• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of pnasPNASInfo for AuthorsSubscriptionsAboutThis Article
Proc Natl Acad Sci U S A. Jan 15, 1992; 89(2): 763–767.

A DNA polymerase alpha pause site is a hot spot for nucleotide misinsertion.


In this study we examined whether the arrest of DNA polymerase alpha (pol alpha)-catalyzed DNA synthesis at template pause sites entails terminal nucleotide misincorporation. An approach was developed to identify the 3'-terminal nucleotide in nascent DNA chains that accumulate at pause sites. A radioactive 5'-end-labeled primer was annealed to a bacteriophage M13mp2 single-stranded DNA template and elongated by pol alpha. Individual DNA chains that were accumulated at pause sites were resolved by sequencing gel electrophoresis, isolated, and purified. These DNA chains were elongated by pol alpha by using four annealed synthetic DNA templates, each of which contained a different nucleotide at the position opposite the 3' terminus of the arrested chain. Owing to the high preference of pol alpha for matched-over-mismatched primer termini, only those templates that contain a nucleotide that is complementary to the 3' terminus of the isolated pause-site chain are copied. Electrophoresis of product DNA showed the extent of copying of each template and thus identified the 3'-terminal nucleotide of the pause-site chains. We found that product DNA chains terminate with a noncomplementary 3'-terminal nucleotide opposite pause sites within the sequence 3'-d(AAAA)-5' at positions 6272-6269 of the M13mp2 genome. pol alpha catalyzed misincorporation of dG or dA into the 3' terminus of nascent chains opposite two of the M13mp2 template dA residues. A similar analysis of a different pause site did not reveal significant misincorporation opposite template dC. These results suggest that some but not all sites at which pol alpha pauses may constitute loci of mutagenesis.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.3M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • de Jong PJ, Grosovsky AJ, Glickman BW. Spectrum of spontaneous mutation at the APRT locus of Chinese hamster ovary cells: an analysis at the DNA sequence level. Proc Natl Acad Sci U S A. 1988 May;85(10):3499–3503. [PMC free article] [PubMed]
  • Barbacid M. ras genes. Annu Rev Biochem. 1987;56:779–827. [PubMed]
  • Levine AJ, Momand J, Finlay CA. The p53 tumour suppressor gene. Nature. 1991 Jun 6;351(6326):453–456. [PubMed]
  • Kunkel TA. The mutational specificity of DNA polymerases-alpha and -gamma during in vitro DNA synthesis. J Biol Chem. 1985 Oct 15;260(23):12866–12874. [PubMed]
  • Kunkel TA, Alexander PS. The base substitution fidelity of eucaryotic DNA polymerases. Mispairing frequencies, site preferences, insertion preferences, and base substitution by dislocation. J Biol Chem. 1986 Jan 5;261(1):160–166. [PubMed]
  • Kunkel TA, Bebenek K. Recent studies of the fidelity of DNA synthesis. Biochim Biophys Acta. 1988 Nov 10;951(1):1–15. [PubMed]
  • Williams KJ, Loeb LA, Fry M. Synthesis of DNA by human immunodeficiency virus reverse transcriptase is preferentially blocked at template oligo(deoxyadenosine) tracts. J Biol Chem. 1990 Oct 25;265(30):18682–18689. [PubMed]
  • Perrino FW, Loeb LA. Differential extension of 3' mispairs is a major contribution to the high fidelity of calf thymus DNA polymerase-alpha. J Biol Chem. 1989 Feb 15;264(5):2898–2905. [PubMed]
  • Mendelman LV, Petruska J, Goodman MF. Base mispair extension kinetics. Comparison of DNA polymerase alpha and reverse transcriptase. J Biol Chem. 1990 Feb 5;265(4):2338–2346. [PubMed]
  • Bebenek K, Joyce CM, Fitzgerald MP, Kunkel TA. The fidelity of DNA synthesis catalyzed by derivatives of Escherichia coli DNA polymerase I. J Biol Chem. 1990 Aug 15;265(23):13878–13887. [PubMed]
  • Fay PJ, Johanson KO, McHenry CS, Bambara RA. Size classes of products synthesized processively by two subassemblies of Escherichia coli DNA polymerase III holoenzyme. J Biol Chem. 1982 May 25;257(10):5692–5699. [PubMed]
  • Kaguni LS, Clayton DA. Template-directed pausing in in vitro DNA synthesis by DNA polymerase a from Drosophila melanogaster embryos. Proc Natl Acad Sci U S A. 1982 Feb;79(4):983–987. [PMC free article] [PubMed]
  • Weaver DT, DePamphilis ML. Specific sequences in native DNA that arrest synthesis by DNA polymerase alpha. J Biol Chem. 1982 Feb 25;257(4):2075–2086. [PubMed]
  • Hillebrand GG, Beattie KL. Influence of template primary and secondary structure on the rate and fidelity of DNA synthesis. J Biol Chem. 1985 Mar 10;260(5):3116–3125. [PubMed]
  • Abbotts J, SenGupta DN, Zon G, Wilson SH. Studies on the mechanism of Escherichia coli DNA polymerase I large fragment. Effect of template sequence and substrate variation on termination of synthesis. J Biol Chem. 1988 Oct 15;263(29):15094–15103. [PubMed]
  • Weisman-Shomer P, Dube DK, Perrino FW, Stokes K, Loeb LA, Fry M. Sequence specificity of pausing by DNA polymerases. Biochem Biophys Res Commun. 1989 Nov 15;164(3):1149–1156. [PubMed]
  • Grosse F, Krauss G. Replication of M13mp7 single-stranded DNA in vitro by the 9-S DNA polymerase alpha from calf thymus. Eur J Biochem. 1984 May 15;141(1):109–114. [PubMed]
  • Fry M, Weisman-Shomer P, Lapidot J, Sharf R. Template-selective stimulation of diverse DNA polymerases by the murine DNA-binding protein factor D. J Biol Chem. 1987 Jun 25;262(18):8861–8867. [PubMed]
  • Sharf R, Weisman-Shomer P, Fry M. Rabbit liver factor D, a poly(thymidine) template stimulatory protein of DNA polymerases: purification and characterization. Biochemistry. 1988 Apr 19;27(8):2990–2997. [PubMed]
  • Asna N, Weisman-Shomer P, Waldman E, Fry M. Factor C from rabbit liver. A new poly(dC) and poly[d(G-C)] template-selective stimulatory protein of DNA polymerases. J Biol Chem. 1989 Mar 25;264(9):5245–5252. [PubMed]
  • LaDuca RJ, Fay PJ, Chuang C, McHenry CS, Bambara RA. Site-specific pausing of deoxyribonucleic acid synthesis catalyzed by four forms of Escherichia coli DNA polymerase III. Biochemistry. 1983 Oct 25;22(22):5177–5188. [PubMed]
  • Kaguni LS, DiFrancesco RA, Lehman IR. The DNA polymerase-primase from drosophila melanogaster embryos. Rate and fidelity of polymerization on single-stranded DNA templates. J Biol Chem. 1984 Jul 25;259(14):9314–9319. [PubMed]
  • Tabor S, Huber HE, Richardson CC. Escherichia coli thioredoxin confers processivity on the DNA polymerase activity of the gene 5 protein of bacteriophage T7. J Biol Chem. 1987 Nov 25;262(33):16212–16223. [PubMed]
  • Huber HE, Tabor S, Richardson CC. Escherichia coli thioredoxin stabilizes complexes of bacteriophage T7 DNA polymerase and primed templates. J Biol Chem. 1987 Nov 25;262(33):16224–16232. [PubMed]
  • Hopfield JJ. The energy relay: a proofreading scheme based on dynamic cooperativity and lacking all characteristic symptoms of kinetic proofreading in DNA replication and protein synthesis. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5248–5252. [PMC free article] [PubMed]
  • Bebenek K, Abbotts J, Roberts JD, Wilson SH, Kunkel TA. Specificity and mechanism of error-prone replication by human immunodeficiency virus-1 reverse transcriptase. J Biol Chem. 1989 Oct 5;264(28):16948–16956. [PubMed]
  • Perrino FW, Preston BD, Sandell LL, Loeb LA. Extension of mismatched 3' termini of DNA is a major determinant of the infidelity of human immunodeficiency virus type 1 reverse transcriptase. Proc Natl Acad Sci U S A. 1989 Nov;86(21):8343–8347. [PMC free article] [PubMed]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


  • PubMed
    PubMed citations for these articles
  • Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...