دانلود رایگان مقاله پردازش فرم ssDNA توسط گروه مرموز RNase H اگزونوکلئاز PF 2046

Abstract

RNase H fold protein PF2046 of Pyrococcus furiosus is a 3′-5′ ssDNA exonuclease that cleaves after the second nucleotide from the 3′ end of ssDNA and prefers poly-dT over poly-dA as a substrate. In our crystal structure of PF2046 complexed with an oligonucleotide of four thymidine nucleotides (dT4), PF2046 accommodates dT4 tightly in a groove and imposes steric hindrance on dT4 mainly by Phe220 such that dT4 assumes the A-form. As poly-dA prefer B-form due to the stereochemical restrictions, the A-form ssDNA binding by PF2046 should disfavor the processing of poly-dA. Phe220 variants display reduced activity toward poly-dA and the A-form appears to be a prerequisite for the processing by PF2046.

5. Conclusion

In conclusion, the crystal structure of the PF2046-dT4 complex should advance the understanding of substrate binding and the reaction mechanism of PF2046. Our study illustrates a peculiar function of the RNase H fold in hyperthermophilic archaea. PF2046 is a full-fledged member of the RNase H family, and our analysis of the A-form of bound ssDNA suggests that PF2046 may also function as a ribonuclease of an ssRNA of unknown identity. Recently, Miyazono et al. reported crystal structures of PhoExo I from Pyrococcus horikoshii, a homolog of PF2046 [5]. Structural comparison between PF2046 and PhoExo I indicated to differ about interaction with ssDNA by key residue such as Thr53 and Ser102. One possibility of the difference is the residual activity of PF2046 processing oligonucleotides into 4-base product to turn out as enzymeproduct complex. Our structural and mutagenesis study suggests the conformational restrain on ssDNA can enable an RNaseH fold protein to function as a deoxyribonuclease. As thermophilic nucleases are attractive tools for particular molecular biology applications, this structure-functional study of PF2046 will be helpful in utilization of this interesting nuclease.