TY - JOUR
T1 - How a B family DNA polymerase has been evolved to copy RNA
AU - Choi, Woo Suk
AU - He, Peng
AU - Pothukuchy, Arti
AU - Gollihar, Jimmy
AU - Ellington, Andrew D.
AU - Yang, Wei
N1 - Funding Information:
We thank Drs. R. Craigie, M. Gellert, and D. J. Leahy for critical reading of the manuscript. This research was supported by National Institute of Diabetes and Digestive and Kidney Disease Intramural Grants DK036144 and DK036146 (to W.Y.), the Welch Foundation (Grant F-1654), and NIH Grant 1R01EB027202-01A0 (to A.D.E.).
Publisher Copyright:
© 2020 National Academy of Sciences. All rights reserved.
PY - 2020/9/1
Y1 - 2020/9/1
N2 - We report here crystal structures of a reverse transcriptase RTX, which was evolved in vitro from the B family polymerase KOD, in complex with either a DNA duplex or an RNA-DNA hybrid. Compared with the apo, binary, and ternary complex structures of the original KOD polymerase, the 16 substitutions that result in the function of copying RNA to DNA do not change the overall protein structure. Only six substitutions occur at the substrate-binding surface, and the others change domain-domain interfaces in the polymerase to enable RNA-DNA hybrid binding and reverse transcription. Most notably, F587L at the Palm and Thumb interface stabilizes the open and apo conformation of the Thumb. The intrinsically flexible Thumb domain seems to play a major role in accommodating the RNA-DNA hybrid product distal to the active site. This is reminiscent of naturally occurring RNA-dependent DNA polymerases, including telomerase, which have a dramatically augmented Thumb domain, and of reverse transcriptase, which extends its Thumb with the RNase H domain.
AB - We report here crystal structures of a reverse transcriptase RTX, which was evolved in vitro from the B family polymerase KOD, in complex with either a DNA duplex or an RNA-DNA hybrid. Compared with the apo, binary, and ternary complex structures of the original KOD polymerase, the 16 substitutions that result in the function of copying RNA to DNA do not change the overall protein structure. Only six substitutions occur at the substrate-binding surface, and the others change domain-domain interfaces in the polymerase to enable RNA-DNA hybrid binding and reverse transcription. Most notably, F587L at the Palm and Thumb interface stabilizes the open and apo conformation of the Thumb. The intrinsically flexible Thumb domain seems to play a major role in accommodating the RNA-DNA hybrid product distal to the active site. This is reminiscent of naturally occurring RNA-dependent DNA polymerases, including telomerase, which have a dramatically augmented Thumb domain, and of reverse transcriptase, which extends its Thumb with the RNase H domain.
KW - 3' to 5' exonuclease
KW - Proofreading
KW - Reverse transcription
KW - Thumb domain
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U2 - 10.1073/pnas.2009415117
DO - 10.1073/pnas.2009415117
M3 - Article
C2 - 32817521
AN - SCOPUS:85090505116
VL - 117
SP - 21274
EP - 21280
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
SN - 0027-8424
IS - 35
ER -