Potential roles of 3′-5′exonuclease activity of NM23-H1 in DNA repair and malignant progression

NM23-H1 is a metastasis suppressor protein that exhibits 3′-5′ exonuclease activity in vitro. As 3′-5′ exonucleases are generally required for maintenance of genome integrity, this activity represents a plausible candidate mediator of the metastasis suppressor properties of the NM23-H1 molecule. Consistent with an antimutator function, ablation of the yeast NM23 homolog, YNK1, results in increased mutation rates following exposure to UV irradiation and exposure to the DNA damaging agents etoposide, cisplatin, and MMS. In human cells, a DNA repair function is further suggested by increased NM23-H1 expression and nuclear translocation following DNA damage. Also, forced expression of NM23-H1 in NM23-deficient and metastatic cell lines results in coordinate downregulation of multiple DNA repair genes, possibly reflecting genomic instability associated with the NM23-deficient state. To assess the relevance of the 3′-5′ exonuclease activity of NM23-H1 to its antimutator and metastasis suppressor functions, a panel of mutants harboring defects in the 3′-5′ exonuclease and other enzymatic activities of the molecule (NDPK, histidine kinase) have been expressed by stable transfection in the melanoma cell line, 1205Lu. Pilot in vivo metastasis assays indicate 1205Lu cells are highly responsive to the metastasis suppressor effects of NM23-H1, thus providing a valuable model for measuring the extent to which the nuclease function opposes metastasis and metastatic progression.     

The metastasis suppressor NM23-H1 possesses 3'-5' exonuclease activity

NM23-H1 belongs to a family of eight gene products in humans that have been implicated in cellular differentiation and development, as well as oncogenesis and tumor metastasis. We have defined NM23-H1 biochemically as a 3'-5' exonuclease by virtue of its ability in stoichiometric amounts to excise single nucleotides in a stepwise manner from the 3' terminus of DNA. The activity is dependent upon the presence of Mg(2+), is most pronounced with single-stranded substrates or mismatched bases at the 3' terminus of double-stranded substrates, and is inhibited by both ATP and the incorporation of cordycepin, a 2'-deoxyadenosine analogue, into the 3'-terminal position. The 3'-5' exonuclease activity was assigned to NM23-H1 by virtue of: 1) precise coelution of enzymatic activity with wild-type and mutant forms of NM23-H1 protein during purification by hydroxylapatite and gel filtration column high performance liquid chromatography and 2) significantly diminished activity exhibited by purified recombinant mutant forms of the proteins. Lysine 12 appears to play an important role in the catalytic mechanism, as evidenced by the significant reduction in 3'-5' exonuclease activity resulting from a Lys(12) to glutamine substitution within the protein. 3'-5' Exonucleases are believed to play an important role in DNA repair, a logical candidate function underlying the putative antimetastatic and oncogenic activities of NM23-H1.     

YNK1, the yeast homolog of human metastasis suppressor NM23, is required for repair of UV radiation- and etoposide-induced DNA damage

In humans, NM23-H1 is a metastasis suppressor whose expression is reduced in metastatic melanoma and breast carcinoma cells, and which possesses the ability to inhibit metastatic growth without significant impact on the transformed phenotype. NM23-H1 exhibits three enzymatic activities in vitro, each with potential to maintain genomic stability, a 3'-5' exonuclease and two kinases, nucleoside diphosphate kinase (NDPK), and protein histidine kinase. Herein we have investigated the potential contributions of NM23 proteins to DNA repair in the yeast, Saccharomyces cerevisiae, which contains a single NM23 homolog, YNK1. Ablation of YNK1 delayed repair of UV- and etoposide-induced nuclear DNA damage by 3-6h. However, YNK1 had no impact upon the kinetics of MMS-induced DNA repair. Furthermore, YNK1 was not required for repair of mitochondrial DNA damage. To determine whether the nuclear DNA repair deficit manifested as an increase in mutation frequency, the CAN1 forward assay was employed. An YNK1 deletion was associated with increased mutation rates following treatment with either UV (2.6x) or MMS (1.6 x). Mutation spectral analysis further revealed significantly increased rates of base substitution and frameshift mutations following UV treatment in the ynk1Delta strain. This study indicates a novel role for YNK1 in DNA repair in yeast, and suggests an anti-mutator function that may contribute to the metastasis suppressor function of NM23-H1 in humans.     

Metastasis suppressor NME1 promotes non-homologous end joining of DNA double-strand breaks

NME1 (also known as NM23-H1) was the first identified tumor metastasis suppressor, which has been reported to link with genomic stability maintenance and cancer. However its underlying mechanisms are still not fully understood. Here we find that NME1 is required for non-homologous end joining (NHEJ) of DNA double-strand breaks (DSBs). Mechanistically, NME1 re-localizes to DNA damage sites in a Ku-XRCC4-dependent manner, and regulates downstream LIG4 recruitment and end joining efficiency. Furthermore, we show that the 3′-5′ exonuclease activity of NME1 is critical for its function in NHEJ. Taken together, our findings identify NME1 as a novel NHEJ factor, and reveal how this metastasis suppressor promotes genome stability.     

Chlamydophila pneumoniae endonuclease IV prefers to remove mismatched 3′ ribonucleotides: Implication in proofreading mismatched 3′-terminal nucleotides in short-patch repair synthesis

DNA polymerase I (DNApolI) catalyzes DNA synthesis during Okazaki fragment maturation, base excision repair, and nucleotide excision repair. Some bacterial DNApolIs are deficient in 3′–5′ exonuclease, which is required for removing an incorrectly incorporated 3′-terminal nucleotide during DNA elongation by DNA polymerase activity. The key amino acid residues in the exonuclease center of Chlamydophila pneumoniae DNApolI (CpDNApolI) are naturally mutated, resulting in the loss of 3′–5′ exonuclease. Hence, the manner by which CpDNApolI proofreads the incorrectly incorporated nucleotide during DNA synthesis warrants clarification. C. pneumoniae encodes three 3′–5′ exonuclease activities: one endonuclease IV and two homologs of the epsilon subunit of replicative DNA polymerase III. The three proteins were biochemically characterized using single- and double-stranded DNA substrate. Among them, C. pneumoniae endonuclease IV (CpendoIV) possesses 3′–5′ exonuclease activity that prefers to remove mismatched 3′-terminal nucleotides in the nick, gap, and 3′ recess of a double-stranded DNA (dsDNA). Finally, we reconstituted the proofreading reaction of the mismatched 3′-terminal nucleotide using the dsDNA with a nick or 3′ recess as substrate. Upon proofreading of the mismatched 3′-terminal nucleotide by CpendoIV, CpDNApolI can correctly reincorporate the matched nucleotide and the nick is further sealed by DNA ligase. Based on our biochemical results, we proposed that CpendoIV was responsible for proofreading the replication errors of CpDNApolI.     

The exonuclease TREX1 is in the SET complex and acts in concert with NM23-H1 to degrade DNA during granzyme A-mediated cell death

Granzyme A (GzmA) activates a caspase-independent cell death pathway with morphological features of apoptosis. Single-stranded DNA damage is initiated when the endonuclease NM23-H1 becomes activated to nick DNA after granzyme A cleaves its inhibitor, SET. SET and NM23-H1 reside in an endoplasmic reticulum-associated complex (the SET complex) that translocates to the nucleus in response to superoxide generation by granzyme A. We now find the 3'-to-5' exonuclease TREX1, but not its close homolog TREX2, in the SET complex. TREX1 binds to SET and colocalizes and translocates with the SET complex. NM23-H1 and TREX1 work in concert to degrade DNA. Silencing NM23-H1 or TREX1 inhibits DNA damage and death of cells treated with perforin (PFN) and granzyme A, but not of cells treated with perforin and granzyme B (GzmB). After granzyme A activates NM23-H1 to make single-stranded nicks, TREX1 removes nucleotides from the nicked 3' end to reduce the possibility of repair by rejoining the nicked ends.     

Multifunctional human apurinic/apyrimidinic endonuclease 1: Role of additional functions

Human apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is a multifunctional enzyme involved in base excision repair (BER). APE1 cleaves DNA 5′ of an AP site to produce a single-strand break with 5′-OH and 3′-deoxyribose phosphate. In addition to its AP-endonucleolytic function, APE1 possesses 3′-phosphodiesterase, 3′–5′ exonuclease, and 3′-phosphatase activities. Independently of its function as a repair protein, APE1 was identified as a redox factor (Ref-1). The review summarizes the published and original data on the role of the additional functions of APE1 in DNA repair and apoptosis and regulation of the BER system via APE1 interaction with DNA and other repair proteins.     

Tumor suppressor NM23-H1 is a granzyme A-activated DNase during CTL-mediated apoptosis,and the nucleosome assembly protein SET is its inhibitor

Granzyme A (GzmA) induces a caspase-independent cell death pathway characterized by single-stranded DNA nicks and other features of apoptosis. A GzmA-activated DNase (GAAD) is in an ER associated complex containing pp32 and the GzmA substrates SET, HMG-2, and Ape1. We show that GAAD is NM23-H1, a nucleoside diphosphate kinase implicated in suppression of tumor metastasis, and its specific inhibitor (IGAAD) is SET. NM23-H1 binds to SET and is released from inhibition by GzmA cleavage of SET. After GzmA loading or CTL attack, SET and NM23-H1 translocate to the nucleus and SET is degraded, allowing NM23-H1 to nick chromosomal DNA. GzmA-treated cells with silenced NM23-H1 expression are resistant to GzmA-mediated DNA damage and cytolysis, while cells overexpressing NM23-H1 are more sensitive.     

Cloning and characterization of NM23-Bbt2 gene from amphioxus Branchiostoma belcheri tsingtauense

A full-length amphioxus (Branchiostoma belcheri tsingtauense) NM23-Bbt2, NM23-H2 homologue, cDNA was isolated from the cDNA library and sequenced. The obtained amphioxus NM23-Bbt2 cDNA contains an open reading frame coding for 171 amino acids. Sequence analysis showed that the amphioxus NM23-Bbt2 was highly conserved with that of other species, and all of them contained highly conserved motifs that play important roles in the function of NM23. RT-PCR revealed that NM23-Bbt2 is expressed in the neuronal tissues and is expressed in all stages during the embryogenesis. Nucleoside kinases are thought to have a critical role in regulatory processes such as signal transduction, proliferation, and differentiation. Taken together, these results suggest that nucleoside diphosphate kinases have an important role to play in embryogenic development in amphioxus. Phylogenetic analysis showed that the amphioxus group 1 NDPKs (Bf1-4) may be precursors of the human group 1 NDPKs, NM23-Bf5, NM23-Bf6, NM23-Bf7 and NM23-Bf8 may be precursors of NM23-H5, NM23-H6, NM23-H7 and M23-H8, respectively. Our finding of nine NM23 genes in Branchiostoma floride, the precursor of vertebrates, strongly suggests that the ancestral gene corresponding to each of vertebrates NM23 genes generated before the appearance of vertebrates. Comparison of the gene structures of NM23-H2 homologue from invertebrates to vertebrates suggests that the locations of three of the four introns are conserved in amphioxus and vertebrates.     

Quantitative parameters of the 3′–5′ exonuclease reaction of human apurinic/apyrimidinic endonuclease 1 with nicked DNA containing dYMP or a modified dCMP analogue

Human apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is a multifunctional enzyme. In addition to its main AP endonuclease activity, that incises DNA 5′ to the AP-site, it possesses other weak enzymatic activities. One of them is 3′–5′ exonuclease activity, which is most effectively exhibited for DNA duplexes containing modified or mismatched nucleotides at the 3′-end of the primer chain. There is a presumption that APE1 can correct the DNA synthesis catalyzed by DNA polymerase β through the base excision repair process. We determined the quantitative parameters of the 3′–5′ exonuclease reaction in dependence on the reaction conditions to reveal the detailed mechanism of this process. The kinetic parameters of APE1 exonuclease excision of mismatched dCMP and dTMP from the 3′ terminus of single-strand DNA and of photoreactive dCMP analogues applied for photoaffinity modification of proteins and DNA in recombinant systems and cell/nuclear extracts were determined.     

The metastasis suppressor Nm23 as a modulator of Ras/ERK signaling

NM23-H1 (also known as NME1) was the first identified metastasis suppressor, which displays a nucleoside diphosphate kinase (NDPK) and histidine protein kinase activity. NDPKs are linked to many processes, such as cell migration, proliferation, differentiation, but the exact mechanism whereby NM23-H1 inhibits the metastatic potential of cancer cells remains elusive. However, some recent data suggest that NM23-H1 may exert its anti-metastatic effect by blocking Ras/ERK signaling. In mammalian cell lines NDPK-mediated attenuation of Ras/ERK signaling occurs through phosphorylation (thus inactivation) of KSR (kinase suppressor of Ras) scaffolds. In this review I summarize our knowledge about KSR’s function and its regulation in mammals and in C. elegans. Genetic studies in the nematode contributed substantially to our understanding of the function and regulation of the Ras pathway (i.e. KSR’s discovery is also linked to the nematode). Components of the RTK/Ras/ERK pathway seem to be highly conserved between mammals and worms. NDK-1, the worm homolog of NM23-H1 affects Ras/MAPK signaling at the level of KSRs, and a functional interaction between NDK-1/NDPK and KSRs was first demonstrated in the worm in vivo. However, NDK-1 is a factor, which is necessary for proper MAPK activation, thus it activates rather than suppresses Ras/MAPK signaling in the worm. The contradiction between results in mammalian cell lines and in the worm regarding NDPKs’ effect exerted on the outcome of Ras signaling might be resolved, if we better understand the function, structure and regulation of KSR scaffolds.     

Excision of gamma-ray damaged thymine by E. coli extracts is due to the 5'leads to 3' exonuclease associated with DNA polymerase I

Extracts of $\text{E. coli pol}$Aexl which contains a temperature sensitive 5′→3′ exonuclease function of polymerase I accomplish the selective excision of products of the 5,6-dihydroxy-dihydrothymine type from γ-irradiated DNA and OsO₄-oxidized polyd(A-T) at the permissive temperature (30°) but not at the nonpermissive temperature (42°). The 5′→3′ exonuclease activity of polymerase I, therefore, acts as a repair exonuclease in γ-ray excision repair.     

Wild-type NM23-H1, but not its S120 mutants, suppresses desensitization of muscarinic potassium current

NM23 (NDP kinase) modulates the gating of muscarinic K+ channels by agonists through a mechanism distinct from GTP regeneration. To better define the function of NM23 in this pathway and to identify sites in NM23 that are important for its role in muscarinic K+ channel function, we utilized MDA-MB-435 human breast carcinoma cells that express low levels of NM23-H1. M2 muscarinic receptors and GIRK1/GIRK4 channel subunits were co-expressed in cells stably transfected with vector only (control), wild-type NM23-H1, or several NM23-H1 mutants. Lysates from all cell lines tested exhibit comparable nucleoside diphosphate (NDP) kinase activity. Whole cell patch clamp recordings revealed a substantial reduction of the acute desensitization of muscarinic K+ currents in cells overexpressing NM23-H1. The mutants NM23-H1P96S and NM23-H1S44A resembled wild-type NM23-H1 in their ability to reduce desensitization. In contrast, mutants NM23-H1S120G and NM23-H1S120A completely abolished the effect of NM23-H1 on desensitization of muscarinic K+ currents. Furthermore, NM23-H1S120G potentiated acute desensitization, indicating that this mutant retains the ability to interact with the muscarinic pathway, but has properties antithetical to those of the wild-type protein. We conclude that NM23 acts as a suppressor of the processes leading to the desensitization of muscarinic K+ currents, and that Ser-120 is essential for its actions.