Multiple Amino Acid Substitutions Allow DNA Polymerases to Synthesize RNA*

DNA and RNA polymerase exhibit similarities in structures and catalytic mechanisms, suggesting that both classes of enzymes are evolutionarily related. To probe the biochemical and structure-function relationship between the two classes of polymerases, a large library (200,000 members) of mutant Thermus aquaticus DNA polymerase I (Taq pol I) was created containing random substitutions within a portion of the dNTP binding site (motif A; amino acids 605–617), and a fraction of all selected active Taq pol I (291 of 8000) was tested for the ability to incorporate successive ribonucleotides; 23 unique mutants that added rNTPs into a growing polynucleotide chain were identified and sequenced. These mutants, each containing one to four substitutions, incorporate ribonucleotides at a efficiency approaching 103-fold greater than that of wild type Taq pol I. Several mutants added successive ribonucleotides and thus can catalyze the synthesis of RNA. Sequence analysis of these mutants demonstrates that at least two amino acid residues are involved in excluding ribonucleotides from the active site. Interestingly, wild type DNA polymerases from several distinct families selectively discriminate against rUTP. This study suggests that current DNA and RNA polymerases could have evolved by divergent evolution from an ancestor that shared a common mechanism for polynucleotide synthesis.

[1]  P H Patel,et al.  DNA polymerase active site is highly mutable: evolutionary consequences. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[2]  L. Blanco,et al.  A single tyrosine prevents insertion of ribonucleotides in the eukaryotic-type phi29 DNA polymerase. , 1999, Journal of molecular biology.

[3]  Gabriel Waksman,et al.  Crystal structures of open and closed forms of binary and ternary complexes of the large fragment of Thermus aquaticus DNA polymerase I: structural basis for nucleotide incorporation , 1998, The EMBO journal.

[4]  G L Verdine,et al.  Structure of a covalently trapped catalytic complex of HIV-1 reverse transcriptase: implications for drug resistance. , 1998, Science.

[5]  C. M. Joyce,et al.  A single side chain prevents Escherichia coli DNA polymerase I (Klenow fragment) from incorporating ribonucleotides. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[6]  S. Doublié,et al.  Crystal structure of a bacteriophage T7 DNA replication complex at 2.2 Å resolution , 1998, Nature.

[7]  M. Costa,et al.  Comparative genomic analysis of isolates belonging to the six species of the genus Thermus using pulsed-field gel electrophoresis and ribotyping , 1997, Archives of Microbiology.

[8]  W A Hendrickson,et al.  Conferring RNA polymerase activity to a DNA polymerase: a single residue in reverse transcriptase controls substrate selection. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[9]  J. Tainer,et al.  A nucleotide-flipping mechanism from the structure of human uracil–DNA glycosylase bound to DNA , 1996, Nature.

[10]  Gerald F. Joyce,et al.  Ribozymes: Building the RNA world , 1996, Current Biology.

[11]  C. Richardson,et al.  A single residue in DNA polymerases of the Escherichia coli DNA polymerase I family is critical for distinguishing between deoxy- and dideoxyribonucleotides. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[12]  P. Pfaffle,et al.  Single-step purification of a thermostable DNA polymerase expressed in Escherichia coli. , 1995, BioTechniques.

[13]  Samuel H. Wilson,et al.  Structures of ternary complexes of rat DNA polymerase beta, a DNA template-primer, and ddCTP. , 1994, Science.

[14]  B. Preston,et al.  Marked infidelity of human immunodeficiency virus type 1 reverse transcriptase at RNA and DNA template ends. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[15]  Yong Je Chung,et al.  Crystal structure of bacteriophage T7 RNA polymerase at 3.3 Å resolution , 1993, Nature.

[16]  T. Steitz,et al.  Structure of DNA polymerase I Klenow fragment bound to duplex DNA , 1993, Science.

[17]  D. Engelke,et al.  Purification of Thermus aquaticus DNA polymerase expressed in Escherichia coli. , 1990, Analytical biochemistry.

[18]  P Argos,et al.  An attempt to unify the structure of polymerases. , 1990, Protein engineering.

[19]  G. F. Joyce.  RNA evolution and the origins of life , 1989, Nature.

[20]  M. Boosalis,et al.  DNA polymerase insertion fidelity. Gel assay for site-specific kinetics. , 1987, The Journal of biological chemistry.

[21]  B. Weiss,et al.  Specific mutator effects of ung (uracil-DNA glycosylase) mutations in Escherichia coli , 1982, Journal of bacteriology.

[22]  E. Friedberg,et al.  An enzyme activity from Escherichia coli that attacks single-stranded deoxyribopolymers and single-stranded deoxyribonucleic acid containing apyrimidinic sites. , 1982, Biochemistry.

[23]  B. Olivera,et al.  DNA intermediates at the Escherichia coli replication fork. II. Studies using dut and ung mutants in vitro. , 1979, Journal of molecular biology.

[24]  W. M. Barnes.  DNA sequencing by partial ribosubstitution. , 1978, Journal of molecular biology.

[25]  B Nyberg,et al.  Heat-induced deamination of cytosine residues in deoxyribonucleic acid. , 1974, Biochemistry.

[26]  P. Arbuthnot,et al.  Rapid purification of recombinant Taq DNA polymerase by freezing and high temperature thawing of bacterial expression cultures. , 1995, Nucleic acids research.

[27]  M. Gefter,et al.  DNA Replication , 2019, Advances in Experimental Medicine and Biology.