Deregulated levels of RUVBL1 induce transcription-dependent replication stress.

Chromatin regulators control transcription and replication, however if and how they might influence the coordination of these processes still is largely unknown. RUVBL1 and the related ATPase RUVBL2 participate in multiple nuclear processes and are implicated in cancer. Here, we report that both the excess and the deficit of the chromatin regulator RUVBL1 impede DNA replication as a consequence of altered transcription. Surprisingly, cells that either overexpressed or were silenced for RUVBL1 had slower replication fork rates and accumulated phosphorylated H2AX, dependent on active transcription. However, the mechanisms of transcription-dependent replication stress were different when RUVBL1 was overexpressed and when depleted. RUVBL1 overexpression led to increased c-Myc-dependent pause release of RNAPII, as evidenced by higher overall transcription, much stronger Ser2 phosphorylation of Rpb1- C-terminal domain, and enhanced colocalization of Rpb1 and c-Myc. RUVBL1 deficiency resulted in increased ubiquitination of Rpb1 and reduced mobility of an RNAP subunit, suggesting accumulation of stalled RNAPIIs on chromatin. Overall, our data show that by modulating the state of RNAPII complexes, RUVBL1 deregulation induces replication-transcription interference and compromises genome integrity during S-phase.

Lytic water dynamics reveal evolutionarily conserved mechanisms of ATP hydrolysis by TIP49 AAA+ ATPases.

Eukaryotic TIP49a (Pontin) and TIP49b (Reptin) AAA+ ATPases play essential roles in key cellular processes. How their weak ATPase activity contributes to their important functions remains largely unknown and difficult to analyze because of the divergent properties of TIP49a and TIP49b proteins and of their homo- and hetero-oligomeric assemblies. To circumvent these complexities, we have analyzed the single ancient TIP49 ortholog found in the archaeon Methanopyrus kandleri (mkTIP49). All-atom homology modeling and molecular dynamics simulations validated by biochemical assays reveal highly conserved organizational principles and identify key residues for ATP hydrolysis. An unanticipated crosstalk between Walker B and Sensor I motifs impacts the dynamics of water molecules and highlights a critical role of trans-acting aspartates in the lytic water activation step that is essential for the associative mechanism of ATP hydrolysis.

Identification of the general transcription factor Yin Yang 1 as a novel and specific binding partner for S6 kinase 2.

S6 kinase is a member of the AGC family of serine/threonine kinases and plays a key role in diverse cellular processes including cell growth and metabolism. Although, the high degree of homology between S6K family members (S6K1 and S6K2) in kinase and kinase-extension domains, the two proteins are highly divergent in the N- and C-terminal regulatory regions, hinting at differential regulation, downstream signalling and cellular function. Deregulated signalling via S6Ks has been linked to various human pathologies, such as diabetes and cancer. Therefore, S6K has emerged as a promising target for drug development. Much of what we know about S6K signalling in health and disease comes from studies of S6K1, as molecular cloning of this isoform was reported a decade earlier than S6K2. In this study, we report for the first time, the identification of the general transcription factor Yin Yang 1 (YY1) as a novel and specific binding partner of S6K2, but not S6K1. The interaction between YY1 and S6K2 was demonstrated by co-immunoprecipitation of transiently overexpressed and endogenous proteins in a number of cell lines, including HEK293, MCF7 and U937. Furthermore, direct association between S6K2 and YY1 was demonstrated by GST pull-down assay using recombinant proteins. A panel of deletion mutants was used to show that the C-terminal regulatory region of S6K2 mediates the interaction with YY1. Interestingly, the complex formation between S6K2 and YY1 can be detected in serum-starved cells, but the interaction is strongly induced in response to mitogenic stimulation. The induction of S6K2/YY1 complex formation in response to serum stimulation is abolished by pre-treatment of cells with the mTOR inhibitor, rapamycin. Furthermore, mTOR is also detected in complex with YY1 and S6K2 in serum-stimulated cells. We utilized size exclusion chromatography along with co-immunoprecipitation analysis to demonstrate the existence of the mTOR/S6K2/YY1 complex in high molecular weight fractions, which might also involve other cellular proteins. The physiological significance of the mTOR/S6K2/YY1 complex, which is induced in response to mitogenic stimulation, remains to be further investigated.

Large-scale conformational flexibility determines the properties of AAA+ TIP49 ATPases.

The TIP49a and TIP49b proteins belong to the family of AAA+ ATPases and play essential roles in vital processes such as transcription, DNA repair, snoRNP biogenesis, and chromatin remodeling. We report the crystal structure of a TIP49b hexamer and the comparative analysis of large-scale conformational flexibility of TIP49a, TIP49b, and TIP49a/TIP49b complexes using molecular modeling and molecular dynamics simulations in a water environment. Our results establish key principles of domain mobility that affect protein conformation and biochemical properties, including a mechanistic basis for the downregulation of ATPase activity upon protein hexamerization. These approaches, applied to the lik-TIP49b mutant reported to possess enhanced DNA-independent ATPase activity, help explain how a three-amino acid insertion remotely affects the structure and conformational dynamics of the ATP binding and hydrolysis pocket while uncoupling ATP hydrolysis from DNA binding. This might be similar to the effects of conformations adopted by TIP49 heterohexamers.

Formation of a stable RuvA protein double tetramer is required for efficient branch migration in vitro and for replication fork reversal in vivo.

In bacteria, RuvABC is required for the resolution of Holliday junctions (HJ) made during homologous recombination. The RuvAB complex catalyzes HJ branch migration and replication fork reversal (RFR). During RFR, a stalled fork is reversed to form a HJ adjacent to a DNA double strand end, a reaction that requires RuvAB in certain Escherichia coli replication mutants. The exact structure of active RuvAB complexes remains elusive as it is still unknown whether one or two tetramers of RuvA support RuvB during branch migration and during RFR. We designed an E. coli RuvA mutant, RuvA2(KaP), specifically impaired for RuvA tetramer-tetramer interactions. As expected, the mutant protein is impaired for complex II (two tetramers) formation on HJs, although the binding efficiency of complex I (a single tetramer) is as wild type. We show that although RuvA complex II formation is required for efficient HJ branch migration in vitro, RuvA2(KaP) is fully active for homologous recombination in vivo. RuvA2(KaP) is also deficient at forming complex II on synthetic replication forks, and the binding affinity of RuvA2(KaP) for forks is decreased compared with wild type. Accordingly, RuvA2(KaP) is inefficient at processing forks in vitro and in vivo. These data indicate that RuvA2(KaP) is a separation-of-function mutant, capable of homologous recombination but impaired for RFR. RuvA2(KaP) is defective for stimulation of RuvB activity and stability of HJ·RuvA·RuvB tripartite complexes. This work demonstrates that the need for RuvA tetramer-tetramer interactions for full RuvAB activity in vitro causes specifically an RFR defect in vivo.

Oligomeric assembly and interactions within the human RuvB-like RuvBL1 and RuvBL2 complexes.

The two closely related eukaryotic AAA+ proteins (ATPases associated with various cellular activities), RuvBL1 (RuvB-like 1) and RuvBL2, are essential components of large multi-protein complexes involved in diverse cellular processes. Although the molecular mechanisms of RuvBL1 and RuvBL2 function remain unknown, oligomerization is likely to be important for their function together or individually, and different oligomeric forms might underpin different functions. Several experimental approaches were used to investigate the molecular architecture of the RuvBL1-RuvBL2 complex and the role of the ATPase-insert domain (domain II) for its assembly and stability. Analytical ultracentrifugation showed that RuvBL1 and RuvBL2 were mainly monomeric and each monomer co-existed with small proportions of dimers, trimers and hexamers. Adenine nucleotides induced hexamerization of RuvBL2, but not RuvBL1. In contrast, the RuvBL1-RuvBL2 complexes contained single- and double-hexamers together with smaller forms. The role of domain II in complex assembly was examined by size-exclusion chromatography using deletion mutants of RuvBL1 and RuvBL2. Significantly, catalytically competent dodecameric RuvBL1-RuvBL2, complexes lacking domain II in one or both proteins could be assembled but the loss of domain II in RuvBL1 destabilized the dodecamer. The composition of the RuvBL1-RuvBL2 complex was analysed by MS. Several species of mixed RuvBL1/2 hexamers with different stoichiometries were seen in the spectra of the RuvBL1-RuvBL2 complex. A number of our results indicate that the architecture of the human RuvBL1-RuvBL2 complex does not fit the recent structural model of the yeast Rvb1-Rvb2 complex.

Involvement of heterogeneous ribonucleoprotein F in the regulation of cell proliferation via the mammalian target of rapamycin/S6 kinase 2 pathway.

The S6 kinases (S6Ks) have been linked to a number of cellular processes, including translation, insulin metabolism, cell survival, and RNA splicing. Signaling via the phosphotidylinositol 3-kinase and mammalian target of rapamycin (mTOR) pathways is critical in regulating the activity and subcellular localization of S6Ks. To date, nuclear functions of both S6K isoforms, S6K1 and S6K2, are not well understood. To better understand S6K nuclear roles, we employed affinity purification of S6Ks from nuclear preparations followed by mass spectrometry analysis for the identification of novel binding partners. In this study, we report that in contrast to S6K1, the S6K2 isoform specifically associates with a number of RNA-binding proteins, including heterogeneous ribonucleoproteins (hnRNPs). We focused on studying the mechanism and physiological relevance of the S6K2 interaction with hnRNP F/H. Interestingly, the S6K2-hnRNP F/H interaction was not affected by mitogenic stimulation, whereas mTOR binding to hnRNP F/H was induced by serum stimulation. In addition, we define a new role of hnRNP F in driving cell proliferation, which could be partially attenuated by rapamycin treatment. S6K2-driven cell proliferation, on the other hand, could be blocked by small interfering RNA-mediated down-regulation of hnRNP F. These results demonstrate that the specific interaction between mTOR and S6K2 with hnRNPs is implicated in the regulation of cell proliferation.

RAD51 foci formation in response to DNA damage is modulated by TIP49.

Chromatin modification plays an important role in modulating the access of homologous recombination proteins to the sites of DNA damage. TIP49 is highly conserved component of chromatin modification/remodeling complexes, but its involvement in homologous recombination repair in mammalian cells has not been examined in details. In the present communication we studied the role of TIP49 in recruitment of the key homologous recombination protein RAD51 to sites of DNA damage. RAD51 redistribution to chromatin and nuclear foci formation induced by double-strand breaks and interstrand crosslinks were followed under conditions of TIP49 depletion by RNA interference. TIP49 silencing reduced RAD51 recruitment to chromatin and nuclear foci formation to about 50% of that of the control. Silencing of TIP48, which is closely related to TIP49, induced a similar reduction in RAD51 foci formation. RAD51 foci reduction in TIP49-silenced cells was not a result of defective DNA damage checkpoint signaling as judged by the normal histone H2AX phosphorylation and cell cycle distribution. Treatment with the histone deacetylase inhibitor sodium butyrate restored RAD51 foci formation in the TIP49-depleted cells. The results suggest that as a constituent of chromatin modification complexes TIP49 may facilitate the access of the repair machinery to the sites of DNA damage.

ruvA Mutants that resolve Holliday junctions but do not reverse replication forks.

RuvAB and RuvABC complexes catalyze branch migration and resolution of Holliday junctions (HJs) respectively. In addition to their action in the last steps of homologous recombination, they process HJs made by replication fork reversal, a reaction which occurs at inactivated replication forks by the annealing of blocked leading and lagging strand ends. RuvAB was recently proposed to bind replication forks and directly catalyze their conversion into HJs. We report here the isolation and characterization of two separation-of-function ruvA mutants that resolve HJs, based on their capacity to promote conjugational recombination and recombinational repair of UV and mitomycin C lesions, but have lost the capacity to reverse forks. In vivo and in vitro evidence indicate that the ruvA mutations affect DNA binding and the stimulation of RuvB helicase activity. This work shows that RuvA's actions at forks and at HJs can be genetically separated, and that RuvA mutants compromised for fork reversal remain fully capable of homologous recombination.

Cell cycle-dependent association of Rad51 with the nuclear matrix.

Progression of the cells through the S phase of the cell cycle is connected with accumulation of stalled and collapsed replication forks that are repaired by homologous recombination. To investigate the temporal order of homologous recombination events during the S phase, HeLa cells synchronized at the G1/S phase boundary with mimosine were released to progress into the S phase and the phosphorylation of the histone variant H2AX, the appearance of Rad51 nuclear foci and the subcellular redistribution of Rad51 were followed. The results showed that there was gradual accumulation of double-strand breaks as judged by the increase in the phosphorylation of H2AX during the S phase. Rad51 nuclear foci did not appear until middle S phase, and this was accompanied by an increase in the chromatin- and nuclear matrix-bound Rad51 in the middle to late S phase. To determine the role of the intra S phase checkpoint in the S phase-dependent redistribution of Rad51 the cells were released in the S phase in the presence of the protein kinase inhibitors caffeine and wortmannin. The results suggest that the association of Rad51 with the nuclear matrix is regulated by activation of the intra S phase ATR-dependent checkpoint pathway.

Dodecameric structure and ATPase activity of the human TIP48/TIP49 complex.

TIP48 and TIP49 are two related and highly conserved eukaryotic AAA(+) proteins with an essential biological function and a critical role in major pathways that are closely linked to cancer. They are found together as components of several highly conserved chromatin-modifying complexes. Both proteins show sequence homology to bacterial RuvB but the nature and mechanism of their biochemical role remain unknown. Recombinant human TIP48 and TIP49 were assembled into a stable high molecular mass equimolar complex and tested for activity in vitro. TIP48/TIP49 complex formation resulted in synergistic increase in ATPase activity but ATP hydrolysis was not stimulated in the presence of single-stranded, double-stranded or four-way junction DNA and no DNA helicase or branch migration activity could be detected. Complexes with catalytic defects in either TIP48 or TIP49 had no ATPase activity showing that both proteins within the TIP48/TIP49 complex are required for ATP hydrolysis. The structure of the TIP48/TIP49 complex was examined by negative stain electron microscopy. Three-dimensional reconstruction at 20 A resolution revealed that the TIP48/TIP49 complex consisted of two stacked hexameric rings with C6 symmetry. The top and bottom rings showed substantial structural differences. Interestingly, TIP48 formed oligomers in the presence of adenine nucleotides, whilst TIP49 did not. The results point to biochemical differences between TIP48 and TIP49, which may explain the structural differences between the two hexameric rings and could be significant for specialised functions that the proteins perform individually.

Activation of the S phase DNA damage checkpoint by mitomycin C.

We have studied the rate of DNA synthesis, cell cycle distribution, formation of gamma-H2AX, and Rad51 nuclear foci and association of Rad51 with the nuclear matrix after treatment of HeLa cells with the interstrand crosslinking agent mitomycin C (MMC) in the presence of the kinase inhibitors caffeine and wortmannin. The results showed that MMC treatment arrested the cells in S-phase and induced the appearance of gamma-H2AX and Rad51 nuclear foci, accompanied with a sequestering of Rad51 to the nuclear matrix. These effects were abrogated by caffeine, which inhibits the Ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR) kinases. However, wortmannin at a concentration that inhibits ATM, but not ATR did not affect cell cycle progression, damage-induced phosphorylation of H2AX and Rad51 foci formation, and association with the nuclear matrix, suggesting that the S-phase arrest induced by MMC is ATR-dependent. These findings were confirmed by experiments with ATR-deficient and AT cells. They indicate that the DNA damage ATR-dependent S-phase checkpoint pathway may regulate the spatiotemporal organization of the process of repair of interstrand crosslinks.

Sub-nuclear localization of Rad51 in response to DNA damage.

The repair of DNA double-strand breaks involves the accumulation of key homologous recombination proteins in nuclear foci at the sites of repair. The organization of these foci in relation to non-chromatin nuclear structures is poorly understood. To address this question, we examined the distribution of several recombination proteins in subcellular fractions following treatment of HeLa cells with ionizing radiation and the crosslinking agent mitomycin C. The results showed association of Rad51, Rad54, BRCA1 and BRCA2, but not Rad51C, with the nuclear matrix fraction in response to double-strand breaks induction. The association of Rad51 with the nuclear matrix correlates with the formation of Rad51 nuclear foci as a result of DNA damage. Fractionation in situ confirmed that Rad51 foci remained firmly immobilized within the chromatin-depleted nuclei. Irs1SF cells that are unable to form Rad51 damage-induced nuclear foci did not show accumulation of Rad51 in the nuclear matrix. Similarly, no accumulation of Rad51 in the nuclear matrix could be observed after treatment of HeLa cells with the kinase inhibitor caffeine, which reduces formation of Rad51 foci. The results were compared to the distribution of the phosphorylated histone variant, gamma-H2AX. These data suggest a dynamic association and tethering of recombination proteins and surrounding chromatin regions to the nuclear matrix.

Relocalization of human chromatin remodeling cofactor TIP48 in mitosis.

TIP48 is a highly conserved eukaryotic AAA+ protein which is an essential cofactor for several complexes involved in chromatin acetylation and remodeling, transcriptional and developmental regulation and nucleolar organization and trafficking. We show that TIP48 abundance in HeLa cells did not change during the cell cycle, nor did its distribution in various biochemical fractions. However, we observed distinct changes in the subcellular localization of TIP48 during M phase using immunofluorescence microscopy. Our studies demonstrate that in interphase cells TIP48 was found mainly in the nucleus and exhibited a distinct localization in the nuclear periphery. As the cells entered mitosis, TIP48 was excluded from the condensing chromosomes but showed association with the mitotic apparatus. During anaphase, some TIP48 was detected in the centrosome colocalizing with tubulin but the strongest staining appeared in the mitotic equator associated with the midzone central spindle. Accumulation of TIP48 in the midzone and the midbody was observed in late telophase and cytokinesis. This redeployment of TIP48 during anaphase and cytokinesis was independent of microtubule assembly. The relocation of endogenous TIP48 to the midzone/midbody under physiological conditions suggests a novel and distinct function for TIP48 in mitosis and possible involvement in the exit of mitosis.

Repair of DNA interstrand crosslinks may take place at the nuclear matrix.

Host cell reactivation assay using Trioxsalen-crosslinked plasmid pEGFP-N1 showed that human cells were able to repair Trioxsalen interstrand crosslinks (ICL). To study the mechanism of this repair pathway, cells were transfected with the plasmids pEGFP-1, which did not contain the promoter of the egfp gene, and with pEGFP-G-, which did not contain the egfp gene. Neither of these plasmids alone was able to express the green fluorescent protein. After cotransfection with the two plasmids, 1%-2% of the cells developed fluorescent signal, which showed that recombination events had taken place in these cells to create DNA constructs containing the promoter and the gene properly aligned. When one or both of the plasmids were crosslinked with Trioxsalen, the recombination rate increased several fold. To identify the nuclear compartment where recombination takes place, cells were transfected with crosslinked pEGFP-N1 and the amount of plasmid DNA in the different nuclear fractions was determined. The results showed that Trioxsalen crosslinking increased the percentage of matrix attached plasmid DNA in a dose-dependent way. Immunoblotting experiments showed that after transfection with Trioxsalen crosslinked plasmids the homologous recombination protein Rad51 also associated with the nuclear matrix fraction. These studies provide a model system for investigating the precise molecular mechanisms that appear to couple repair of DNA ICL with nuclear matrix attachment.

Biochemical characterization and DNA repair pathway interactions of Mag1-mediated base excision repair in Schizosaccharomyces pombe.

The Schizosaccharomyces pombe mag1 gene encodes a DNA repair enzyme with sequence similarity to the AlkA family of DNA glycosylases, which are essential for the removal of cytotoxic alkylation products, the premutagenic deamination product hypoxanthine and certain cyclic ethenoadducts such as ethenoadenine. In this paper, we have purified the Mag1 protein and characterized its substrate specificity. It appears that the substrate range of Mag1 is limited to the major alkylation products, such as 3-mA, 3-mG and 7-mG, whereas no significant activity was found towards deamination products, ethenoadducts or oxidation products. The efficiency of 3-mA and 3-mG removal was 5-10 times slower for Mag1 than for Escherichia coli AlkA whereas the rate of 7-mG removal was similar to the two enzymes. The relatively low efficiency for the removal of cytotoxic 3-methylpurines is consistent with the moderate sensitivity of the mag1 mutant to methylating agents. Furthermore, we studied the initial steps of Mag1-dependent base excision repair (BER) and genetic interactions with other repair pathways by mutant analysis. The double mutants mag1 nth1, mag1 apn2 and mag1 rad2 displayed increased resistance to methyl methanesulfonate (MMS) compared with the single mutants nth1, apn2 and rad2, respectively, indicating that Mag1 initiates both short-patch (Nth1-dependent) and long-patch (Rad2-dependent) BER of MMS-induced damage. Spontaneous intrachromosomal recombination frequencies increased 3-fold in the mag1 mutant suggesting that Mag1 and recombinational repair (RR) are both involved in repair of alkylated bases. Finally, we show that the deletion of mag1 in the background of rad16, nth1 and rad2 single mutants reduced the total recombination frequencies of all three double mutants, indicating that abasic sites formed as a result of Mag1 removal of spontaneous base lesions are substrates for nucleotide excision repair, long- and short-patch BER and RR.

The role of RuvA octamerization for RuvAB function in vitro and in vivo.

RuvA plays an essential role in branch migration of the Holliday junction by RuvAB as part of the RuvABC pathway for processing Holliday junctions in Escherichia coli. Two types of RuvA-Holliday junction complexes have been characterized: 1) complex I containing a single RuvA tetramer and 2) complex II in which the junction is sandwiched between two RuvA tetramers. The functional differences between the two forms are still not clear. To investigate the role of RuvA octamerization, we introduced three amino acid substitutions designed to disrupt the E. coli RuvA tetramer-tetramer interface as identified by structural studies. The mutant RuvA was tetrameric and interacted with both RuvB and junction DNA but, as predicted, formed complex I only at protein concentrations up to 500 nm. We present biochemical and surface plasmon resonance evidence for functional and physical interactions of the mutant RuvA with RuvB and RuvC on synthetic junctions. The mutant RuvA with RuvB showed DNA helicase activity and could support branch migration of synthetic four-way and three-way junctions. However, junction binding and the efficiency of branch migration of four-way junctions were affected. The activity of the RuvA mutant was consistent with a RuvAB complex driven by one RuvB hexamer only and lead us to propose that one RuvA tetramer can only support the activity of one RuvB hexamer. Significantly, the mutant failed to complement the UV sensitivity of E. coli DeltaruvA cells. These results indicate strongly that RuvA octamerization is essential for the full biological activity of RuvABC.

A tetramer-octamer equilibrium in Mycobacterium leprae and Escherichia coli RuvA by analytical ultracentrifugation.

In the context of the bacterial RuvABC system, RuvA protein binds to and is involved in the subsequent processing of a four-way DNA structure called Holliday junction that is formed during homologous recombination. Four crystal structures of RuvA from Escherichia coli (EcoRuvA) showed that it was tetrameric, while neutron scattering and two other crystal structures for RuvA from Mycobacterium leprae (MleRuvA) and EcoRuvA showed that it was an octamer. To clarify this discrepancy, sedimentation equilibrium experiments by analytical ultracentrifugation were carried out and the results showed that MleRuvA existed as a tetramer-octamer equilibrium between 0.2-0.5 mg/ml in 0.1 M NaCl with a dissociation constant of 4 muM, and is octameric at higher concentrations. The same experiments in 0.3 M NaCl showed that MleRuvA is a tetramer up to 3.5 mg/ml, indicating that salt bridges are involved in octamer formation. Sedimentation equilibrium experiments with EcoRuvA showed that it was tetrameric at low concentration in both salt buffers but the protein was insoluble at high-protein concentrations in 0.1 M NaCl. It is concluded that free RuvA exists in an equilibrium between tetrameric and octameric forms in the typical concentration range and buffer found in bacterial cells.

Functional dissection of the Schizosaccharomyces pombe Holliday junction resolvase Ydc2: in vivo role in mitochondrial DNA maintenance.

The crystal structure of the Schizosaccharomyces pombe Holliday junction resolvase Ydc2 revealed significant structural homology with the Escherichia coli resolvase RuvC but Ydc2 contains a small triple helical bundle that has no equivalent in RuvC. Two of the alpha-helices that form this bundle show homology to a putative DNA-binding motif known as SAP. To investigate the biochemical function of the triple-helix domain, truncated Ydc2 mutants were expressed in E. coli and in fission yeast. Although the truncated proteins retained all amino-acid residues that map to the structural core of RuvC including the catalytic site, deletion of the SAP motif alone or the whole triple-helix domain of Ydc2 resulted in the complete loss of resolvase activity and impaired significantly the binding of Ydc2 to synthetic junctions in vitro. These results are in full agreement with our proposal for a DNA-binding role of the triple-helix motif [Ceschini et al. (2001) EMBO J. 20, 6601-6611]. The biological effect of Ydc2 on mtDNA in yeast was probed using wild-type and several Ydc2 mutants expressed in Deltaydc2 S. pombe. The truncated mutants were shown to localize exclusively to yeast mitochondria ruling out a possible role of the helical bundle in mitochondrial targeting. Cells that lacked Ydc2 showed a significant depletion of mtDNA content. Plasmids expressing full-length Ydc2 but not the truncated or catalytically inactive Ydc2 mutants could rescue the mtDNA 'phenotype'. These results provide evidence that the Holliday junction resolvase activity of Ydc2 is required for mtDNA transmission and affects mtDNA content in S. pombe.

A new Schizosaccharomyces pombe base excision repair mutant, nth1, reveals overlapping pathways for repair of DNA base damage.

Endonuclease III (Nth) enzyme from Escherichia coli is involved in base excision repair of oxidised pyrimidine residues in DNA. The Schizosaccharomyces pombe Nth1 protein is a sequence and functional homologue of E. coli Nth, possessing both DNA glycosylase and apurinic/apyrimidinic (AP) lyase activity. Here, we report the construction and characterization of the S. pombe nth1 mutant. The nth1 mutant exhibited no enhanced sensitivity to oxidising agents, UV or gamma-irradiation, but was hypersensitive to the alkylating agent methyl methanesulphonate (MMS). Analysis of base excision from DNA exposed to [3H]methyl-N-nitrosourea showed that the purified Nth1 enzyme did not remove alkylated bases such as 3-methyladenine and 7-methylguanine whereas methyl-formamidopyrimidine was excised efficiently. The repair of AP sites in S. pombe has previously been shown to be independent of Apn1-like AP endonuclease activity, and the main reason for the MMS sensitivity of nth1 cells appears to be their lack of AP lyase activity. The nth1 mutant also exhibited elevated frequencies of spontaneous mitotic intrachromosomal recombination, which is a phenotype shared by the MMS-hypersensitive DNA repair mutants rad2, rhp55 and NER repair mutants rad16, rhp14, rad13 and swi10. Epistasis analyses of nth1 and these DNA repair mutants suggest that several DNA damage repair/tolerance pathways participate in the processing of alkylation and spontaneous DNA damage in S. pombe.