DNA-repair reactions by purified HeLa DNA polymerases and exonucleases.

Randahl, Håkan; Elliott, George C.; Linn, Stuart

doi:10.1016/s0021-9258(18)37744-5

Cited by 46 publications

(7 citation statements)

References 15 publications

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“…DNA polymerase ,B is a vertebrate DNA repair enzyme considered to be involved in 'gap-filling' DNA synthesis during nucleotide excision DNA repair." [3][4][5][6][7][8][9][10][11][12][13][14][15] The purified enzyme conducts gap-filling synthesis on single-stranded (ss) DNA templates in vitro,2' 16 and this reaction is consistent with 'very short patch' synthesis during DNA repair.4-7 Inhibitor studies had implicated (-pol in some pathways of mammalian cell DNA repair,8-12 and other recent studies also have implicated (3-pol in gap-filling synthesis involved in mismatch repair by a HeLa cell nuclear extract,13 in repair of UV-damaged DNA'4 and abasic lesions'5 by Xenopus 1. oocyte extract, and in correction of damaged residues following exposure of human cells to monofunctional DNA damaging agents, such as MNNG or MMS. '12 In cultured CHO cells, (B-pol mRNA level is induced severalfold after exposure to MNNG or MMS.17 In addition, a transfected ,B-pol promoter-CAT fusion gene responded to MNNG treatment of CHO cells by exhibiting a strong transcriptional activation (c.f., Figure 3, ref.…”

Section: Introductionmentioning

confidence: 99%

DNA damage response of cloned DNA β-polymerase promoter is blocked in mutant cell lines deficient in protein kinase A

Englander

Wilson

1992

Nucl Acids Res

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DNA beta-polymerase (beta-pol), one of the recognized DNA polymerizing enzymes in vertebrates, has a role in 'very short patch' gap-filling synthesis during nucleotide excision DNA repair. In human and mouse, the enzyme is encoded by a single-copy gene located on the short arm of chromosome 8 near the centromere. In a series of studies, we have found that the cloned human beta-pol promoter is regulated by signals acting through the single ATF/CRE palindrome in the core promoter. These signals include transactivation by: adenovirus E1a/E1b proteins; activated p21ras; and in CHO cells, treatment with the DNA damaging agent MNNG. Hence, several types of stimulatory signals are mediated through the single ATF/CRE site, including DNA damage induction. To understand the mechanism of beta-pol promoter activation by MNNG in CHO cells, we asked whether induction of the cAMP/protein kinase A pathway can increase transcription of the cloned promoter in this system. Agents that increase cellular cAMP levels (8-BrcAMP; forskolin and IBMx) activated the beta-pol promoter fusion gene in transient expression experiments, and a mutation in the ATF/CRE palindrome blocked this response. Thus, the ATF/CRE site appears to be cAMP responsive in the CHO cell system. We found that the activation of the cloned beta-pol promoter by MNNG does not occur with two mutant CHO cell lines that are deficient in protein kinase A activity. Further, simultaneous treatment of wild-type CHO cells, with MNNG and to elevate cAMP, failed to result in an additive effect for activation of the beta-pol promoter. Thus, these effectors may act through a common pathway. These results suggest that the activation of the cloned beta-pol promoter in CHO cells following MNNG treatment is mediated through the cAMP/protein kinase A signal transduction pathway.

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Section: Introductionmentioning

confidence: 99%

DNA damage response of cloned DNA β-polymerase promoter is blocked in mutant cell lines deficient in protein kinase A

Englander

Wilson

1992

Nucl Acids Res

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“…Finally, although it has been observed that pol 2 Unlike BER, where only a single-nucleotide gap is left to be filled in by pol β, other DNA repair mechanisms, particularly those invoked for NER, have been shown to leave gaps in the DNA that can be as long as 30-50 nucleotides. Taking into consideration the observation that pol , unlike pol β, may not be capable of filling in DNA gaps to completion at physiological ionic strengths (Randahl et al, 1988), a working hypothesis has emerged for these other repair mechanisms whereby a relatively large DNA gap may be partially filled in by pol , followed by pol β taking over to fill the smaller gap to completion. Although this is an attractive hypothesis, it is as yet not clear whether pol β is directly involved in NER (Horton et al, 1995;Sobol et al, 1996), nor is it clear that pol is incapable of filling in DNA gaps to completion in vivo (Mozzherin & Fisher, 1996).…”

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Crystal Structures of Human DNA Polymerase β Complexed with DNA: Implications for Catalytic Mechanism, Processivity, and Fidelity^,

et al. 1996

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Mammalian DNA polymerase beta (pol beta) is a small (39 kDa) DNA gap-filling enzyme that comprises an amino-terminal 8-kDa domain and a carboxy-terminal 31-kDa domain. In the work reported here, crystal structures of human pol beta complexed with blunt-ended segments of DNA show that, although the crystals belong to a different space group, the DNA is nevertheless bound in the pol beta binding channel in the same way as the DNA in previously reported structures of rat pol beta complexed with a template-primer and ddCTP [Pelletier, H., Sawaya, M. R., Kumar, A., Wilson, S. H., & Kraut, J. (1994) Science 264, 1891-1903]. The 8-kDa domain is in one of three previously observed positions relative to the 31-kDa domain, suggesting that the 8-kDa domain may assume only a small number of stable conformations. The thumb subdomain is in a more open position in the human pol beta-DNA binary complex than it is in the rat pol beta-DNA-ddCTP ternary complex, and a closing thumb upon nucleotide binding could represent the rate-limiting conformational change that has been observed in pre-steady-state kinetic studies. Intermolecular contacts between the DNA and the 8-kDa domain of a symmetry-related pol beta molecule reveal a plausible binding site on the 8-kDa domain for the downstream oligonucleotide of a gapped-DNA substrate; in addition to a lysine-rich binding pocket that accommodates a 5'-PO4 end group, the 8-kDa domain also contains a newly discovered helix-hairpin-helix (HhH) motif that binds to DNA in the same way as does a structurally and sequentially homologous HhH motif in the 31-kDa domain. DNA binding by both HhH motifs is facilitated by a metal ion. In that HhH motifs have been identified in other DNA repair enzymes and DNA polymerases, the HhH-DNA interactions observed in pol beta may be applicable to a broad range of DNA binding proteins. The sequence similarity between the HhH motif of endonuclease III from Escherichia coli and the HhH motif of the 8-kDa domain of pol beta is particularly striking in that all of the conserved residues are clustered in one short sequence segment, LPGVGXK, where LPGV corresponds to a type II beta-turn (the hairpin turn), and GXK corresponds to a part of the HhH motif that is proposed to be critical for DNA binding and catalysis for both enzymes. These results suggest that endonuclease III and the 8-kDa domain of pol beta may employ a similar mode of DNA binding and may have similar catalytic mechanisms for their respective DNA lyase activities. A model for productive binding of pol beta to a gapped-DNA substrate requires a 90 degrees bend in the single-stranded template, which could enhance nucleotide selectivity during DNA repair or replication.

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“…Caron et al (1985) suggested that a complex of UvrBCD and pol I exists as a "repairosome". Short-patch excision repair in eukaryotes has been modeled with pol ft and exonuclease V (Mosbaugh & Linn, 1983; Randahl et al, 1988). It has been suggested that pol« (Nishida et al, 1988) or pol a (Mosbaugh & Linn, 1984) may participate in long-patch repair.…”

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DNA helicase E and DNA polymerase .epsilon. functionally interact for displacement synthesis

Turchi¹,

Siegal²,

Bambara³

1992

Biochemistry

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A functional interaction between DNA helicase E and DNA polymerase epsilon from calf thymus has been detected which results in the extension of an upstream 3' OH through a downstream primer to the end of a synthetic template. DNA synthesis resulting in full-length extension products was dependent on the addition of DNA helicase E and hydrolysis of ATP, suggesting that displacement of the downstream primer was required. Identical reactions using DNA polymerases alpha and delta in place of DNA polymerase epsilon showed no full-length products dependent on helicase E, indicating that polymerases alpha and delta were incapable of functionally interacting with the helicase. The reaction leading to full-length extension products was time dependent and dependent on the concentration of added polymerase epsilon and helicase E. Exonucleolytic degradation of the downstream primer, or ligation of the downstream primer to the upstream 3' OH, were not responsible for the full-length products observed. Displacement of the downstream primer by DNA helicase E was not affected by the addition of polymerase epsilon to the reactions. Template dilution experiments demonstrated that DNA polymerase epsilon and helicase E were acting in concert to perform displacement synthesis. Additional evidence for functional coordination was obtained by demonstration that DNA helicase E stimulated DNA polymerase epsilon in a standard DNA synthetic assay using dA3000.dT16 as the template-primer. The results presented are consistent with the hypothesis that DNA helicase E and DNA polymerase epsilon are capable of coordinated activities that result in displacement synthesis. A functional interaction of this sort may be involved at the eukaryotic replication fork or in DNA repair.

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DNA-repair reactions by purified HeLa DNA polymerases and exonucleases.

Cited by 46 publications

References 15 publications

DNA damage response of cloned DNA β-polymerase promoter is blocked in mutant cell lines deficient in protein kinase A

DNA damage response of cloned DNA β-polymerase promoter is blocked in mutant cell lines deficient in protein kinase A

Crystal Structures of Human DNA Polymerase β Complexed with DNA: Implications for Catalytic Mechanism, Processivity, and Fidelity^,

DNA helicase E and DNA polymerase .epsilon. functionally interact for displacement synthesis

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DNA-repair reactions by purified HeLa DNA polymerases and exonucleases.

Cited by 46 publications

References 15 publications

DNA damage response of cloned DNA β-polymerase promoter is blocked in mutant cell lines deficient in protein kinase A

DNA damage response of cloned DNA β-polymerase promoter is blocked in mutant cell lines deficient in protein kinase A

Crystal Structures of Human DNA Polymerase β Complexed with DNA: Implications for Catalytic Mechanism, Processivity, and Fidelity,

DNA helicase E and DNA polymerase .epsilon. functionally interact for displacement synthesis

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Product

Resources

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Crystal Structures of Human DNA Polymerase β Complexed with DNA: Implications for Catalytic Mechanism, Processivity, and Fidelity^,