Translesion synthesis by yeast DNA polymerase from templates containing lesions of ultraviolet radiation and acetylaminofluorene

In the yeast Saccharomyces cerevisiae, DNA polymerase zeta (Polzeta) is required in a major lesion bypass pathway. To help understand the role of Polzeta in lesion bypass, we have performed in vitro biochemical analyses of this polymerase in response to several DNA lesions. Purified yeast Polzeta performed limited translesion synthesis opposite a template TT (6-4) photoproduct, incorporating A or T with similar efficiencies (and less frequently G) opposite the 3' T, and predominantly A opposite the 5' T. Purified yeast Polzeta predominantly incorporated a G opposite an acetylaminofluorene (AAF)-adducted guanine. The lesion, however, significantly inhibited subsequent extension. Furthermore, yeast Polzeta catalyzed extension DNA synthesis from primers annealed opposite the AAF-guanine and the 3' T of the TT (6-4) photoproduct with varying efficiencies. Extension synthesis was more efficient when A or C was opposite the AAF-guanine, and when G was opposite the 3' T of the TT (6-4) photoproduct. In contrast, the 3' T of a cis-syn TT dimer completely blocked purified yeast Polzeta, whereas the 5' T was readily bypassed. These results support the following dual-function model of Polzeta. First, Polzeta catalyzes nucleotide incorporation opposite AAF-guanine and TT (6-4) photoproduct with a limited efficiency. Secondly, more efficient bypass of these lesions may require nucleotide incorporation by other DNA polymerases followed by extension DNA synthesis by Polzeta.

[1]  R. Fuchs,et al.  N-2-aminofluorene and N-2 acetylaminofluorene adducts: the local sequence context of an adduct and its chemical structure determine its replication properties. , 1995, Journal of molecular biology.

[2]  A. Grollman,et al.  Mutagenic specificity of (acetylamino)fluorene-derived DNA adducts in mammalian cells. , 1998, Biochemistry.

[3]  Yanbin Zhang,et al.  Error-prone lesion bypass by human DNA polymerase eta. , 2000, Nucleic acids research.

[4]  C. Lawrence,et al.  Deoxycytidyl transferase activity of yeast REV1 protein , 1996, Nature.

[5]  Klaus Rajewsky,et al.  Disruption of the Rev3l-encoded catalytic subunit of polymerase ζ in mice results in early embryonic lethality , 2000, Current Biology.

[6]  Robert E. Johnson,et al.  hRAD30 mutations in the variant form of xeroderma pigmentosum. , 1999, Science.

[7]  Roger Woodgate,et al.  Roles of E. coli DNA polymerases IV and V in lesion-targeted and untargeted SOS mutagenesis , 2000, Nature.

[8]  L. Prakash,et al.  RAD6 gene of Saccharomyces cerevisiae encodes a protein containing a tract of 13 consecutive aspartates. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[9]  J P McDonald,et al.  Misinsertion and bypass of thymine–thymine dimers by human DNA polymerase ι , 2000, The EMBO journal.

[10]  C A Smith,et al.  Preparation and characterization of a set of deoxyoligonucleotide 49-mers containing site-specific cis-syn, trans-syn-I, (6-4), and Dewar photoproducts of thymidylyl(3'-->5')-thymidine. , 1993, The Journal of biological chemistry.

[11]  F. Fabre,et al.  A similar defect in UV-induced mutagenesis conferred by the rad6 and rad18 mutations of Saccharomyces cerevisiae. , 1991, Mutation research.

[12]  R. P. P. Fuchs,et al.  Analysis of Damage Tolerance Pathways in Saccharomyces cerevisiae: a Requirement for Rev3 DNA Polymerase in Translesion Synthesis , 1998, Molecular and Cellular Biology.

[13]  Satya Prakash,et al.  Fidelity of Human DNA Polymerase η* , 2000, The Journal of Biological Chemistry.

[14]  M. Yamaizumi,et al.  Dysfunction of human Rad18 results in defective postreplication repair and hypersensitivity to multiple mutagens. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[15]  Robert E. Johnson,et al.  Fidelity and Processivity of Saccharomyces cerevisiae DNA Polymerase η* , 1999, The Journal of Biological Chemistry.

[16]  Alexander Varshavsky,et al.  The yeast DNA repair gene RAD6 encodes a ubiquitin-conjugating enzyme , 1987, Nature.

[17]  L. Prakash,et al.  The Saccharomyces cerevisiae RAD18 gene encodes a protein that contains potential zinc finger domains for nucleic acid binding and a putative nucleotide binding sequence. , 1988, Nucleic acids research.

[18]  J. Hoeijmakers,et al.  Structural and functional conservation of two human homologs of the yeast DNA repair gene RAD6. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[19]  W. Sumanasekera,et al.  The human RAD18 gene product interacts with HHR6A and HHR6B. , 2000, Nucleic acids research.

[20]  P E Gibbs,et al.  Evidence for a second function for Saccharomyces cerevisiae Rev1p , 2000, Molecular microbiology.

[21]  J. Lemontt,et al.  REV3, a Saccharomyces cerevisiae gene whose function is required for induced mutagenesis, is predicted to encode a nonessential DNA polymerase , 1989, Journal of bacteriology.

[22]  Fenghua Yuan,et al.  Response of human DNA polymerase ι to DNA lesions , 2001 .

[23]  F. Hanaoka,et al.  Error-prone bypass of certain DNA lesions by the human DNA polymerase kappa. , 2000, Genes & development.

[24]  Robert E. Johnson,et al.  Efficient bypass of a thymine-thymine dimer by yeast DNA polymerase, Poleta. , 1999, Science.

[25]  F. Larimer,et al.  The REV1 gene of Saccharomyces cerevisiae: isolation, sequence, and functional analysis , 1989, Journal of bacteriology.

[26]  Z. Wang,et al.  A full-length cDNA of hREV3 is predicted to encode DNA polymerase zeta for damage-induced mutagenesis in humans. , 1999, Mutation research.

[27]  C. Croce,et al.  A Human REV7 Homolog That Interacts with the Polymerase ζ Catalytic Subunit hREV3 and the Spindle Assembly Checkpoint Protein hMAD2* , 2000, The Journal of Biological Chemistry.

[28]  R. Chanet,et al.  Potential DNA-binding domains in the RAD18 gene product of Saccharomyces cerevisiae. , 1988, Gene.

[29]  Fenghua Yuan,et al.  Preferential Incorporation of G Opposite Template T by the Low-Fidelity Human DNA Polymerase ι , 2000, Molecular and Cellular Biology.

[30]  Fenghua Yuan,et al.  Error-free and error-prone lesion bypass by human DNA polymerase κ in vitro , 2000 .

[31]  Charles R.scriver,et al.  The Metabolic basis of inherited disease , 1989 .

[32]  Chikahide Masutani,et al.  The XPV (xeroderma pigmentosum variant) gene encodes human DNA polymerase η , 1999, Nature.

[33]  R. D. Wood,et al.  Disruption of the developmentally regulated Rev3l gene causes embryonic lethality , 2000, Current Biology.

[34]  Errol C. Friedberg,et al.  Deletion of the Saccharomyces cerevisiae gene RAD30 encoding an Escherichia coli DinB homolog confers UV radiation sensitivity and altered mutability , 1998, Molecular and General Genetics MGG.

[35]  E. G. Frank,et al.  UmuD'(2)C is an error-prone DNA polymerase, Escherichia coli pol V. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[36]  Z. Livneh,et al.  The Mutagenesis Protein UmuC Is a DNA Polymerase Activated by UmuD′, RecA, and SSB and Is Specialized for Translesion Replication* , 1999, The Journal of Biological Chemistry.

[37]  S. Creighton,et al.  Gel fidelity assay measuring nucleotide misinsertion, exonucleolytic proofreading, and lesion bypass efficiencies. , 1995, Methods in enzymology.

[38]  P E Gibbs,et al.  A human homolog of the Saccharomyces cerevisiae REV3 gene, which encodes the catalytic subunit of DNA polymerase zeta. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[39]  Yanbin Zhang,et al.  The human REV1 gene codes for a DNA template-dependent dCMP transferase. , 1999, Nucleic acids research.

[40]  P. Sung,et al.  Specific complex formation between yeast RAD6 and RAD18 proteins: a potential mechanism for targeting RAD6 ubiquitin-conjugating activity to DNA damage sites. , 1994, Genes & development.

[41]  Satya Prakash,et al.  Eukaryotic polymerases ι and ζ act sequentially to bypass DNA lesions , 2000, Nature.

[42]  William J. Feaver,et al.  Purification and Characterization of polκ, a DNA Polymerase Encoded by the Human DINB1 Gene* , 2001, The Journal of Biological Chemistry.

[43]  C. Lawrence,et al.  Thymine-Thymine Dimer Bypass by Yeast DNA Polymerase ζ , 1996, Science.

[44]  Fenghua Yuan,et al.  Specificity of DNA Lesion Bypass by the Yeast DNA Polymerase η* , 2000, The Journal of Biological Chemistry.

[45]  R. D. Gietz,et al.  New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. , 1988, Gene.

[46]  P E Gibbs,et al.  The function of the human homolog of Saccharomyces cerevisiae REV1 is required for mutagenesis induced by UV light. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[47]  M. Neuberger,et al.  Disruption of mouse polymerase ζ (Rev3) leads to embryonic lethality and impairs blastocyst development in vitro , 2000, Current Biology.

[48]  J P McDonald,et al.  Is Dna Damage Inducible and Functions in a Novel Error-free Postreplication Repair Mechanism , 1997 .

[49]  Chikahide Masutani,et al.  Low fidelity DNA synthesis by human DNA polymerase-η , 2000, Nature.

[50]  F. Hanaoka,et al.  Xeroderma pigmentosum variant (XP‐V) correcting protein from HeLa cells has a thymine dimer bypass DNA polymerase activity , 1999, The EMBO journal.