Genetic instability induced by overexpression of DNA ligase I in budding yeast

Recombination and microsatellite mutation in humans contribute to disorders including cancer and trinucleotide repeat (TNR) disease. TNR expansions in wild-type yeast may arise by flap ligation during lagging-strand replication. Here we show that overexpression of DNA ligase I (CDC9) increases the rates of TNR expansion, of TNR contraction, and of mitotic recombination. Surprisingly, this effect is observed with catalytically inactive forms of Cdc9p protein, but only if they possess a functional PCNA-binding site. Furthermore, in vitro analysis indicates that the interaction of PCNA with Cdc9p and Rad27p (Fen1) is mutually exclusive. Together our genetic and biochemical analysis suggests that, although DNA ligase I seals DNA nicks during replication, repair, and recombination, higher than normal levels can yield genetic instability by disrupting the normal interplay of PCNA with other proteins such as Fen1.     

Two distinct DNA ligase activities in mitotic extracts of the yeast Saccharomyces cerevisiae.

Four biochemically distinct DNA ligases have been identified in mammalian cells. One of these enzymes, DNA ligase I, is functionally homologous to the DNA ligase encoded by the Saccharomyces cerevisiae CDC9 gene. Cdc9 DNA ligase has been assumed to be the only species of DNA ligase in this organism. In the present study we have identified a second DNA ligase activity in mitotic extracts of S. cerevisiae with chromatographic properties different from Cdc9 DNA ligase, which is the major DNA joining activity. This minor DNA joining activity, which contributes 5-10% of the total cellular DNA joining activity, forms a 90 kDa enzyme-adenylate intermediate which, unlike the Cdc9 enzyme-adenylate intermediate, reacts with an oligo (pdT)/poly (rA) substrate. The levels of the minor DNA joining activity are not altered by mutation or by overexpression of the CDC9 gene. Furthermore, the 90 kDa polypeptide is not recognized by a Cdc9 antiserum. Since this minor species does not appear to be a modified form of Cdc9 DNA ligase, it has been designated as S. cerevisiae DNA ligase II. Based on the similarities in polynucleotide substrate specificity, this enzyme may be the functional homolog of mammalian DNA ligase III or IV.     

DNA ligation during excision repair in yeast cell-free extracts is specifically catalyzed by the CDC9 gene product

Excision repair of DNA is an important cellular response to DNA damage induced by radiation and many chemicals. In eukaryotes, base excision repair (BER) and nucleotide excision repair (NER) are two major excision repair pathways which are completed by a DNA ligation step. Using a cell-free system, we have determined the DNA ligase requirement during BER and NER of the yeast S. cerevisiae. Under nonpermissive conditions in extracts of the cdc9-2 temperature-sensitive mutant, DNA ligation in both BER and NER pathways was defective, and the repair patches were enlarged. At the permissive temperature (23 degrees C), DNA ligation during excision repair was only partially functional in the mutant extracts. In contrast, deleting the DNA ligase IV gene did not affect DNA ligation of BER or NER. Defective DNA ligation of BER and NER in cdc9-2 mutant extracts was complemented in vitro by purified yeast Cdc9 protein, but not by DNA ligase IV even when overexpressed. These results demonstrate that the ligation step of excision repair in yeast cell-free extracts is catalyzed specifically by the Cdc9 protein, the homologue of mammalian DNA ligase I.     

An error-prone viral DNA ligase

Our recent demonstration that DNA polymerase X (Pol X), the DNA repair polymerase encoded by the African swine fever virus (ASFV), is extremely error prone during single-nucleotide gap filling led us to hypothesize that it might contribute to genetic variability in ASFV. For the infidelity of Pol X to be relevant, however, the DNA ligase working downstream of it would need to be capable of sealing nicks containing 3'-OH mismatches. We therefore examined the nick ligation capabilities of the ASFV-encoded DNA ligase and here report the first complete 3' fidelity analysis, employing catalytic parameters, for any DNA ligase. The catalytic efficiency of nick sealing by both ASFV DNA ligase and bacteriophage T4 DNA ligase was determined in the steady state for substrates containing all 16 possible matched and mismatched base pair combinations at the 3' side of a nick. Our results indicate that ASFV DNA ligase is the lowest-fidelity DNA ligase ever reported, capable of ligating a 3' C:T mismatched nick (where C and T are the templating and nascent nucleotides, respectively) more efficiently than nicks containing Watson-Crick base pairs. Comparison of the mismatch specificity of Pol X with that of ASFV DNA ligase suggests that the latter may have evolved toward low fidelity for the purpose of generating the broadest possible spectrum of sealed mismatches. These findings are discussed in light of the genetic and antigenic variability observed among some ASFV isolates. Two novel assays for determining the concentration of active DNA ligase are also reported.     

Lif1p targets the DNA ligase Lig4p to sites of DNA double-strand breaks

DNA ligases catalyse the joining of DNA single- and double-strand breaks. Saccharomyces cerevisiae Cdc9p is a homologue of mammalian DNA ligase I and is required for DNA replication, recombination and single-strand break repair. The other yeast ligase, Lig4p/Dnl4p, is a homologue of mammalian DNA ligase IV, and functions in the non-homologous end-joining (NHEJ) pathway of DNA double-strand break repair [1] [2] [3] [4]. Lig4p interacts with Lif1p, the yeast homologue of the human ligase IV-associated protein, XRCC4 [5]. This interaction takes place through the carboxy-terminal domain of Lig4p and is required for Lig4p stability. We show that the carboxy-terminal interaction region of Lig4p is necessary for NHEJ but, when fused to Cdc9p, is insufficient to confer NHEJ function to Cdc9p. Also, Lif1p stimulates the in vitro catalytic activity of Lig4p in adenylation and DNA ligation. Nevertheless, Lig4p is inactive in NHEJ in the absence of Lif1p in vivo, even when Lig4p is stably expressed. We show that Lif1p binds DNA in vitro and, through in vivo cross-linking and chromatin immuno precipitation assays, demonstrate that it targets Lig4p to chromosomal DNA double-strand breaks. Furthermore, this targeting requires another key NHEJ protein, Ku.     

Identification of Saccharomyces cerevisiae DNA ligase IV: involvement in DNA double-strand break repair.

DNA ligases catalyse the joining of single and double-strand DNA breaks, which is an essential final step in DNA replication, recombination and repair. Mammalian cells have four DNA ligases, termed ligases I-IV. In contrast, other than a DNA ligase I homologue (encoded by CDC9), no other DNA ligases have hitherto been identified in Saccharomyces cerevisiae. Here, we report the identification and characterization of a novel gene, LIG4, which encodes a protein with strong homology to mammalian DNA ligase IV. Unlike CDC9, LIG4 is not essential for DNA replication, RAD52-dependent homologous recombination nor the repair of UV light-induced DNA damage. Instead, it encodes a crucial component of the non-homologous end-joining (NHEJ) apparatus, which repairs DNA double-strand breaks that are generated by ionizing radiation or restriction enzyme digestion: a function which cannot be complemented by CDC9. Lig4p acts in the same DNA repair pathway as the DNA end-binding protein Ku. However, unlike Ku, it does not function in telomere length homeostasis. These findings indicate diversification of function between different eukaryotic DNA ligases. Furthermore, they provide insights into mechanisms of DNA repair and suggest that the NHEJ pathway is highly conserved throughout the eukaryotic kingdom.     

Repair of DNA strand gaps and nicks containing 3'-phosphate and 5'-hydroxyl termini by purified mammalian enzymes.

A putative role for mammalian polynucleotide kinases that possess both 5'-phosphotransferase and 3'-phosphatase activity is the restoration of DNA strand breaks with 5'-hydroxyl termini or 3'-phosphate termini, or both, to a form that supports the subsequent action of DNA repair polymerases and DNA ligases, i.e. 5'-phosphate and 3'-hydroxyl termini. To further assess this possibility, we compared the activity of the 3'-phosphatase of purified calf thymus polynucleotide kinase towards a variety of substrates. The rate of removal of 3'-phosphate groups from nicked or short (1 nt) gapped sites in double-stranded DNA was observed to be similar to that of 3'-phosphate groups from single-stranded substrates. Thus this activity of polynucleotide kinase does not appear to be influenced by steric accessibility of the phosphate group. We subsequently demonstrated that the concerted reactions of polynucleotide kinase and purified human DNA ligase I could efficiently repair DNA nicks possessing 3'-phosphate and 5'-hydroxyl termini, and similarly the combination of these two enzymes together with purified rat DNA polymerase beta could seal a strand break with a 1 nt gap. With a substrate containing a nick bounded by 3'- and 5'-OH termini, the rate of gap filling by polymerase beta was significantly enhanced in the presence of polynucleotide kinase and ATP, indicating the positive influence of 5'-phosphorylation. The reaction was further enhanced by addition of DNA ligase I to the reaction mixture. This is due, at least in part, to an enhancement by DNA ligase I of the rate of 5'-phosphorylation catalyzed by polynucleotide kinase.     

Improving the fidelity of Thermus thermophilus DNA ligase.

The DNA ligase from Thermus thermophilus (Tth DNA ligase) seals single-strand breaks (nicks) in DNA duplex substrates. The specificity and thermostability of this enzyme are exploited in the ligase chain reaction (LCR) and ligase detection reaction (LDR) to distinguish single base mutations associated with genetic diseases. Herein, we describe a quantitative assay using fluorescently labeled substrates to study the fidelity of Tth DNA ligase. The enzyme exhibits significantly greater discrimination against all single base mismatches on the 3'-side of the nick in comparison with those on the 5'-side of the nick. Among all 12 possible single base pair mismatches on the 3'-side of the nick, only T-G and G-T mismatches generated a quantifiable level of ligation products after 23 h incubation. The high fidelity of Tth DNA ligase can be improved further by introducing a mismatched base or a universal nucleoside analog at the third position of the discriminating oligonucleotide. Finally, two mutant Tth DNA ligases, K294R and K294P, were found to have increased fidelity using this assay.     

Mitochondrial DNA ligase function in Saccharomyces cerevisiae

The Saccharomyces cerevisiae CDC9 gene encodes a DNA ligase protein that is targeted to both the nucleus and the mitochondria. While nuclear Cdc9p is known to play an essential role in nuclear DNA replication and repair, its role in mitochondrial DNA dynamics has not been defined. It is also unclear whether additional DNA ligase proteins are present in yeast mitochondria. To address these issues, mitochondrial DNA ligase function in S.cerevisiae was analyzed. Biochemical analysis of mitochondrial protein extracts supported the conclusion that Cdc9p was the sole DNA ligase protein present in this organelle. Inactivation of mitochondrial Cdc9p function led to a rapid decline in cellular mitochondrial DNA content in both dividing and stationary yeast cultures. In contrast, there was no apparent defect in mitochondrial DNA dynamics in a yeast strain deficient in Dnl4p (Deltadnl4). The Escherichia coli ECO:RI endonuclease was targeted to yeast mitochondria. Transient expression of this recombinant ECO:RI endonuclease led to the formation of mitochondrial DNA double-strand breaks. While wild-type and Deltadnl4 yeast were able to rapidly recover from this mitochondrial DNA damage, clones deficient in mitochondrial Cdc9p were not. These results support the conclusion that yeast rely upon a single DNA ligase, Cdc9p, to carry out mitochondrial DNA replication and recovery from both spontaneous and induced mitochondrial DNA damage.     

Kinetic mechanism of human DNA ligase I reveals magnesium-dependent changes in the rate-limiting step that compromise ligation efficiency

DNA ligase I (LIG1) catalyzes the ligation of single-strand breaks to complete DNA replication and repair. The energy of ATP is used to form a new phosphodiester bond in DNA via a reaction mechanism that involves three distinct chemical steps: enzyme adenylylation, adenylyl transfer to DNA, and nick sealing. We used steady state and pre-steady state kinetics to characterize the minimal mechanism for DNA ligation catalyzed by human LIG1. The ATP dependence of the reaction indicates that LIG1 requires multiple Mg(2+) ions for catalysis and that an essential Mg(2+) ion binds more tightly to ATP than to the enzyme. Further dissection of the magnesium ion dependence of individual reaction steps revealed that the affinity for Mg(2+) changes along the reaction coordinate. At saturating concentrations of ATP and Mg(2+) ions, the three chemical steps occur at similar rates, and the efficiency of ligation is high. However, under conditions of limiting Mg(2+), the nick-sealing step becomes rate-limiting, and the adenylylated DNA intermediate is prematurely released into solution. Subsequent adenylylation of enzyme prevents rebinding to the adenylylated DNA intermediate comprising an Achilles' heel of LIG1. These ligase-generated 5'-adenylylated nicks constitute persistent breaks that are a threat to genomic stability if they are not repaired. The kinetic and thermodynamic framework that we have determined for LIG1 provides a starting point for understanding the mechanism and specificity of mammalian DNA ligases.     

Modulation of the 3'-->5'-exonuclease activity of human apurinic endonuclease (Ape1) by its 5'-incised Abasic DNA product

The major abasic endonuclease of human cells, Ape1 protein, is a multifunctional enzyme with critical roles in base excision repair (BER) of DNA. In addition to its primary activity as an apurinic/apyrimidinic endonuclease in BER, Ape1 also possesses 3'-phosphodiesterase, 3'-phosphatase, and 3'-->5'-exonuclease functions specific for the 3' termini of internal nicks and gaps in DNA. The exonuclease activity is enhanced at 3' mismatches, which suggests a possible role in BER for Ape1 as a proofreading activity for the relatively inaccurate DNA polymerase beta. To elucidate this role more precisely, we investigated the ability of Ape1 to degrade DNA substrates that mimic BER intermediates. We found that the Ape1 exonuclease is active at both mismatched and correctly matched 3' termini, with preference for mismatches. In our hands, the exonuclease activity of Ape1 was more active at one-nucleotide gaps than at nicks in DNA, even though the latter should represent the product of repair synthesis by polymerase beta. However, the exonuclease activity was inhibited by the presence of nearby 5'-incised abasic residues, which result from the apurinic/apyrimidinic endonuclease activity of Ape1. The same was true for the recently described exonuclease activity of Escherichia coli endonuclease IV. Exonuclease III, the E. coli homolog of Ape1, did not discriminate among the different substrates. Removal of the 5' abasic residue by polymerase beta alleviated the inhibition of the Ape1 exonuclease activity. These results suggest roles for the Ape1 exonuclease during BER after both DNA repair synthesis and excision of the abasic deoxyribose-5-phosphate by polymerase beta.     

Characterization of an ATP-dependent type I DNA ligase from Arabidopsis thaliana

Here we report the purification and biochemical characterization of recombinant Arabidopsis thaliana DNA ligase I. We show that this ligase requires ATP as a source for adenylation. The calculated K _m [ATP] for ligation is 3 M. This enzyme is able to ligate nicks in oligo(dT)/poly(dA) and oligo(rA)/poly(dT) substrates, but not in oligo(dT)/poly(rA) substrates. Double-stranded DNAs with cohesive or blunt ends are also good substrates for the ligase. These biochemical features of the purified enzyme show the characteristics typical of a type I DNA ligase. Furthermore, this DNA ligase is able to perform the reverse reaction (relaxation of supercoiled DNA) in an AMP-dependent and PPi-stimulated manner.     

RNA nicking activity associated with DNA ligase of T4 infected E. coli: properties and influence on in vitro reactions of ligase.

Highly purified DNA ligase from T4 infected E. coli displays an RNA nicking activity which cleaves endonucleolytically the RNA of ribo-desoxy-and ribo-ribo type doublestranded structures to oligonucleotides with 5'phosphoryl-and 3'hydroxy termini. In the presence of ATP the generated nicks are repaired by the ligase except at the ends of the doublestranded regions where some short oligonucleotides are released before ligation can occur. As judged from its behaviour during the various purification steps and from some of its properties, the nicking activity seems to be different from known nicking enzymes.     

ATP for the DNA ligation step in base excision repair is generated from poly(ADP-ribose)

In mammalian cells, the base excision repair (BER) pathway is the main route to counteract the mutagenic effects of DNA lesions. DNA nicks induce, among others, DNA polymerase activities and the synthesis of poly(ADP-ribose). It is shown here that poly(ADP-ribose) serves as an energy source for the final and rate-limiting step of BER, DNA ligation. This conclusion was drawn from experiments in which the fate of [(32)P]poly(ADP-ribose) or [(32)P]NAD added to HeLa nuclear extracts was systematically followed. ATP was synthesized from poly(ADP-ribose) in a pathway that strictly depended on nick-induced DNA synthesis. NAD was used for the synthesis of poly(ADP-ribose), which, in turn, was converted to ATP by pyrophosphorylytic cleavage utilizing the pyrophosphate generated from dNTPs during DNA synthesis. The adenylyl moiety was then preferentially used to adenylate DNA ligase III, from which it was transferred to the 5'-phosphoryl end of the nicked DNA. Finally, ligation to the 3'-OH end resulted in the release of AMP. When using NAD, but not poly(ADP-ribose), in the presence of 3-aminobenzamide, the entire process was blocked, confirming poly(ADP-ribosyl)ation to be the essential initial step. Thus, poly(ADP-ribose) polymerase-1, DNA polymerase beta, and ligase III interact with x-ray repair cross-complementing protein-1 within the BER complex, which ensures that ATP is generated and specifically used for DNA ligation.     

Enzyme action at 3' termini of ionizing radiation-induced DNA strand breaks

gamma-Irradiation of DNA in vitro produces two types of single strand breaks. Both types of strand breaks contain 5'-phosphate DNA termini. Some strand breaks contain 3'-phosphate termini, some contain 3'-phosphoglycolate termini (Henner, W.D., Rodriguez, L.O., Hecht, S. M., and Haseltine, W. A. (1983) J. Biol. Chem. 258, 711-713). We have studied the ability of prokaryotic enzymes of DNA metabolism to act at each of these types of gamma-ray-induced 3' termini in DNA. Neither strand breaks that terminate with 3'-phosphate nor 3'-phosphoglycolate are substrates for direct ligation by T4 DNA ligase. Neither type of gamma-ray-induced 3' terminus can be used as a primer for DNA synthesis by either Escherichia coli DNA polymerase or T4 DNA polymerase. The 3'-phosphatase activity of T4 polynucleotide kinase can convert gamma-ray-induced 3'-phosphate but not 3'-phosphoglycolate termini to 3'-hydroxyl termini that can then serve as primers for DNA polymerase. E. coli alkaline phosphatase is also unable to hydrolyze 3'-phosphoglycolate groups. The 3'-5' exonuclease actions of E. coli DNA polymerase I and T4 DNA polymerase do not degrade DNA strands that have either type of gamma-ray-induced 3' terminus. E. coli exonuclease III can hydrolyze DNA with gamma-ray-induced 3'-phosphate or 3'-phosphoglycolate termini or with DNase I-induced 3'-hydroxyl termini. The initial action of exonuclease III at 3' termini of ionizing radiation-induced DNA fragments is to remove the 3' terminal phosphate or phosphoglycolate to yield a fragment of the same nucleotide length that has a 3'-hydroxyl terminus. These results suggest that repair of ionizing radiation-induced strand breaks may proceed via the sequential action of exonuclease, DNA polymerase, and DNA ligase. The possible role of exonuclease III in repair of gamma-radiation-induced strand breaks is discussed.     

Dual mode of inhibition of purified DNA ligase I from human cells by 9-beta-D-arabinofuranosyl-2-fluoroadenine triphosphate.

9-beta-D-Arabinofuranosyl-2-fluoroadenine (F-ara-A) is an analogue of adenosine and deoxyadenosine with potent anti-tumor activity. The mechanism of action for this compound has been elucidated as the inhibition of DNA and RNA synthesis, induction of DNA fragmentation, and genetic damage. This study demonstrated that DNA ligase I, an enzyme involved in DNA replication, is a target for the drug action. F-ara-adenine triphosphate (F-ara-ATP) at 80 microM inhibited the activity of DNA ligase I by more than 90%. In contrast, eight other related nucleoside analogues showed no effect on the enzyme activity at 200 microM. F-ara-ATP inhibited DNA ligation in two distinct ways. First, F-ara-ATP directly interacted with DNA ligase I and inhibited the formation of the ligase-AMP complex. This inhibition could not be reversed when free F-ara-ATP was eliminated from the treated enzyme; however, the addition of pyrophosphate, followed by gel filtration chromatography, restored enzyme activity, indicating that F-ara-ATP bound to the enzyme and altered the AMP-binding site. Secondly, the activity of DNA ligase I was inhibited when F-ara-ATP was incorporated into the 3' terminus of the DNA substrate. The dual mode of inhibition of DNA ligase I by F-ara-ATP indicates that its effect on DNA ligation may be important in the inhibition of DNA synthesis and the cytotoxicity of F-ara-A.     

Actions of aprataxin in multiple DNA repair pathways

Mutations in the Aptx gene lead to a neurological disorder known as ataxia oculomotor apraxia-1. The product of Aptx is Aprataxin (Aptx), a DNA-binding protein that resolves abortive DNA ligation intermediates. Aprataxin catalyzes the nucleophilic release of adenylate groups covalently linked to 5' phosphate termini, resulting in termini that can again serve as substrates for DNA ligases. Here we show that Aprataxin acts preferentially on adenylated nicks and double-strand breaks rather than on single-stranded DNA. Moreover, we show that whereas the catalytic activity of Aptx resides within the HIT domain, the C-terminal zinc finger domain provides stabilizing contacts that lock the enzyme onto its high affinity AMP-DNA target site. Both domains are therefore required for efficient AMP-DNA hydrolase activity. Additionally, we find a role for Aprataxin in base excision repair, specifically in the removal of adenylates that arise from abortive ligation reactions that take place at incised abasic sites in DNA. We suggest that Aprataxin may have a general proofreading function in DNA repair, removing DNA adenylates as they arise during single-strand break repair, double-strand break repair, and in base excision repair.     

DNA ligase I and proliferating cell nuclear antigen form a functional complex

DNA ligase I is responsible for joining Okazaki fragments during DNA replication. An additional proposed role for DNA ligase I is sealing nicks generated during excision repair. Previous studies have shown that there is a physical interaction between DNA ligase I and proliferating cell nuclear antigen (PCNA), another important component of DNA replication and repair. The results shown here indicate that human PCNA enhances the reaction rate of human DNA ligase I up to 5-fold. The stimulation is specific to DNA ligase I because T4 DNA ligase is not affected. Electrophoretic mobility shift assays indicate that PCNA improves the binding of DNA ligase I to the ligation site. Increasing the DNA ligase I concentration leads to a reduction in PCNA stimulation, consistent with PCNA-directed improvement of DNA ligase I binding to its DNA substrate. Two experiments show that PCNA is required to encircle duplex DNA to enhance DNA ligase I activity. Biotin-streptavidin conjugations at the ends of a linear substrate inhibit PCNA stimulation. PCNA cannot enhance ligation on a circular substrate without the addition of replication factor C, which is the protein responsible for loading PCNA onto duplex DNA. These results show that PCNA is responsible for the stable association of DNA ligase I to nicked duplex DNA.     

Determination of the free-energy change for repair of a DNA phosphodiester bond

The repair of phosphodiester bonds in nicked DNA is catalyzed by DNA ligases. Ligation is coupled to cleavage of a phosphoanhydride bond in a nucleotide cofactor resulting in a thermodynamically favorable process. A free energy value for phosphodiester bond formation was calculated using the reversibility of the T4 DNA ligase reaction. The relative number of DNA nicks to phosphodiester bonds in a circular plasmid DNA, formed during this reaction at fixed concentrations of ATP to AMP and PP(i), was quantified. At 25 degrees C, pH 7, the equilibrium constant (K(eq)) for the ligation reaction is 3.89 x 10(4) m. This value corresponds to a standard free energy (DeltaG degrees ') of -6.3 kcal mol(-1). By subtracting the known energy contribution due to hydrolysis of ATP to AMP and PP(i), DeltaG degrees ' for the hydrolysis of a DNA phosphodiester bond is -5.3 kcal mol(-1).