Enhancement of DNA, cDNA synthesis and fidelity at high temperatures by a dimeric single-stranded DNA-binding protein.
Bacterial single-stranded DNA-binding proteins (SSBs) are required for DNA replication and repair. We have over-expressed and purified the native form and two His-tagged fusions of the SSB from Thermus thermophilus (TthSSB). The three proteins were found as dimers in solution. They bound in vitro to single-stranded DNA specifically over a temperature range of 4-80 degrees C, and the wild-type protein could withstand incubation at 94 degrees C for 2 min. Addition of TthSSB to PCR halved the elongation time required for the DNA polymerases of T.thermophilus (Tth) and Pyrococcus furiosus (Pfu) to synthesise DNA fragments in PCRs. The presence of TthSSB increased the fidelity of the proof- reading-free DNA polymerase of T.thermophilus. TthSSB was also able to bind single-stranded RNA, allowing a dramatic enhancement of the reverse transcription activity of its cognate Tth DNA polymerase during cDNA synthesis.
Perales
Celia
C
Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Campus de Cantoblanco, 28049 Madrid, Spain.
Cava
Felipe
F
Meijer
Wilfried J J
WJ
Berenguer
José
J
D003001:Cloning, Molecular / Q000379:methods; D004269:DNA, Bacterial / Q000235:genetics* / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D019281:Dimerization; D024202:Electrophoretic Mobility Shift Assay; D008969:Molecular Sequence Data; D011485:Protein Binding; D012329:RNA, Bacterial / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013696:Temperature; D016963:Thermus thermophilus / Q000235:genetics*
D004269:DNA, Bacterial; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D012329:RNA, Bacterial; D004259:DNA-Directed DNA Polymerase