The archaea are a group of microorganisms
The archaea are a group of microorganisms, and many belong to extremophiles, living in extreme environments, such as those with high temperatures , . Methanocaldococcus jannaschii is a methane-producing archaea . It grows at pressures of up to more than 200atm and at an optimum temperature of 85°C. High temperature and nitrosative stress result in high frequency of Heparin deamination and other types of DNA damage. DNA ligases seal the matured Okazaki fragments in DNA replication and the generated nick in DNA repair. Some DNA ligases from thermophilic bacteria and archaea have been studied , , , , , , . DNA ligase is an important enzyme in molecular biology. Aside from ligating recombinant DNA, thermostable DNA ligases have been used in single nucleotide polymorphism (SNP) genotyping , , . SNP is the most abundant form of genetic variation. SNP is important marker that link sequence variations to phenotypic changes. Because of the importance of SNP in the life and medical sciences, a great deal of effort has been devoted to developing accurate, rapid, and cost-effective technologies for SNP analysis. DNA ligases show preferential ligation of matched base pairs, and this then forms the molecular basis of SNP determination , , . Duo to the thermostability, DNA ligases from thermophilic microbe, such as Taq and Tth DNA ligases , , have some advantage over mesothermic DNA ligases, such as T4 DNA ligase , in SNP genotyping.
In this paper, we cloned the lig gene from M. jannaschii and characterized its biochemical properties. M. jannaschii DNA ligase is a typical ATP-dependent ligase. To apply M. jannaschii DNA ligase in SNP genotyping, the ability of M. jannaschii DNA ligase to discriminate terminal base mismatches on the nick was systematically characterized. We determined the best strategy for achieving the highest precision in distinguishing mismatch by M. jannaschii DNA ligase; that is, improving discriminative ability via the introduction of an additional mismatch into the DNA duplex. Our results provide useful data for the potential application of M. jannaschii DNA ligase in SNP genotyping.
Materials and methods
Discussion DNA ligase plays an important role in genome replication and DNA repair. M. jannaschii inhabits high temperature environments and suffers from serious DNA damages compared with mesophilic microbes. M. jannaschii encodes one uracil-DNA glycosylase , AP endonuclease, family B DNA polymerase, and other proteins involved in base excision repair. However, the results regarding uracil-DNA glycosylase are different , . We cloned all potential genes involved in base excision repair. M. jannaschii DNA ligase is the only DNA ligase in M. jannaschii. SNP is the most abundant form of genetic variation that occurs once every 100–300 bases, and more than 3 million SNPs are found in the human genome . Thermostable DNA ligases have been used in SNP genotyping because of their high base specificity in ligation , . We confirm that ligated M. jannaschii DNA ligase matched oligonucleotide duplexes more efficiently than it did mismatched duplexes, and the introduction of another mismatch near the nick could improve the ability of the ligase to discriminate terminal mismatches. Successfully joining the nick requires a stable duplex surrounding the nick. The introduction of an additional mismatch into the oligonucleotide duplex further disrupts the stability of the duplex with a mismatched nick and leads to the separation of two oligonucleotide strands and the failure of ligation of two short oligonucleotides. Therefore, for SNP genotyping based on ligase, improving discriminative ability is useful and easily carried out by introducing an additional mismatch surrounding the nick.
Introduction DNA ligases are ubiquitous proteins involved in DNA replication and repair. The ligation reaction consists of three conserved steps that involve an enzyme–adenylate and a DNA–adenylate complex . Step one involves the transfer of AMP to an active site lysine, step two consists of the transfer of the AMP moiety to the 5′-PO4 of nicked DNA strand and step three consists of the assisted attack of the 3′-OH strand of the nick to the 5′-PO4 strand . According to the AMP donor moiety, DNA ligases can be classified as ATP or NAD+ dependent. ATP-dependent DNA ligases are present in viruses, archea and eukaryotic organisms, whereas NAD+ ligases are predominantly found in bacteria . However, bacteria like Haemophilus influenzae, Mycobacterium tubercolisis, Bacillus subtilis, and Neisseria meningitidis also contain ATP-dependent DNA ligases , , .