Production of UV-induced Frameshift Mutations in Vitro by DNA Polymerases Deficient in 3′ → 5′ Exonuclease Activity

Sagher, Daphna; Turkington, Edith; Acharya, Sonia; Strauss, B.

doi:10.1006/jmbi.1994.1437

Cited by 11 publications

(6 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…5). These results are in accord with the observation that synthesis opposite irradiated templates was increased when exo Ϫ KF and T7 DNA polymerase were used in place of the wild-type enzymes (75). Recently, elimination of the 3Ј 3 5Ј exonuclease activity of KF, has been shown to greatly accelerate bypass of an abasic site analog (33).…”

Section: Discussionsupporting

confidence: 79%

The Ability of a Variety of Polymerases to Synthesize Past Site-specific cis-syn, trans-syn-II, (6–4), and Dewar Photoproducts of Thymidylyl-(3′→5′)-thymidine

Smith

Baeten

Taylor

1998

Journal of Biological Chemistry

View full text Add to dashboard Cite

The role of photoproduct structure, 3 3 5 exonuclease activity, and processivity on polynucleotide synthesis past photoproducts of thymidylyl-(3 3 5)-thymidine was investigated. Both Moloney murine leukemia virus reverse transcriptase and 3 3 5 exonuclease-deficient (exo ؊ ) Vent polymerase were blocked by all photoproducts, whereas Taq polymerase could slowly bypass the cis-syn dimer. T7 RNA polymerase was able to bypass all the photoproducts in the order cis-syn > Dewar > (6 -4) > trans-syn-II. Klenow fragment could not bypass any of the photoproducts, but an exo ؊ mutant could bypass the cis-syn dimer to a greater extent than the others. Likewise T7 DNA polymerase, composed of the T7 gene 5 protein and Escherichia coli thioredoxin, was blocked by all the photoproducts, but the exo ؊ mutant Sequenase 2.0 was able to bypass them all in the order cissyn > Dewar > trans-syn-II > (6 -4). No bypass occurred with an exo ؊ gene 5 protein in the absence of the thioredoxin processivity factor. Bypass of the cis-syn and trans-syn-II products by Sequenase 2.0 was essentially non-mutagenic, whereas about 20% dTMP was inserted opposite the 5-T of the Dewar photoproduct. A mechanism involving a transient abasic site is proposed to account for the preferential incorporation of dAMP opposite the 3-T of the photoproducts.Dipyrimidine sites are the major sites of UV-induced photoproducts and mutations (1-7). The four main classes of photoproducts formed by ultraviolet light at dipyrimidine sites (shown in Fig. 1 for a TpT 1 site) are the cis-syn and trans-syn (trans-syn-I (8) and trans-syn-II (9)) cyclobutane dimers and the (6 -4) pyrimidine-pyrimidone photoproducts and their Dewar valence isomers (10 -13). All of these photoproducts have been found to lead to mutations in Escherichia coli under SOS conditions by use of site-specific photoproduct-containing bacteriophage vectors, but the (6 -4) and Dewar photoproducts are far more mutagenic than either the cis-syn and trans-syn isomers (14 -18). The extent to which a particular photoproduct contributes to UV-induced mutations at a particular site not only depends on its mutagenicity, but also depends on its rate of induction, repair, and DNA synthesis bypass (7,12). At the moment, the relative contribution of individual DNA photoproducts to UV-induced mutations is not known, nor are the detailed mechanisms by which DNA photoproducts are repaired or bypassed. Recently, we have prepared homogeneous 49-mer oligonucleotides containing the four major photoproduct classes of TpT (19) 2 for use as substrates for the necessary in vitro and in vivo mechanistic studies. Herein, we report the use of these 49-mers and 72-mers containing a T7 promoter to study the role of photoproduct structure and the 3Ј 3 5Ј exonuclease activity and processivity of polymerases on DNA and RNA synthesis past these photoproducts. EXPERIMENTAL PROCEDURESEnzymes, Reagents, and Equipment-The preparation of the photoproduct-containing 49-mers has been reported elsewhere (19).2 Other oligonucleotides were purchas...

show abstract

Section: Discussionsupporting

confidence: 79%

The Ability of a Variety of Polymerases to Synthesize Past Site-specific cis-syn, trans-syn-II, (6–4), and Dewar Photoproducts of Thymidylyl-(3′→5′)-thymidine

Smith

Baeten

Taylor

1998

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…This observation supports the proposed role of Bs Pol I in assisting the TLS reaction catalysed by the Y‐DNA polymerases. Indeed, proofreading activity is expected to oppose translesion DNA synthesis by preventing the stable incorporation of a mispaired nucleotide opposite DNA lesions (Sagher et al ., 1994; Khare and Eckert, 2002). In E. coli , Pol I, which is proficient for 3′−5′ proofreading activity, does not participate in spontaneous or in UV‐targeted mutagenesis in vivo (Wagner and Nohmi, 2000).…”

Section: Discussionmentioning

confidence: 99%

DNA polymerase I acts in translesion synthesis mediated by the Y‐polymerases in Bacillus subtilis

Duigou

Ehrlich

Naveilhan

et al. 2005

Molecular Microbiology

View full text Add to dashboard Cite

“…We previously demonstrated a role for proofreading exonuclease activity in UV-induced frameshift mutagenesis at repeated sequences in vitro (4). There is also in vitro evidence that proofreading exonuclease is important in the surveillance of spontaneous frameshift mutations (5).…”

Section: Introductionmentioning

confidence: 99%

Role of proofreading and mismatch repair in maintaining the stability of nucleotide repeats in DNA

Strauss

Sagher

Acharya

1997

Nucleic Acids Research

View full text Add to dashboard Cite

The role of the proofreading exonuclease in maintaining the stability of multiply repeated units in DNA was studied in Escherichia coli. Reversion of plasmids in which the beta-galactosidase alpha complementing sequence was moved +2 out of frame by inserts containing (CA)14, (CA)5, (CA)2 or (TA)6 or +1 by creating a run of 8 C was compared in mutS and mutSdnaQ strains. Proofreading corrects at least half of the frameshift errors for all the plasmids and at least 99% of the errors in the (CA)2 plasmid. The (CA)2 plasmid reverts mostly by +1 frameshifts in the restriction sites flanking the insert. With the (CA)14, (TA)6, (CA)5 and 8C plasmids, reversion is mainly by loss of a repeat unit. The data support the hypothesis that the dnaQgene product recognizes frameshifts close to the DNA growing point. Frameshifts distal to the growing point are mainly corrected by mismatch repair. We speculate that mismatches in mononucleotide repeats are susceptible to proofreading because they can either migrate to a point where they are recognized by the exonuclease or, alternatively, because single nucleotide distortions are more readily detected than dinucleotides.

show abstract

Production of UV-induced Frameshift Mutations in Vitro by DNA Polymerases Deficient in 3′ → 5′ Exonuclease Activity

Cited by 11 publications

References 0 publications

The Ability of a Variety of Polymerases to Synthesize Past Site-specific cis-syn, trans-syn-II, (6–4), and Dewar Photoproducts of Thymidylyl-(3′→5′)-thymidine

The Ability of a Variety of Polymerases to Synthesize Past Site-specific cis-syn, trans-syn-II, (6–4), and Dewar Photoproducts of Thymidylyl-(3′→5′)-thymidine

DNA polymerase I acts in translesion synthesis mediated by the Y‐polymerases in Bacillus subtilis

Role of proofreading and mismatch repair in maintaining the stability of nucleotide repeats in DNA

Contact Info

Product

Resources

About