The phospholipase D (PLD) superfamily is a diverse group of proteins that includes enzymes involved in phospholipid metabolism, a bacterial toxin, poxvirus envelope proteins, and bacterial nucleases. Based on sequence comparisons, we show here that the tyrosyl-DNA phosphodiesterase (Tdp1) that has been implicated in the repair of topoisomerase I covalent complexes with DNA contains two unusual HKD signature motifs that place the enzyme in a distinct class within the PLD superfamily. Mutagenesis studies with the human enzyme in which the invariant histidines and lysines of the HKD motifs are changed confirm that these highly conserved residues are essential for Tdp1 activity. Furthermore, we show that, like other members of the family for which it has been examined, the reaction involves the formation of an intermediate in which the cleaved substrate is covalently linked to the enzyme. These results reveal that the hydrolytic reaction catalyzed by Tdp1 occurs by the phosphoryl transfer chemistry that is common to all members of the PLD superfamily.T he members of the phospholipase D (PLD) superfamily comprise a highly diverse group of proteins that include plant, mammalian and bacterial PLDs, bacterial phosphatidylserine and cardiolipin synthases, a bacterial toxin, several poxvirus envelope proteins, and some bacterial nucleases (1-3). Sequence alignments reveal that with the exception of two nucleases (1) the proteins arose as a result of a gene duplication event with each half of the protein containing four repeated motifs. Motifs 3 and 4 contain the highly conser ved HxK(x) 4 D(x) 6 GSxN sequence, termed the HKD motif (4), which has been implicated in the catalytic mechanism (see below). The crystal structure of the Salmonella typhimurium Nuc protein (4), one of the bacterial nucleases, shows that the active form of the enzyme is a dimer with the HKD motifs contributed by each subunit organized in roughly the same spatial arrangement as the two HKD motifs found in the crystal structure of the monomeric PLD from Streptomyces sp. strain PMF (5).For those members of the superfamily known to have catalytic activity, the enzymatic reactions all involve phosphoryl transfer from a donor to an acceptor that is either an alcohol or water. In the cases of the phospholipid synthases, a phosphatidyl moiety is transferred either from a cytidine 5Ј-diphosphate-diacylglycerol donor to serine or from phosphatidylglycerol to another phosphatidylglycerol to synthesize phosphatidylserine or cardiolipin, respectively. The PLDs either hydrolyze the phosphodiester bond in the phospholipid to produce phosphatidic acid and a free head group (often choline) or catalyze the exchange of one head group for another (transphosphatidylation). The nucleases appear to simply catalyze the hydrolysis of DNA phosphodiester bonds.The similar chemistry underlying these reactions suggests that the enzymes in the superfamily share a similar catalytic mechanism. Early evidence showing retention of configuration at the substrate phosphorous in the re...
Cds1, a serine/threonine kinase, enforces the S-M checkpoint in the fission yeast Schizosaccharomyces pombe. Cds1 is required for survival of replicational stress caused by agents that stall replication forks, but how Cds1 performs these functions is largely unknown. Here we report that the forkhead-associated-1 (FHA1) proteindocking domain of Cds1 interacts with Mus81, an evolutionarily conserved damage tolerance protein. Mus81 has an endonuclease homology domain found in the XPF nucleotide excision repair protein. Inactivation of mus81 reveals a unique spectrum of phenotypes. Mus81 enables survival of deoxynucleotide triphosphate starvation, UV radiation, and DNA polymerase impairment. Mus81 is essential in the absence of Bloom's syndrome Rqh1 helicase and is required for productive meiosis. Genetic epistasis studies suggest that Mus81 works with recombination enzymes to properly replicate damaged DNA. Inactivation of Mus81 triggers a checkpoint-dependent delay of mitosis. We propose that Mus81 is involved in the recruitment of Cds1 to aberrant DNA structures where Cds1 modulates the activity of damage tolerance enzymes.Genome integrity is vulnerable during DNA replication. The act of replication can convert a relatively benign single-strand DNA break to a cytotoxic double-strand break. A cyclobutane dimer, formed by UV irradiation, is sufficient to block the progression of DNA polymerases and to cause replication fork collapse (17). To cope with these problems, eukaryotic organisms have developed mechanisms for replicating DNA through and around damage in ways that cause minimal genome instability. Notable among the proteins involved in these processes are lesion bypass polymerases that can replicate through cyclobutane dimers (45). Recombination and double-strand break repair enzymes are also important, participating in the direct repair of DNA structure abnormalities that arise during DNA replication or permitting continued replication around sites of damage (17). These systems, and perhaps others that remain undiscovered, collectively form a genome defense system that allows tolerance of damage during DNA replication.In the fission yeast Schizosaccharomyces pombe, the checkpoint kinase Cds1 is thought to regulate DNA damage tolerance systems (26,28,31). Cds1 is the presumptive homolog of Rad53 in the budding yeast Saccharomyces cerevisiae and Cds1 (also called Chk2) in humans (7,9,16,27). One of its functions is to delay the onset of mitosis when genome replication encounters difficulties (8,26,47). Cds1 enforces this S-M checkpoint by regulating the Cdc25 and Mik1 mitotic control proteins (2,8,18,47). In addition, Cds1 regulates the way in which DNA is replicated under conditions that cause replicational stress. For example, Cds1 is required to slow the replication of DNA that has been damaged by UV irradiation (26,34). A similar function has been ascribed to Rad53 in budding yeast (32). Slowing of DNA replication partly involves the suppression of late-firing replication origins (35, 37). Activation of C...
Human tyrosyl-DNA phosphodiesterase (Tdp1) hydrolyzes the phosphodiester bond between a DNA 3 end and a tyrosyl moiety. In eukaryotic cells, this type of linkage is found in stalled topoisomerase I-DNA covalent complexes, and Tdp1 has been implicated in the repair of such complexes in vivo. We confirm here that the Tdp1 catalytic cycle involves a covalent reaction intermediate in which a histidine residue is connected to a DNA 3-phosphate through a phosphoamide linkage. Most surprisingly, this linkage can be hydrolyzed by Tdp1, and unlike a topoisomerase I-DNA complex, which requires modification to be an efficient substrate for Tdp1, the native form of Tdp1 can be removed from the DNA. The spinocerebellar ataxia with axonal neuropathy neurodegenerative disease is caused by the H493R mutant form of Tdp1, which shows reduced enzymatic activity and accumulates the Tdp1-DNA covalent intermediate. The ability of wild type Tdp1 to remove the stalled mutant protein from the DNA likely explains the recessive nature of spinocerebellar ataxia with axonal neuropathy. In addition to its activity on phosphotyrosine and phosphohistidine substrates, Tdp1 also possesses a limited DNA and RNA 3-exonuclease activity in which a single nucleoside is removed from the 3-hydroxyl end of the substrate. Furthermore, Tdp1 also removes a 3 abasic site and an artificial 3-biotin adduct from the DNA. In combination with earlier data showing that Tdp1 can use 3-phosphoglycolate as a substrate, these data suggest that Tdp1 may function to remove a variety of 3 adducts from DNA during DNA repair.Tyrosyl-DNA phosphodiesterase (Tdp1) 2 was discovered as an enzymatic activity from Saccharomyces cerevisiae that specifically hydrolyzes the phosphodiester linkage between the O-4 atom of a tyrosine and a DNA 3Ј-phosphate (1). This type of linkage is typical of the covalent reaction intermediate produced when a type IB topoisomerase cleaves one strand of DNA. Type IB topoisomerases are ubiquitous enzymes that perform essential functions in key cellular processes such as replication, recombination, and transcription (2-6). Following cleavage and DNA relaxation, the topoisomerase normally religates the DNA strand and dissociates from the DNA (4). After cleavage near certain DNA lesions or modified nucleotides, or in the presence of the anticancer drug camptothecin, religation is blocked, and the topoisomerase becomes covalently trapped on the 3Ј end of the cleaved DNA strand (7-10). At the time of the discovery of Tdp1, Yang et al.(1) suggested that the enzyme might be involved in the removal of such covalently stalled topoisomerase I molecules from the DNA. At present, extensive genetic, biochemical, and cell biological data support the view that Tdp1 is involved in the repair of topoisomerase I-DNA covalent lesions in eukaryotic cells (11)(12)(13)(14)(15)(16).Tdp1 is a member of the phospholipase D (PLD) superfamily, which is characterized by two "HKD" motifs that provide the conserved active site residues (17). The diverse members of this family ful...
Tyrosyl-DNA phosphodiesterase (Tdp1) catalyzes the hydrolysis of the tyrosyl-3 0 phosphate linkage found in topoisomerase I-DNA covalent complexes. The inherited disorder, spinocerebellar ataxia with axonal neuropathy (SCAN1), is caused by a H493R mutation in Tdp1. Contrary to earlier proposals that this disease results from a loss-of-function mutation, we show here that this mutation reduces enzyme activity B25-fold and importantly causes the accumulation of the Tdp1-DNA covalent reaction intermediate. Thus, the attempted repair of topoisomerase I-DNA complexes by Tdp1 unexpectedly generates a new protein-DNA complex with an apparent half-life of B13 min that, in addition to the unrepaired topoisomerase I-DNA complex, may interfere with transcription and replication in human cells and contribute to the SCAN1 phenotype. The analysis of Tdp1 mutant cell lines derived from SCAN1 patients reveals that they are hypersensitive to the topoisomerase I-specific anticancer drug camptothecin (CPT), implicating Tdp1 in the repair of CPT-induced topoisomerase I damage in human cells. This finding suggests that inhibitors of Tdp1 could act synergistically with CPT in anticancer therapy.
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