Structural basis for uracil recognition by archaeal family B DNA polymerases

Deamination of cytosine to uracil in a G-C base pair is a major promutagenic event, generating G-C→A-T mutations if not repaired before DNA replication. Archaeal family B DNA polymerases are uniquely able to recognize unrepaired uracil in a template strand and stall polymerization upstream of the lesion, thereby preventing the irreversible fixation of an A-T mutation. We have now identified a 'pocket' in the N-terminal domains of archaeal DNA polymerases that is positioned to interact with the template strand and provide this ability. The structure of this pocket provides interacting groups that discriminate uracil from the four normal DNA bases (including thymine). These groups are conserved in archaeal polymerases but absent from homologous viral polymerases that are unable to recognize uracil. Using site-directed mutagenesis, we have confirmed the biological role of this pocket and have engineered specific mutations in the Pfu polymerase that confer the ability to read through template-strand uracils and carry out PCR with dUTP in place of dTTP.

[1]  W. Franklin,et al.  Thermostable uracil-DNA glycosylase from Thermotoga maritima a member of a novel class of DNA repair enzymes , 1999, Current Biology.

[2]  T. Lindahl.  An N-glycosidase from Escherichia coli that releases free uracil from DNA containing deaminated cytosine residues. , 1974, Proceedings of the National Academy of Sciences of the United States of America.

[3]  L. Pearl,et al.  A read-ahead function in archaeal DNA polymerases detects promutagenic template-strand uracil. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[4]  H. Hogrefe,et al.  Archaeal dUTPase enhances PCR amplifications with archaeal DNA polymerases by preventing dUTP incorporation , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[5]  H. Fritz,et al.  Counteracting the mutagenic effect of hydrolytic deamination of DNA 5‐methylcytosine residues at high temperature: DNA mismatch N‐glycosylase Mig.Mth of the thermophilic archaeon Methanobacterium thermoautotrophicum THF. , 1996, The EMBO journal.

[6]  H. Krokan,et al.  DNA glycosylases in the base excision repair of DNA. , 1997, The Biochemical journal.

[7]  Jeffrey H. Miller,et al.  Biochemical Characterization of Uracil Processing Activities in the Hyperthermophilic Archaeon Pyrobaculum aerophilum * , 2001, The Journal of Biological Chemistry.

[8]  Y. Kai,et al.  Crystal structure of DNA polymerase from hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1. , 2001, Journal of molecular biology.

[9]  L. Beese,et al.  Crystal structure of a pol alpha family DNA polymerase from the hyperthermophilic archaeon Thermococcus sp. 9 degrees N-7. , 2000, Journal of molecular biology.

[10]  Thomas A. Steitz,et al.  Structure of the Replicating Complex of a Pol α Family DNA Polymerase , 2001, Cell.

[11]  J. Tainer,et al.  Envisioning the molecular choreography of DNA base excision repair. , 1999, Current opinion in structural biology.

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

[13]  E. Marcotte,et al.  Characterization of a Thermostable DNA Glycosylase Specific for U/G and T/G Mismatches from the Hyperthermophilic ArchaeonPyrobaculum aerophilum , 2000, Journal of bacteriology.

[14]  W. Franklin,et al.  Characterization of the full length uracil-DNA glycosylase in the extreme thermophile Thermotoga maritima. , 2001, Mutation research.

[15]  W. Franklin,et al.  Uracil-DNA Glycosylase in the Extreme Thermophile Archaeoglobus fulgidus * , 2000, The Journal of Biological Chemistry.

[16]  R. Huber,et al.  Crystal structure of a thermostable type B DNA polymerase from Thermococcus gorgonarius. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[17]  T. Steitz,et al.  Crystal Structure of a pol α Family Replication DNA Polymerase from Bacteriophage RB69 , 1997, Cell.

[18]  J. Kuriyan,et al.  Crystal structure of an archaebacterial DNA polymerase. , 1999, Structure.

[19]  J. Thompson,et al.  The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. , 1997, Nucleic acids research.

[20]  L. Pearl,et al.  Structure and function in the uracil-DNA glycosylase superfamily. , 2000, Mutation research.

[21]  Richard D. Wood,et al.  Human DNA Repair Genes , 2001, Science.

[22]  S. Ho,et al.  Site-directed mutagenesis by overlap extension using the polymerase chain reaction. , 1989, Gene.

[23]  G. Cantoni,et al.  Procedures in nucleic acid research , 1966 .

[24]  A. Shinohara,et al.  Homologous recombination and the roles of double-strand breaks. , 1995, Trends in biochemical sciences.

[25]  M. Goodman,et al.  Coping with replication 'train wrecks' in Escherichia coli using Pol V, Pol II and RecA proteins. , 2000, Trends in biochemical sciences.

[26]  F. Young.  Biochemistry , 1955, The Indian Medical Gazette.

[27]  T. Lindahl.  Instability and decay of the primary structure of DNA , 1993, Nature.

[28]  R. Fuchs,et al.  Lesions in DNA: hurdles for polymerases. , 2000, Trends in biochemical sciences.

[29]  L. Pearl,et al.  The problem with pyrimidines , 1996, Nature Structural Biology.

[30]  R. Lasken,et al.  Archaebacterial DNA Polymerases Tightly Bind Uracil-containing DNA* , 1996, The Journal of Biological Chemistry.

[31]  D. Grogan.  The question of DNA repair in hyperthermophilic archaea. , 2000, Trends in microbiology.

[32]  P. Nyman,et al.  Crystal structure of the Escherichia coli dUTPase in complex with a substrate analogue (dUDP) , 1996, Nature Structural Biology.

[33]  A. Ronen,et al.  Human DNA repair genes , 2001, Environmental and molecular mutagenesis.

[34]  T. Steitz,et al.  Crystal structures of an NH2-terminal fragment of T4 DNA polymerase and its complexes with single-stranded DNA and with divalent metal ions. , 1996, Biochemistry.

[35]  B. Connolly,et al.  Binding and recognition of GATATC target sequences by the EcoRV restriction endonuclease: a study using fluorescent oligonucleotides and fluorescence polarization. , 2001, Biochemistry.