is Mediated By The 7 C-mentioning

confidence: 99%

“…We thus focused our efforts on host 35 proteins which reside or consistently interact with DNA on the lagging strand. Recently, we identified a binding interaction between Redβ and a C-terminal peptide of E. coli SSB (singlestranded DNA-binding protein) 22 . Phage SSBs have been shown to play important roles in phage replication and recombination pathways 23 , and SSBs have been used to increase recombineering efficiency in E. coli 24 .…”

mentioning

confidence: 99%

Characterizing the portability of RecT-mediated oligonucleotide recombination

Filsinger

Wannier

Pedersen

et al. 2020

Preprint

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Bacterial genome editing methods are used to engineer strains for biotechnology and fundamental research. Homologous recombination (HR) is the most versatile method of genome editing, but traditional techniques using endogenous RecA-mediated pathways are inefficient and 5 laborious. Phage encoded RecT proteins can improve HR over 1000-fold, but these proteins have limited portability between species. Using Escherichia coli, Lactococcus lactis, Mycobacterium smegmatis, Lactobacillus rhamnosus, and Caulobacter crescentus we investigated the hostlimited functionality of RecTs. We find that these proteins specifically recognize the 7 Cterminal amino acids of the bacterial single-stranded DNA-binding protein (SSB), and are 10 portable between species only if compatibility with this host domain is maintained. Furthermore, in some species, we find that co-expressing otherwise incompatible RecTs with a paired bacterial SSB is sufficient to establish functionality. Finally, we demonstrate that high-efficiency HR surpasses the mutational capacity of more widely used error-prone methods for genome diversification, and can be used to identify exceptional phenotypes inaccessible through 15 sequential nucleotide conversions.

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Crystal structure of the Redβ C-terminal domain in complex with λ Exonuclease reveals an unexpected homology with λ Orf and an interaction withEscherichia colisingle stranded DNA binding protein

Cited by 29 publications

References 60 publications

The efficiency for recombineering is dependent on the source of the phage recombinase function unit

The efficiency for recombineering is dependent on the source of the phage recombinase function unit

High-efficiency multi-site genomic editing (HEMSE) ofPseudomonas putidathrough thermoinducible ssDNA recombineering

Characterizing the portability of RecT-mediated oligonucleotide recombination

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