[OEC] p45 enzyme is incorrect

[jcmolloy]

The p45 enzyme from the pOPEN collection (https://www.addgene.org/165570/) is described as a dUTPase capable of significantly improving maximum amplicon size and PCR yields for Family B polymerases.

A user has reported a discrepancy between the expected biochemical properties and the provided sequence:

Molecular Weight Discrepancy: The literature for the Pyrococcus furiosus PCR-enhancing dUTPase describes a protein of approximately 18 kDa. However, the pOPEN p45 clone encodes a protein of approximately 45 kDa.

BLAST Results: A BLAST analysis of the pOPEN sequence aligns with a bifunctional phosphopantothenoylcysteine decarboxylase (CoaC) / phosphopantothenate synthase (CoaB), involved in Coenzyme A biosynthesis, rather than a dUTPase.

This confusion appears to stem from the original characterization of the P. furiosus PCR-enhancing complex. According to Hogrefe et al. (PNAS, 2002), the activity was found to reside solely in an 18 kDa dUTPase (GenBank AY066005). This dUTPase was originally co-purified with a 45 kDa flavoprotein (GenBank AY066006), also known as CoaBC (or dfp gene), which the authors confirmed had no effect on PCR performance.

It seems the current pOPEN "p45" clone might contain the sequence for the 45 kDa CoaBC protein instead of the active 18 kDa dUTPase.

References for verification:

Hogrefe, H. H., et al. (2002). "Archaeal dUTPase enhances PCR amplifications with archaeal DNA polymerases by preventing dU insertion." PNAS, 99(2), 596-601.

GenBank AY066005 (18 kDa dUTPase) vs. GenBank AY066006 (45 kDa CoaBC).

Actions

• Identify the correct sequence and prepare for synthesis as part of the next extension/wishlist synthesis order
• Alert Addgene to the error and have them deactivate the entry for now

[kvgaona]

Starting from the pOPEN p45 vector sequence, a sequence alignment analysis was performed against the two proteins reported in the literature: the 18 kDa dUTPase from Pyrococcus furiosus (GenBank: AY066005) and the ~45 kDa bifunctional CoaBC protein (GenBank: AY066006). This analysis confirmed that the sequence originally present in the vector corresponds to CoaBC rather than the dUTPase, thereby validating the previously described discrepancy.

A directed assembly was designed and successfully implemented using Golden Gate, incorporating the correct 18 kDa dUTPase sequence (AY066005). The assembly strategy included optimization of overhangs through primer redesign to ensure compatibility and to eliminate sequence redundancies.

The construct retains flanking BsaI restriction sites, enabling its reuse in future modular Golden Gate assemblies.

The primers used for the assembly are attached for reference.
Backbone-F: CGACATGGTCTCCtaggccgatctggtggttggt
Backbone-R: CGACATGGTCTCCagagacccgagggtcgtc
Insert-F: CGACATGGTCTCCctctatgctacttccagactgga
Insert-R: CGACATGGTCTCCcctagagtttctttctctttgaaaac