Purification and substrate specificity of polydeoxyribonucleotide kinases isolated from calf thymus and rat liver

Karimi‐Busheri, Feridoun; Weinfeld, Michael

doi:10.1002/(sici)1097-4644(199702)64:2<258::aid-jcb9>3.0.co;2-w

Cited by 34 publications

(48 citation statements)

References 30 publications

(37 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…a 3Ј-phosphate and 5Ј-hydroxyl. Indeed, the preferred substrate of mammalian PNK is a DNA nick (18), and its enzyme activities are stimulated by interaction with the XRCC1 repair protein (19), strongly suggesting a role in BER/single-strand break repair.…”

mentioning

confidence: 99%

Uncoupling of 3′-Phosphatase and 5′-Kinase Functions in Budding Yeast

Vance

Wilson

2001

Journal of Biological Chemistry

View full text Add to dashboard Cite

Polynucleotide kinase is a bifunctional enzyme containing both DNA 3-phosphatase and 5-kinase activities seemingly suited to the coupled repair of singlestrand nicks in which the phosphate has remained with the 3-base. We show that the yeast Saccharomyces cerevisiae is able to repair transformed dephosphorylated linear plasmids by non-homologous end joining with considerable efficiency independently of the end-processing polymerase Pol4p. Homology searches and biochemical assays did not reveal a 5-kinase that would account for this repair, however. Instead, open reading frame YMR156C (here named TPP1) is shown to encode only a polynucleotide kinase-type 3-phosphatase. Tpp1p bears extensive similarity to the ancient L-2-haloacid dehalogenase and DDDD phosphohydrolase superfamilies, but is specific for double-stranded DNA. It is present at high levels in cell extracts in a functional form and so does not represent a pseudogene. Moreover, the phosphatase-only nature of this gene is shared by Saccharomyces mikatae YMR156C and Arabidopsis thaliana K15M2.3. Repair of 3-phosphate and 5-hydroxyl lesions is thus uncoupled in budding yeast as compared with metazoans. Repair of transformed dephosphorylated plasmids, and 5-hydroxyl blocking lesions more generally, likely proceeds by a cycle of base removal and resynthesis.Oxidative damage to DNA can result from endogenously generated reactive oxygen species or from exposure to exogenous agents such as ionizing radiation or anticancer agents such as bleomycin and neocarzinostatin (reviewed in Ref. 1). Such damage and the enzymes involved in its repair frequently produce fragmentation of the deoxyribose sugar backbone, resulting in DNA strand breaks bearing abnormal structures at the 3Ј and 5Ј termini. These are termed "blocking lesions" because they prevent the reactions necessary to achieve final repair of the damaged strand, namely polymerization and ligation. Since both single-and double-strand lesions can occur with potential consequences that include replication failure and genomic rearrangement, the resolution of blocking lesions is of major importance in genome maintenance. Although many chemical forms are possible, important blocking lesions on 5Ј termini include hydroxyls and deoxyribose phosphates. As an example of the redundancies in end processing, deoxyribose phosphate moieties can be removed in short-patch base excision repair (BER) 1 by the lyase function of DNA polymerase ␤ (2, 3) or certain glycosylases (4, 5) or in long-patch BER by flap excision and resynthesis (6). The extensive 5Ј-resection that occurs in the first steps of recombination also likely removes blocking lesions at double-strand breaks (7,8). Common blocking lesions on 3Ј termini include phosphates, ␣,␤-unsaturated aldehydes resulting from ␤-elimination reactions (9), and phosphoglycolate moieties that are the primary product of bleomycin action (10). The most potent 3Ј-processing enzymes are the apurinic-apyrimidinic endonucleases, which, in addition to cleaving strands at abasic sites, po...

show abstract

mentioning

confidence: 99%

Uncoupling of 3′-Phosphatase and 5′-Kinase Functions in Budding Yeast

Vance

Wilson

2001

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Omega Pnkp is catholic in its ability to label all three types of ends with similar efficacy. Note that none of the phage Pnkp enzymes evinced the bias against a 5Ј single strand overhang and the accompanying preference for a recessed 5Ј-OH end that are characteristic of the mammalian DNA-specific kinase (32). The crystal structure of the T4 kinase reveals a tunnel-like aperture leading to the 3Ј-phosphate binding site of the phosphate acceptor that appears to be too narrow to allow facile ingress of duplex nucleic acid but can readily accommodate a single-stranded polynucleotide (15,16,56).…”

Section: Discussionmentioning

confidence: 99%

“…T4 Pnkp prefers to phosphorylate RNAs or DNAs with 5Ј single strand extensions and is poorly active on blunt duplex 5Ј-OH DNA termini or 5Ј-OH DNA termini that are recessed within duplex regions or at the junction of a 3Ј single strand tail. In contrast, the mammalian kinase preferentially phosphorylates 5Ј-ends recessed within duplex DNA structures (32).…”

Section: Omega and Cjw1 Pnkp Have Distinct Preferences For Protrudingmentioning

confidence: 99%

“…Mycobacteriophage Pnkp Proteins Phosphorylate 5Ј-OH RNAs-The 5Ј-RNA kinase activity of Cjw1 Pnkp was demonstrated by the transfer of 32 P i from [␥- 32 P]ATP to the 5Ј-OH terminus of a synthetic RNA oligonucleotide to form a 5Ј-32 Plabeled RNA product that was resolved from free ATP by polyacrylamide gel electrophoresis (Fig. 7).…”

Section: New Bacteriophage Homologs Of T4 Pnkp-mentioning

confidence: 99%

“…The mammalian RNA kinase has an alkaline pH optimum (30), whereas the DNA-specific kinase has a characteristic acidic pH optimum (27)(28)(29). Whereas the human RNA kinase does not have an associated 3Ј-phosphatase (30), the eukaryal DNA kinases do have an intrinsic DNA-specific 3Ј-phosphatase function (31)(32)(33). In metazoans and fission yeast, such bifunctional DNA-specific Pnkp enzymes assist in the repair of DNA damage induced by oxidation, radiation, and topoisomerase I poisons (34 -37).…”

mentioning

confidence: 99%

See 2 more Smart Citations

Characterization of Polynucleotide Kinase/Phosphatase Enzymes from Mycobacteriophages Omega and Cjw1 and Vibriophage KVP40

Zhu

Yin

Shuman

2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

Coliphage T4 Pnkp is a bifunctional polynucleotide 5 -kinase/3 -phosphatase that catalyzes the end-healing steps of a RNA repair pathway. Here we show that mycobacteriophages Omega and Cjw1 and vibriophage KVP40 also encode bifunctional Pnkp enzymes consisting of a proximal 5 -kinase module with an essential P-loop motif, GXGK(S/T), and a distal 3 -phosphatase module with an essential acyl-phosphatase motif, DX-DGT. Biochemical characterization of the viral Pnkp proteins reveals several shared features, including an alkaline pH optimum for the kinase component, an intrinsic RNA kinase activity, and a homotetrameric or homodimeric quaternary structure, that distinguish them from the monomeric DNA-specific phosphatase/ kinase enzymes found in mammals and fission yeast. Whereas the phage 5 -kinases differ from each other in their preferences for phosphorylation of 5 overhangs, blunt ends, or recessed ends, none of them displays the preference for recessed ends reported for mammalian DNA kinase. We hypothesize that Pnkp provides phages that have it with a means to evade an RNA-damaging antiviral host response. Genetic complementation of the essential end-healing steps of yeast tRNA splicing by the Omega and Cjw1 Pnkp enzymes establishes their capacity to perform RNA repair reactions in vivo. A supportive correlation is that Omega and Cjw1, which are distinguished from other mycobacteriophages by their possession of a Pnkp enzyme, are also unique among the mycobacteriophages in their specification of putative RNA ligases.Breaks in the phosphodiester backbone of genomic DNA or essential RNA molecules can lead to cell death if not repaired. Such breaks can be triggered by physical or chemical insults, enzymatic hydrolysis, exposure to cytotoxic drugs, etc. Although DNA repair pathways have been the focus of much attention in light of the connections between DNA damage, mutagenesis, and cancer, there is an emerging appreciation that distinctive pathways exist to maintain or manipulate RNA structure in response to breakage events. Viruses provided the initial sources for the identification and characterization of enzymes that rectify DNA and RNA breaks (1-12) and viral model systems continue to illuminate the mechanisms, atomic structures, and biological roles of such enzymes (13-17).When breakage of a 3Ј-5Ј phosphodiester results in the formation of 5Ј-PO 4 and 3Ј-OH termini at the break site, the ends can be rejoined to each other (or to novel partner strands) by the action of DNA-specific or RNA-specific polynucleotide ligases. However, when breakage occurs with the opposite polarity, resulting in 5Ј-OH and 3Ј-PO 4 (or 2Ј,3Ј-cyclic PO 4 ) termini, the broken ends must be "healed" before they can be sealed. Healing entails two steps: (i) hydrolysis of the 3Ј-PO 4 (or 2Ј,3Ј-cyclic phosphate) to form a 3Ј-OH and (ii) phosphorylation of the 5Ј-OH to form a 5Ј-PO 4 end.The bacteriophage T4 proteins polynucleotide kinase/phosphatase (Pnkp) and RNA ligase 1 (Rnl1) are the prototypal RNA healing and sealing enzymes (1-3, 8 -1...

show abstract

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

et al. 1999

View full text Add to dashboard Cite

Mammalian polynucleotide kinases (PNKs) carry out 5'-phosphorylation of nucleic acids. Although the cellular function(s) of these enzymes remain to be delineated, important suggestions have included a role in DNA repair and, more recently, in DNA replication. Like T4 PNK, some preparations of mammalian PNKs have been reported to have an associated 3'-phosphatase activity. Previously, we have identified in calf thymus glands an apparently novel PNK with a neutral to alkaline pH optimum that lacked 3'-phosphatase activity. In this report, we describe purification of another bovine PNK, SNQI-PNK, with a slightly acidic pH optimum that copurifies with a 3'-phosphatase activity. The enzyme appears to be a monomer of 60 kDa. Mammalian DNA replication reactions were supplemented with T4 PNK or SNQI-PNK, and no significant effect on DNA replication in vitro was observed. Database searches support the earlier mapping of the 3'-phosphatase activity of T4 PNK to the C-terminus and suggest that the 3'-phosphatase domain of T4 PNK is related to the protein superfamily of L-2-haloacid dehalogenases. Exopeptidase digestion experiments were carried out to compare the SNQI-PNK enzyme with T4 PNK and led to the inference that the domain organization of the bovine polypeptide may differ from that of the T4 enzyme.

show abstract

Purification and substrate specificity of polydeoxyribonucleotide kinases isolated from calf thymus and rat liver

Cited by 34 publications

References 30 publications

Uncoupling of 3′-Phosphatase and 5′-Kinase Functions in Budding Yeast

Uncoupling of 3′-Phosphatase and 5′-Kinase Functions in Budding Yeast

Characterization of Polynucleotide Kinase/Phosphatase Enzymes from Mycobacteriophages Omega and Cjw1 and Vibriophage KVP40

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

Contact Info

Product

Resources

About