Abstract: The utility of Topo V's HhH motifs for modulating DNA binding properties of a DNA processing enzyme, such as Taq DNA polymerase or PfuDNA polymerase, is demonstrated. In one embodiment, an amino acid residue comprising one or more HhH domains derived from Topoisomerase V, linked to either the NH2-terminus or COOH-terminus of a Taq polymerase fragment significantly broadens the processivity and/or salt concentration range of polymerase activity. The specific activities of the chimeric polymerases are not affected by added HhH motifs. Depending on the type of the construct, the thermal stability of chimeric polymerases increases or remains the same as that of Taq DNA polymerase or its Stoffel fragment. This invention further provides a method of increasing the salt tolerance of Taq polymerases. The methods of this invention may be applied to all pol A-type DNA polymerases, as well as to other DNA processing enzymes.

Abstract: We have determined the complete 1,694,969 nucleotide sequence of the GC-rich genome of Methanopyrus kandleri using a novel approach. It is based on unlinking genomic DNA with the ThermoFidelase version of M. kandleri topoisomerase V and cycle sequencing directed by 2?-modified oligonucleotides (Fimers). 3.3× sequencing redundancy was sufficient to assemble the genome with <1 error per 40 kb. Using a combination of sequence database searches and coding potential prediction, 1692 protein-coding genes and 39 genes for structural RNAs were identified. M. kandleri proteins show an unusually high content of negatively charged amino acids, which might be an adaptation to its high intracellular salinity. Previous phylogenetic analysis of 16S RNA suggested that M. kandleri belonged to a very deep branch, close to the root of the archaeal tree. However, genome comparisons, using both trees constructed from concatenated alignments of ribosomal proteins and trees based on gene content, indicate that M.