Measurement of DNA polymerase β in skin fibroblast cell lines from patients with ataxia telangiectasia

Mitchell, James B.; Karawya, Essam; Kinsella, Timothy J.; Wilson, Samuel H.

doi:10.1016/0167-8817(85)90071-9

Cited by 10 publications

(7 citation statements)

References 20 publications

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“…bases added in 3-base (triplet) steps ( Fig 12 -3Ј (shown in bold) and a CAG-Nick strand with the sequence 5Ј-(CTG) 2 -Hb-(CAG) 12 -3Ј. The annealed duplex of the Nick construct contains a total 14 CTG/CAG repeats with 10 repeats between and 2 repeats downstream of the two resultant strand breaks.…”

Section: Resultsmentioning

confidence: 99%

“…DNA sequences, 5Ј-(CAG) 2 -Ha-(CTG) 12 -3Ј and 5Ј-(CTG) 2 -Hb-(CAG) 12 -3Ј, referred to as CTG-Nick and CAG-Nick strands, respectively, were used to construct a double-stranded DNA molecule with a CTG/CAG repeat duplex having a nick between CTG repeats on one side (CTG Nick) and a nick between CAG repeats on the other side (CAG Nick), as illustrated (Fig. 1A, unslipped , TCC TTG GCC TCG CTG CTG CGA GGC CAA GGA and Hb, ACG GCA GTT GTC CTG CTG GAC AAC TGC CGT, where the underlined 4 bases form a favorable even-numbered hairpin bend (24,25), enclosed by a stable duplex formed by correct base pairing between the complementary 13-base sequences on the two sides of the bend.…”

Section: Dna Sequences Used For Nick and Gap Constructs-two 72-mermentioning

confidence: 99%

“…Human DNA polymerase ␤ (pol ␤), 1 having no proofreading (3Ј-exonuclease activity), is a logical choice for study in such contexts, since it has a well established role in DNA repair, notably base excision repair (BER) (11). Cells express pol ␤ independently of the cell cycle (12) but show tissue specific differences in pol ␤ expression, with particularly high levels of expression occurring in testis (13). During BER, following glycosylase and AP endonuclease activity, human pol ␤ carries out DNA repair synthesis by filling single nucleotide gaps (short patch BER) or by strand displacement to create flaps of several nucleotides (long patch BER) (14).…”

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confidence: 99%

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Weak Strand Displacement Activity Enables Human DNA Polymerase β to Expand CAG/CTG Triplet Repeats at Strand Breaks

Hartenstine

Goodman

Petruska

2002

Journal of Biological Chemistry

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Using synthetic DNA constructs in vitro, we find that human DNA polymerase ␤ effectively catalyzes CAG/ CTG triplet repeat expansions by slippage initiated at nicks or 1-base gaps within short (14 triplet) repeat tracts in DNA duplexes under physiological conditions. In the same constructs, Escherichia coli DNA polymerase I Klenow Fragment exo ؊ is much less effective in expanding repeats, because its much stronger strand displacement activity inhibits slippage by enabling rapid extension through two downstream repeats into flanking non-repeat sequence. Polymerase ␤ expansions of CAG/CTG repeats, observed over a 32-min period at rates of ϳ1 triplet added per min, reveal significant effects of break type (nick versus gap), strand composition (CTG versus CAG), and dNTP substrate concentration, on repeat expansions at strand breaks. At physiological substrate concentrations (1-10 M of each dNTP), polymerase ␤ expands triplet repeats with the help of weak strand displacement limited to the two downstream triplet repeats in our constructs. Such weak strand displacement activity in DNA repair at strand breaks may enable short tracts of repeats to be converted into longer, increasingly mutable ones associated with neurological diseases.Lengthy expansions of triplet repeats in human DNA result in several neurological diseases (1). Triplets of bases repeated in tandem can form a variety of slipped structures (2), including single-strand hairpin loops observed in vitro (3-5) and in vivo (6). The slippery nature of tandemly repeated triplets may contribute to repeat expansions catalyzed by eukaryotic enzymes in DNA replication (7,8), repair (9), or recombination (10). The degree to which a given process or enzyme contributes to expansion mutations in humans is not entirely clear but likely depends on the repeat type involved. Most studies to date have concentrated on the role that hairpin folding may play in slippage-expansion with DNA polymerase during DNA replication where single-stranded regions are involved.The present study focuses on repeat expansions in doublestranded DNA repair contexts, at strand breaks in the form of simple nicks, and 1-base gaps. Human DNA polymerase ␤ (pol ␤), 1 having no proofreading (3Ј-exonuclease activity), is a logical choice for study in such contexts, since it has a well established role in DNA repair, notably base excision repair (BER) (11). Cells express pol ␤ independently of the cell cycle (12) but show tissue specific differences in pol ␤ expression, with particularly high levels of expression occurring in testis (13). During BER, following glycosylase and AP endonuclease activity, human pol ␤ carries out DNA repair synthesis by filling single nucleotide gaps (short patch BER) or by strand displacement to create flaps of several nucleotides (long patch BER) (14). As observed in vitro under physiological salt conditions, pol ␤ itself has weak strand displacement DNA synthesis activity (15, 16) that is stimulated in a cooperative fashion by poly(ADP-ribose) polymerase-1 (PARP-1) and f...

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Section: Resultsmentioning

confidence: 99%

Section: Dna Sequences Used For Nick and Gap Constructs-two 72-mermentioning

confidence: 99%

mentioning

confidence: 99%

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Weak Strand Displacement Activity Enables Human DNA Polymerase β to Expand CAG/CTG Triplet Repeats at Strand Breaks

Hartenstine

Goodman

Petruska

2002

Journal of Biological Chemistry

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“…P-polymerase mRNA level was highest 4 h after exposure of cells to MMS and remained elevated for several hours after No P-polymerase mRNA increase was found; an example of this with UV irradiation is shown in Table 2. Finally, we examined the effect of MNNG and MMS treatment on the level of ,-polymerase protein in the crude cell extract by using activity gel analysis (12,14). During the 12-h period after the addition of MNNG (Fig.…”

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confidence: 99%

Induction of beta-polymerase mRNA by DNA-damaging agents in Chinese hamster ovary cells.

Fornace¹,

Zmudzka²,

Hollander³

et al. 1989

Mol. Cell. Biol.

151

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Studies of mammalian cells with DNA polymerase inhibitors have suggested that 3-polymerase has a role in DNA repair (1,3,13,18,24), and the in vitro activity of purified ,-polymerase points to an in vivo role in resynthesis of short gaps after damaged residues have been excised (4,9,15,19,21,22). Lesions produced by several DNA-damaging agents are repaired by single-base excision or short-gap excision followed by resynthesis to fill the gap (8). It is unknown whether regulation of P-polymerase gene expression is correlated with DNA repair, although it is clear that levels of 3-polymerase mRNA do not correlate with stages of the cell cycle or with replication of genomic DNA (25a). A rodent cDNA for ,-polymerase was isolated recently (26), and we have used this DNA as a probe to study regulation of P-polymerase mRNA in Chinese hamster ovary (CHO) cells after treatment with a variety of DNA-damaging agents.We found that the level of ,B-polymerase mRNA in exponentially growing CHO cells is elevated three-to fivefold after treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methyl methanesulfonate (MMS), or N-acetoxy-2-acetylaminofluorene (AAAF) (Fig. 1). The results of quantitative RNA dot blot analyses (6, 7) after treatment with high levels of these and other agents are summarized in Table 1. For reference, dot blots also were probed with

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“…There are differences in the properties of the , polymerases in various eukaryotes. For example, the polypeptide chain size of the enzyme from mammalian cells is 35-40 kDa, whereas the Drosophila, plant, and fungal enzymes range from about 70 to 110 kDa (7,8); the level of polymerase (3 activity in mammalian cells in culture remains constant during periods of change in growth rate and replication of genomic DNA (1,9), whereas levels of the Drosophila, plant, and fungal,8 polymerases are regulated as a function of developmental stage (7).…”

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confidence: 99%

Structure of rat DNA polymerase beta revealed by partial amino acid sequencing and cDNA cloning.

Zmudzka¹,

Sengupta²,

Matsukage³

et al. 1986

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

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A cDNA library of newborn rat brain poly(A)+ RNA in phage Xgtll was screened with a polyconal antibody against chicken DNA polymerase (3. One positive phage was isolated and purified after testing 2 x 107 recombinants. This phage, designated Xpolf8-10, contained an 1197-base-pair cDNA insert that corresponded to a mRNA with a poly(A) sequence at the 3' terminus and a single, long openreading frame of 957 bases. The open-reading frame, starting 44 residues from the 5' end of the cDNA, predicted a 36,375-Da protein of 318 amino acids. Comparison of this deduced amino acid sequence with the partial sequence obtained with purified polymerase (3 revealed a match of six tryptic peptides, involving a total of 47 amino acid residues. This confirmed the identity of the cDNA. Blot-hybridization analysis of newborn rat brain poly(A)+ RNA revealed a mRNA species of approximately the same size as the cDNA insert; in addition, a second mRNA species -4000 bases long was detected. Computer-derived secondary structure analysis of the enzyme predicted seven regions of a-helix distributed throughout and three regions of P-sheet.DNA polymerase A is the simplest naturally occurring DNA polymerase known. The purified enzyme is unable to conduct processive DNA synthesis, lacks associated nuclease activity, and does not catalyze detectable levels of pyrophosphate exchange, pyrophosphorolysis or dNMP turnover; the enzyme from vertebrate sources is purified as a holoenzyme composed of a single -40-kDa polypeptide chain (1-3). Although DNA polymerase ( is typified by the mammalian enzymes from calf and rodent tissues, polymerase-,(-like enzymes are present throughout the animal kingdom (4), and tryptic peptide mapping and immunological studies indicate that the primary structure of polymerase 3 in vertebrates is highly conserved (5, 6). In addition to animals, polymerase-(3-like enzymes have been found in plants, fungi, and protozoa (for discussion, see ref. 7). There are differences in the properties of the , polymerases in various eukaryotes. For example, the polypeptide chain size of the enzyme from mammalian cells is 35-40 kDa, whereas the Drosophila, plant, and fungal enzymes range from about 70 to 110 kDa (7,8); the level of polymerase (3 activity in mammalian cells in culture remains constant during periods of change in growth rate and replication of genomic DNA (1, 9), whereas levels of the Drosophila, plant, and fungal,8 polymerases are regulated as a function of developmental stage (7).The cellular role of DNA polymerase (3 is not fully understood. As noted above, levels of the enzyme do not correlate with replication of genomic DNA in a mammalian cell, and the enzyme has been described as constitutive (1, 4). This implies that polymerase A is involved in cellular maintenance, such as DNA repair, rather than in replicative DNA synthesis. Indeed, several groups have reported that after DNA damage of mammalian cells by bleomycin or neocarzinostatin, polymerase 8 has a synthetic role in DNA repair (10, 11). Purified polymer...

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Measurement of DNA polymerase β in skin fibroblast cell lines from patients with ataxia telangiectasia

Cited by 10 publications

References 20 publications

Weak Strand Displacement Activity Enables Human DNA Polymerase β to Expand CAG/CTG Triplet Repeats at Strand Breaks

Weak Strand Displacement Activity Enables Human DNA Polymerase β to Expand CAG/CTG Triplet Repeats at Strand Breaks

Induction of beta-polymerase mRNA by DNA-damaging agents in Chinese hamster ovary cells.

Structure of rat DNA polymerase beta revealed by partial amino acid sequencing and cDNA cloning.

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