Novel Thermostable Y-Family DNA Polymerases


Y-family polymerases are able to bypass lesions in DNA that would otherwise block replication by high fidelity DNA polymerases and are key to the effective study of ancient DNA and for use in forensic medicine. These enzymes are ubiquitous and are found in all kingdoms of life: bacteria, archaea and eukaryotes. The number of proteins related to the Y-family polymerases is well over 200 orthologs and despite being closely related at the phylogenetic level, the few polymerases now characterized, each show a unique set of properties including processivity, fidelity, and the ability to bypass certain types of DNA. Y-family polymerases from thermostable organisms are of particular interest because the enzymes isolated from such species tend to be more stable, easy to work with and may have more utility in assays at higher temperatures, such as Polymerase Chain Reaction (PCR). For example, the thermostable archeal Sulfolobus solfataricus DinB-like polymerase Dpo4 can bypass lesions by generally inserting the correct complementary nucleotide opposite a variety of damaged bases and can, under appropriate conditions substitute for Taq polymerase in PCR applications. Additionally, functional and structural organization of this family of polymerases permits domain swapping designed to optimize specific properties of use in novel applications.

Dr. Woodgate's group at the National Institute of Child Health and Development have expanded their earlier work and have now discovered several additional thermostable dpo4 homologs from other strains found in the Sulfolobaceae family, some of which have optimal growth temperatures higher than 80oC. These novel DinB-like proteins have thermostable DNA polymerase activity and are capable of: 1) PCR amplifications over 1kb in length, 2) replication past DNA lesions such as abasic sites and CPD (cis-syn cyclobutane pyrimidine dimer) lesions and 3) incorporation of several different labeled DNA nucleotides into DNA during replication. These enzymes may therefore be a good substitute for Taq polymerase in applications utilizing fluorescent nucleoside triphosphate derivatives. These lesion-bypassing Dpo4-like polymerases could also be included along with a conventional thermostable polymerase in a PCR protocol designed to amplify old or damaged DNA samples which could greatly increase recoverability, accuracy and length of products. Other applications could include labelling or tagging DNA, real-time PCR, detection of SNPs, mismatches or DNA lesions, mutagenic PCR, directed-evolution methods and expanding the "DNA alphabet" utilizing non-natural nucleotides.

Available for licensing are seven novel Y-family polymerases. Claims are directed to these sequences and chimeras, as well as to methods of identifying other Y-family polymerases and generating other chimeric Y-family polymerases and methods of use. These enzymes and methods of identifying and generating novel Y-family polymerases should be of interest to forensic DNA service companies as well as to research reagent companies pursuing novel thermophlic enzymes for use in ancient and damaged DNA analysis and for novel applications with modified nucleotides.

Inventors:

Roger Woodgate (NICHD)  ➽ more inventions...

John Mcdonald (NICHD)  ➽ more inventions...

Wei Yang (NIDDK)  ➽ more inventions...


Intellectual Property:
U.S. Pat: 7,745,188 issued 2010-06-29
U.S. Pat: 8,114,653 issued 2012-02-14
US Application No. 12/776,603

Publications:
Boudsocq F, et al. PMID 11713310
Boudsocq F, et al. PMID 15155753

Licensing Contact:
Admin. Licensing Specialist (ALS),
Email:
Phone:

OTT Reference No: E-166-2004/2
Updated: Jul 31, 2012