Purification of a polynucleotide kinase from calf thymus, comparison of its 3′-phosphatase domain with T4 polynucleotide kinase, and investigation of its effect on DNA replication in vitro

Jilani, Arshad; Slack, Carolyn; Matheos, Diamanto; Zannis‐Hadjopoulos, Maria; Lasko, Dana D.

doi:10.1002/(sici)1097-4644(19990501)73:2<188::aid-jcb5>3.0.co;2-h

Cited by 22 publications

(5 citation statements)

References 55 publications

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“…The striking difference is that none of the eukaryotic Pnk proteins contains a basic side chain at the position corresponding to Arg38 of T4 Pnk, an essential residue that makes a bidentate contact with the 3′ phosphate of the terminal nucleotide of the phosphate acceptor. The existence of a dedicated electrostatic interaction with the 3′ phosphate almost certainly accounts for the ability of T4 Pnk to phosphorylate 3′ NMPs, whereas the lack of an equivalent arginine in the mammalian Pnk suffices to explain its failure to utilize 3′ dNMP as a substrate (Jilani et al ., 1999b). Indeed, mammalian Pnk requires that the phosphate acceptor have a minimum chain length of approximately eight deoxynucleotides for the kinase reaction to occur (Karimi‐Busheri and Weinfeld, 1997).…”

Section: Resultsmentioning

confidence: 99%

“…Other fundamental differences in the phosphate acceptor specificities of T4 versus mammalian Pnks are clarified by the structure of the T4 domain. Although T4 Pnk is widely used to label DNA 5′ ends in vitro , it is actually quite selective in phosphorylating DNAs with 5′ single‐strand extensions; T4 Pnk is poorly active or inactive on blunt duplex 5′ OH termini or 5′ OH termini that are recessed within duplex regions or at the junction of a 3′ single‐strand tail (Jilani et al ., 1999b). The crystal structure of the T4 kinase domain indicates that the tunnel aperture leading to the 3′ phosphate binding site of the phosphate acceptor (Figure 3) is too narrow to allow facile ingress of duplex nucleic acid, but can readily accommodate a single‐stranded polynucleotide.…”

Section: Resultsmentioning

confidence: 99%

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Structure and mechanism of T4 polynucleotide kinase: an RNA repair enzyme

2002

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T4 polynucleotide kinase (Pnk), in addition to being an invaluable research tool, exempli®es a family of bifunctional enzymes with 5¢-kinase and 3¢-phosphatase activities that play key roles in RNA and DNA repair. T4 Pnk is a homotetramer composed of a C-terminal phosphatase domain and an N-terminal kinase domain. The 2.0 A Ê crystal structure of the isolated kinase domain highlights a tunnel-like active site through the heart of the enzyme, with an entrance on the 5¢ OH acceptor side that can accommodate a single-stranded polynucleotide. The active site is composed of essential side chains that coordinate the b phosphate of the NTP donor and the 3¢ phosphate of the 5¢ OH acceptor, plus a putative general acid that activates the 5¢ OH. The structure rationalizes the different speci®cities of T4 and eukaryotic Pnk and suggests a model for the assembly of the tetramer.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Structure and mechanism of T4 polynucleotide kinase: an RNA repair enzyme

2002

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“…Because Ku has been implicated in mammalian DNA replication (de Vries et al, 1989;Toth et al, 1993;Araujo et al, 1999;Ruiz et al, 1999;our unpublished results), in vitro DNA replication experiments were performed with the use of extracts prepared from both the Ku80 ϩ/ϩ or Ku80 Ϫ/Ϫ MEFs ( Figure 7C) in a mammalian in vitro replication system (Pearson et al, 1991;Zannis-Hadjopoulos et al, 1994;Diaz-Perez et al, 1996, 1998Matheos et al, 1998;Jilani et al, 1999;Ruiz et al, 1999). Approximately a 70% decrease in in vitro DNA replication was observed when the Ku80 Ϫ/Ϫ extracts were used, compared with the Ku80 ϩ/ϩ extracts.…”

Section: Replication Activity and Ku Immunoprecipitation From Ku80 ؉/mentioning

confidence: 99%

In Vivo Association of Ku with Mammalian Origins of DNA Replication

Novac

Matheos

Araujo

et al. 2001

MBoC

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Ku is a heterodimeric (Ku70/86-kDa) nuclear protein with known functions in DNA repair, V(D)J recombination, and DNA replication. Here, the in vivo association of Ku with mammalian origins of DNA replication was analyzed by studying its association with ors8 and ors12, as assayed by formaldehyde cross-linking, followed by immunoprecipitation and quantitative polymerase chain reaction analysis. The association of Ku with ors8 and ors12 was also analyzed as a function of the cell cycle. This association was found to be approximately fivefold higher in cells synchronized at the G1/S border, in comparison with cells at G0, and it decreased by approximately twofold upon entry of the cells into S phase, and to near background levels in cells at G2/M phase. In addition, in vitro DNA replication experiments were performed with the use of extracts from Ku80(+/+) and Ku80(-/-) mouse embryonic fibroblasts. A decrease of approximately 70% in in vitro DNA replication was observed when the Ku80(-/-) extracts were used, compared with the Ku80(+/+) extracts. The results indicate a novel function for Ku as an origin binding-protein, which acts at the initiation step of DNA replication and dissociates after origin firing.

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“…This processing likely reflects the action of PNKP, which is recruited to DSBs by XRCC4 (40), and whose phosphatase activity is highly specific for 3′ DNA ends (41). However, the surprising lack of effect of PNKP knockdown and inhibition raises the possibility of an alternative mode of phosphate removal.…”

Section: Discussionmentioning

confidence: 99%

Tracking the processing of damaged DNA double-strand break ends by ligation-mediated PCR: increased persistence of 3′-phosphoglycolate termini in SCAN1 cells

Akopiants

Mohapatra

Menon

et al. 2013

Nucleic Acids Research

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To track the processing of damaged DNA double-strand break (DSB) ends in vivo, a method was devised for quantitative measurement of 3′-phosphoglycolate (PG) termini on DSBs induced by the non-protein chromophore of neocarzinostatin (NCS-C) in the human Alu repeat. Following exposure of cells to NCS-C, DNA was isolated, and labile lesions were chemically stabilized. All 3′-phosphate and 3′-hydroxyl ends were enzymatically capped with dideoxy termini, whereas 3′-PG ends were rendered ligatable, linked to an anchor, and quantified by real-time Taqman polymerase chain reaction. Using this assay and variations thereof, 3′-PG and 3′-phosphate termini on 1-base 3′ overhangs of NCS-C-induced DSBs were readily detected in DNA from the treated lymphoblastoid cells, and both were largely eliminated from cellular DNA within 1 h. However, the 3′-PG termini were processed more slowly than 3′-phosphate termini, and were more persistent in tyrosyl-DNA phosphodiesterase 1-mutant SCAN1 than in normal cells, suggesting a significant role for tyrosyl-DNA phosphodiesterase 1 in removing 3′-PG blocking groups for DSB repair. DSBs with 3′-hydroxyl termini, which are not directly induced by NCS-C, were formed rapidly in cells, and largely eliminated by further processing within 1 h, both in Alu repeats and in heterochromatic α-satellite DNA. Moreover, absence of DNA-PK in M059J cells appeared to accelerate resolution of 3′-PG ends.

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Purification of a polynucleotide kinase from calf thymus, comparison of its 3′-phosphatase domain with T4 polynucleotide kinase, and investigation of its effect on DNA replication in vitro

Cited by 22 publications

References 55 publications

Structure and mechanism of T4 polynucleotide kinase: an RNA repair enzyme

Structure and mechanism of T4 polynucleotide kinase: an RNA repair enzyme

In Vivo Association of Ku with Mammalian Origins of DNA Replication

Tracking the processing of damaged DNA double-strand break ends by ligation-mediated PCR: increased persistence of 3′-phosphoglycolate termini in SCAN1 cells

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