DNA replication and UV-induced DNA repair synthesis in human fibroblasts are much less sensitive than DNA polymerase α to inhibition by butylphenyl-deoxyguanosine triphosphate

Dresler, Steven L.; Frattini, Mark G.

doi:10.1093/nar/14.17.7093

Cited by 89 publications

(46 citation statements)

References 20 publications

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“…The sucrose gradients of Fig. 1 show that (14,15,27). The difference in amount of residual synthesis observed in poll mutants and in the presence of DNA polymerase a inhibitors can best be explained by the fact that the inhibitors functionally inactivate DNA polymerase a, leaving the polypeptide intact.…”

Section: Discussionmentioning

confidence: 92%

“…At least one form of DNA polymerase 8 may also be required. The evidence, while compelling, is indirect in that it relies on the use of differential inhibitors in permeabilized cells and on the requirement for an accessory subunit, rather than the catalytic subunit of DNA polymerase 8, in simian virus 40 DNA replication in vitro (4,14,32,41). One major difference between these b-type polymerases and DNA polymerase a is the existence of an apparently intrinsic 3'-to-5' "proofreading" exonuclease in each of the b-like enzymes.…”

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confidence: 99%

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DNA polymerase I is required for premeiotic DNA replication and sporulation but not for X-ray repair in Saccharomyces cerevisiae.

et al. 1989

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We have used a set of seven temperature-sensitive mutants in the DNA polymerase I gene of Saccharomyces cerevisiae to investigate the role of DNA polymerase I in various aspects of DNA synthesis in vivo. Previously, we showed that DNA polymerase I is required for mitotic DNA replication. Here we extend our studies to several stages of meiosis and repair of X-ray-induced damage. We find that sporulation is blocked in all of the DNA polymerase temperature-sensitive mutants and that premeiotic DNA 81, and 82 (8, 42). Although these enzymes were first identified and purified many years ago, the precise role of each polymerase in various aspects of DNA metabolism, such as replication, repair, and recombination, is controversial (8). Studies of this question have been limited due to the absence of mutants totally defective in any of the polymerases. Saccharomyces cerevisiae contain analogs of both DNA polymuerases a and the b-type enzymes and offer an ideal genetic system in which to explore the cellular functions of these polymerases (8).A comparison of the metazoan and yeast polymerases is shown in Table 1. It is now clear that DNA polymerase I of S. rerevisiae is the counterpart of DNA polymerase a, the conserved subunit structure being the key reason for concluding this (8). DNA polymerase a has been implicated in replication because it is inhibited by aphidicolin, an inhibitor of cellular replication, and because a mouse carcinoma cell line containing a thermolabile DNA polymerase a is also temperature sensitive for DNA replication (16). We have confirmed the requirement for DNA polymerase a in chromosomal replication by cloning the yeast POLI gene, showing the gene disruptions are lethal, and deriving conditional lethal mutants which show gross defects in DNA synthesis (5,22).Recent evidence suggests that DNA polymerase a may not be the sole polymerase involved in eucaryotic DNA replication, however. At least one form of DNA polymerase 8 may also be required. The evidence, while compelling, is indirect in that it relies on the use of differential inhibitors in permeabilized cells and on the requirement for an accessory subunit, rather than the catalytic subunit of DNA polymerase 8, in simian virus 40 DNA replication in vitro (4, 14, 32, 41). One major difference between these b-type polymerases and DNA polymerase a is the existence of an apparently intrinsic 3'-to-5' "proofreading" exonuclease in each of the b-like enzymes. The putative yeast analogs of DNA poly-* Corresponding author. merase 8 are DNA polymerases 11 (9,43,44) and III (7), though neither DNA polymerase b nor DNA polymerase II or III has been well enough characterized yet to warrant a definitive statement on the extent to which the species correspond (7, 13, 25, 42; M. E. Budd and J. L. Campbell, unpublished data). Thus, a critical question in eucaryotic DNA replication today is to. what extent each of these polymerases participates in mitotic replication, meiotic replication, repair, and recombination; yeast cells should be useful in answerin...

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Section: Discussionmentioning

confidence: 92%

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confidence: 99%

DNA polymerase I is required for premeiotic DNA replication and sporulation but not for X-ray repair in Saccharomyces cerevisiae.

et al. 1989

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“…These include similar molecular weight and shape, associated DNA primase activity, identical inhibition by aphidicolin, and similar processivity and stimulation by ATP (16,17,21). Recent selective inhibitor studies with diploid human fibroblasts suggest the involvement of pol 8 in the processes of semiconservative DNA replication and repair of damage by UV irradiation (35). The well-known difficulties in defining the subunit composition of pol a, often ascribed to proteolysis, led to the interesting speculation that pol a may be derived from pol 8 or vice versa.…”

Section: Discussionmentioning

confidence: 95%

Exonucleolytic proofreading by calf thymus DNA polymerase delta.

Kunkel¹,

Sabatino²,

Bambara³

1987

Proc. Natl. Acad. Sci. U.S.A.

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The fidelity of DNA synthesis by calf thymus DNA polymerase 6 (po 6) in vitro has been determined using an M13lacZa nonsense codon reversion assay. Pol 6 is highly accurate, producing on average <1 single-base substitution error for each 106 nucleotides polymerized. This accuracy is 10-and 500-fold greater than that of DNA polymerases a and (3, respectively, in the same assay. Three observations suggest that this higher fidelity results in part from proofreading of misinserted bases by the 3' to 5' exonuclease associated with pol 6. First, the exonuclease efficiently excises terminally mismatched bases. Second, both terminal mismatch excision and the fidelity of DNA synthesis by po1 6 are reduced with increasing concentration of deoxynucleoside triphosphates in the synthesis reaction. These effects result from increasing the rate of polymerization relative to the rate of exonucleolytic excision and are hallmarks of exonuclease proofreading. Third, both terminal mismatch excision and fidelity decrease upon addition to the reaction mixture of adenosine monophosphate, a compound known to selectively inhibit the exonuclease but not the polymerase activity of po0 6.These results suggest that 3' to 5' exonuclease-dependent proofreading enhances the fidelity of DNA synthesis by a mammalian DNA polymerase in vitro.The high base-substitution fidelity of prokaryotic DNA polymerases results from discrimination at the base insertion step and from exonucleolytic proofreading of incorrectly inserted bases during synthesis (for review, see ref.

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“…It had been postulated that DNA polymerase a is responsible for replication of SV40 DNA in vivo (7) and for replication of SV40-based DNAs in the in vitro system of Li and Kelly (15). However, recently it has been suggested that DNA polymerase 8 may also play an important role in DNA replication (6). Since DNA polymerase 8 shares many properties with DNA polymerase a, it may be difficult to distinguish the roles of the two enzymes (26) We have compared our results, in which the pZ189 shuttle vector has been used to measure replication fidelity, with those of Kunkel (12,13) We have not ruled out the possibility that the higher fidelity we observe in the in vitro system may be due in part to the presence of a mismatch correction system (8,9,20).…”

Section: Discussionmentioning

confidence: 99%

Fidelity of DNA synthesis in a mammalian in vitro replication system.

Hauser¹,

Levine²,

Dixon³

1988

Mol. Cell. Biol.

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We have used the simian virus 40 (SV40)-based shuttle vector pZ189 in a forward-mutation assay to determine the fidelity of DNA replication in the in vitro DNA replication system developed by J. J. Li and T. J. Kelly (Proc. Natl. Acad. Sci. USA 81:6973-6977, 1984). We find that very few base substitution errors (approximately 1/180,000 bases incorporated) are made during in vitro replication of the pZ189 vector in a system derived from CV-1 monkey cells. This replication is completely dependent on added SV40 T antigen and presumably reflects synthesis that is initiated at the SV40 replication origin. The observed level of fidelity is far greater than that reported for in vitro replication of DNA by conventionally purified eucaryotic DNA polymerases a and P1. Thus, there must be additional cellular factors in the crude in vitro system that serve to enhance the fidelity of DNA replication.Replication of mammalian chromosomal DNA apparently occurs with a high level of fidelity. Error rates for misincorporation of nucleotides in vivo have been estimated to be on the order of 1/109 (5). However, purified DNA polymerases derived from a variety of mammalian sources exhibit very high error rates for in vitro DNA synthesis (13,17). Thus, it has been postulated that there are additional factors in mammalian cells that serve to enhance replication fidelity in vivo. In order to study DNA replication in an in vitro system that more closely mimics the in vivo DNA replication conditions, a number of laboratories (1,15,25,27,29) have recently developed crude in vitro DNA replication systems that will replicate exogenous simian virus 40 (SV40) DNA. Replication in these systems is dependent on added SV40 T antigen and requires an SV40 origin of replication in the template DNA. Replication is bidirectional, and completely replicated closed circular molecules are produced.Here we report the use of the SV40-based shuttle vector, pZ189 (23), to measure the fidelity of DNA replication in a crude in vitro system prepared from CV-1 monkey cells, as previously described by Li and Kelly (15,16). The pZ189 vector ( Fig. 1) contains the early region of SV40, including the viral origin of replication. In addition, the vector contains the pBR327 origin and replication functions, as well as the 1-lactamase gene, to allow growth and selection in bacteria. The suipF gene, which codes for a tRNA suppressor, is included in the vector to provide a selectable target for measuring mutagenesis. We have used this vector previously to characterize spontaneous and UV-induced mutations arising during DNA replication in primate cells (10,11,18). and forms white colonies if the suppressor is inactive. E. coli G717 (darn) was used to produce pZ189 lacking methylated adenine at GATC sites. The pZ189 vector (23) contains the pBR327 origin of replication and the ,B-lactamase gene for replication and selection in bacteria, the SV40 early region for replication in monkey cells, and the bacterial suipF suppressor tRNA gene for mutation detection.Preparation of c...

show abstract

DNA replication and UV-induced DNA repair synthesis in human fibroblasts are much less sensitive than DNA polymerase α to inhibition by butylphenyl-deoxyguanosine triphosphate

Cited by 89 publications

References 20 publications

DNA polymerase I is required for premeiotic DNA replication and sporulation but not for X-ray repair in Saccharomyces cerevisiae.

DNA polymerase I is required for premeiotic DNA replication and sporulation but not for X-ray repair in Saccharomyces cerevisiae.

Exonucleolytic proofreading by calf thymus DNA polymerase delta.

Fidelity of DNA synthesis in a mammalian in vitro replication system.

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