Polynucleotide kinase/phosphatase, Pnk1, is involved in base excision repair in Schizosaccharomyces pombe.

We previously reported that Schizosaccharomyces pombe pnk1 cells are more sensitive than wild-type cells to γ-radiation and camptothecin, indicating that Pnk1 is required for DNA repair. Here, we report that pnk1pku70 and pnk1rhp51 double mutants are more sensitive to γ-radiation than single mutants, from which we infer that Pnk1's primary role is independent of either homologous recombination or non-homologous end joining mechanisms. We also report that pnk1 cells are more sensitive than wild-type cells to oxidizing and alkylating agents, suggesting that Pnk1 is involved in base excision repair. Mutational analysis of Pnk1 revealed that the DNA 3'-phosphatase activity is necessary for repair of DNA damage, whereas the 5'-kinase activity is dispensable. A role for Pnk1 in base excision repair is supported by genetic analyses which revealed that pnk1apn2 is synthetically lethal, suggesting that Pnk1 and Apn2 may function in parallel pathways essential for the repair of endogenous DNA damage. Furthermore, the nth1pnk1apn2 and tdp1pnk1apn2 triple mutants are viable, implying that single-strand breaks with 3'-blocked termini produced by Nth1 and Tdp1 contribute to synthetic lethality. We also examined the sensitivity to methyl methanesulfonate of all single and double mutant combinations of nth1, apn2, tdp1 and pnk1. Together, our results support a model where Tdp1 and Pnk1 act in concert in an Apn2-independent base excision repair pathway to repair 3'-blocked termini produced by Nth1; and they also provide evidence that Pnk1 has additional roles in base excision repair.

Conserved Orb6 phosphorylation sites are essential for polarized cell growth in Schizosaccharomyces pombe.

The Ndr-related Orb6 kinase is a key regulator of polarized cell growth in fission yeast, however the mechanism of Orb6 activation is unclear. Activation of other Ndr kinases involves both autophosphorylation and phosphorylation by an upstream kinase. Previous reports suggest that the Nak1 kinase functions upstream from Orb6. Supporting this model, we show that HA-Orb6 overexpression partially restored cell polarity in nak1 ts cells. We also demonstrated by coimmunoprecipitation and in vitro binding assays that Nak1 and Orb6 physically interact, and that the Nak1 C-terminal region is required for Nak1/Orb6 complex formation in vivo. However, results from in vitro kinase assays did not show phosphorylation of recombinant Orb6 by HA-Nak1, suggesting that Orb6 activation may not involve direct phosphorylation by Nak1. To investigate the role of Orb6 phosphorylation and activity, we substituted Ala at the ATP-binding and conserved phosphorylation sites. Overexpression of kinase-dead HA-Orb6(K122A) in wild-type cells resulted in a loss of cell polarity, suggesting that it has a dominant-negative effect, and it failed to rescue the polarity defect of nak1 or orb6 ts mutants. Recombinant GST-Orb6(S291A) did not autophosphorylate in vitro suggesting that Ser291 is the primary autophosphorylation site. HA-Orb6(S291A) overexpression only partially rescued the orb6 polarity defect and failed to rescue the nak1 defect, suggesting that autophosphorylation is important for Orb6 function. GST-Orb6(T456A) autophosphorylated in vitro, indicating that the conserved phosphorylation site at Thr456 is not essential for kinase activity. However, HA-Orb6(T456A) overexpression had similar effects as overexpressing kinase-dead HA-Orb6(K122A), suggesting that Thr456 is essential for Orb6 function in vivo. Also, we found that both phosphorylation site mutations impaired the ability of Myc-Nak1 to coimmunoprecipitate with HA-Orb6. Together, our results suggest a model whereby autophosphorylation of Ser291 and phosphorylation of Thr456 by an upstream kinase promote Nak1/Orb6 complex formation and Orb6 activation.

ING1a expression increases during replicative senescence and induces a senescent phenotype.

The ING family of tumor suppressor proteins affects cell growth, apoptosis and response to DNA damage by modulating chromatin structure through association with different HAT and HDAC complexes. The major splicing isoforms of the ING1 locus are ING1a and INGlb. While INGlb plays a role in inducing apoptosis, the function of ING1a is currently unknown. Here we show that alternative splicing of the ING1 message alters the INGla:INGlb ratio by approximately 30-fold in senescent compared to low passage primary fibroblasts. INGla antagonizes INGlb function in apoptosis, induces the formation of structures resembling senescence-associated heterochromatic foci containing heterochromatin protein 1 gamma, the accumulation of senescence-associated beta-galactosidase activity and promotes senescent cell morphology and cell cycle arrest. Phenotypic effects may result from differential effects on gene expression since ING1a increases levels of both retinoblastoma and the p16 cyclin-dependent kinase inhibitor and ING1a and ING1b have opposite effects on the expression of proliferating nuclear cell antigen (PCNA), which is required for cell growth. Gene expression appears to be altered by targeting of HDAC complexes to gene promoters since INGla associates with several-fold higher levels of HDAC1 in senescent, compared to replication-competent cells and ING1 is found on the PCNA promoter by chromatin immunoprecipitation analysis. These data demonstrate a novel role for the ING1 proteins in differentially regulating senescence-associated chromatin remodeling vs. apoptosis and support the idea that altered ratios of the ING1 splicing isoforms may contribute to establishing the senescent phenotype through HDAC and HAT complex-mediated effects on chromatin structure.

Raf60, a novel component of the Rpd3 histone deacetylase complex required for Rpd3 activity in Saccharomyces cerevisiae.

The Rpd3 histone deacetylase complex contains several previously characterized proteins, including Rpd3, Sin3, Sds3, Sap30, and Pho23. We purified the Rdp3 complex to near homogeneity using the tandem affinity purification method. Mass spectrometric analysis revealed the presence of a novel component, which we named Raf60. We showed that Myc-Raf60 co-fractionated with Rpd3-TAP by gel filtration chromatography, and both Myc-Rpd3 and Sin3 co-immunoprecipitated with HA-Raf60. In addition, HA-Raf60 immunoprecipitates displayed Rpd3-dependent histone deacetylase activity, and raf60 deletion resulted in loss of Rpd3 complex activity, as measured by in vitro assays. Furthermore, we found that raf60Delta cells exhibited phenotypes similar to those of rpd3Delta cells, including derepression of secreted acid phosphatase (Pho5), hypersensitivity to cycloheximide, and hypersensitivity to heat shock. Also, we found by reverse transcription-PCR that raf60Delta cells, similar to rpd3Delta cells, displayed elevated levels of PHO5 and INO1 mRNA. Our results demonstrate that Raf60 is a component of the Rpd3 histone deacetylase complex and that it is required for normal Rpd3 complex activity and repression of gene expression.

Nak1 interacts with Hob1 and Wsp1 to regulate cell growth and polarity in Schizosaccharomyces pombe.

We have previously reported that Nak1, a group-II germinal center (GC) kinase, is essential for polarized growth in Schizosaccharomyces pombe. Here, we provide evidence that Nak1 regulates cell growth and polarity, in part, through its interactions with Hob1 (an Rvs167/amphiphysin homolog) and Wsp1 (Wiskott-Aldrich-syndrome-protein homolog). We found that Nak1, Hob1 and Wsp1 interact physically, and that both Hob1/green-fluorescent-protein (Hob1-GFP) and Wsp1-GFP fusion proteins localized to F-actin patches at growing cell ends and medial division sites. Hob1-GFP was dissociated from patches in cells lacking Wsp1. Also, Hob1 overexpression dissociated Wsp1-GFP from foci, inhibited Wsp1-directed F-actin formation in vitro and partially restored polarity defects associated with Wsp1 overexpression or nak1 repression. Furthermore, loss of both Wsp1 and Hob1 resulted in rounded cells, slow growth and multiple septae. Together, these observations suggest that Hob1 and Wsp1 cooperate to mediate cell polarity, growth and division. Repression of nak1 resulted in a random redistribution of Hob1-GFP and Wsp1-GFP foci, and inhibition of Wsp1-directed F-actin formation in vitro. Furthermore, hob1delta and wsp1delta mutants exhibited synthetic growth defects in combination with nak1 repression, suggesting that Nak1 has redundant functions with Hob1 and Wsp1. Collectively, our results suggest that Nak1 both regulates and cooperates with Hob1 and Wsp1 to promote F-actin formation and polarized cell growth.

Nak1, an essential germinal center (GC) kinase regulates cell morphology and growth in Schizosaccharomyces pombe.

We have identified and characterized Nak1, a 652- amino acid NH(2)-terminal kinase belonging to the group II germinal center kinase (GCK) family, in Schizosaccharomyces pombe. We found that nak1 is essential for cell proliferation. Furthermore, partial repression of nak1, under regulation of an integrated nmt1 promoter, resulted in an aberrant round cellular morphology, actin and microtubule mislocalization, slow growth, and cell division defects. Overexpression of either a kinase-inactive mutant (Nak1(K39R)) or the non-catalytic domain resulted in similar phenotypes, suggesting dominant-negative effects. By deletion analysis, we mapped the region responsible for this dominant-negative effect to the COOH-terminal 99 residues. Furthermore, we found that deletion of the COOH-terminal 99 residues inhibited Nak1 autophosphorylation, and expression of a partially inactive (Nak1(T171A)) or truncated (Nak1(1-562)) protein only weakly suppressed morphological and growth phenotypes, indicating that both kinase and COOH-terminal regions are important for Nak1 function. GFP-Nak1 localized uniformly throughout the cytoplasm, unlike many other proteins which influence cell polarity that preferentially localize to cell ends. Together, our results implicate Nak1 in the regulation of cell polarity, growth, and division and suggest that the COOH-terminal end plays an important role in the regulation of this kinase.

Human ING1 proteins differentially regulate histone acetylation.

ING1 proteins are nuclear, growth inhibitory, and regulate apoptosis in different experimental systems. Here we show that similar to their yeast homologs, human ING1 proteins interact with proteins associated with histone acetyltransferase (HAT) activity, such as TRRAP, PCAF, CBP, and p300. Human ING1 immunocomplexes contain HAT activity, and overexpression of p33(ING1b), but not of p47(ING1a), induces hyperacetylation of histones H3 and H4, in vitro and in vivo at the single cell level. p47(ING1a) inhibits histone acetylation in vitro and in vivo and binds the histone deacetylase HDAC1. Finally, we present evidence indicating that p33(ING1b) affects the degree of physical association between proliferating cell nuclear antigen (PCNA) and p300, an association that has been proposed to link DNA repair to chromatin remodeling. Together with the finding that human ING1 proteins bind PCNA in a DNA damage-dependent manner, these data suggest that ING1 proteins provide a direct linkage between DNA repair, apoptosis, and chromatin remodeling via multiple HAT.ING1.PCNA protein complexes.

Pnk1, a DNA kinase/phosphatase required for normal response to DNA damage by gamma-radiation or camptothecin in Schizosaccharomyces pombe.

We report the characterization of Pnk1, a 45-kDa homolog of the human polynucleotide kinase PNKP in Schizosaccharomyces pombe. Recombinant Pnk1 like human PNKP exhibits both 5'-DNA kinase and 3'-DNA phosphatase activities in vitro. Furthermore, we detected 3'-DNA phosphatase activity with a single-stranded substrate in extracts from wild-type yeast, but no activity was detected in pnk1delta strains. We have shown that GFP-tagged Pnk1 like mammalian PNKP localizes to the nucleus. Deletion of pnk1 does not affect cell growth under normal conditions but results in significant hypersensitivity to gamma-radiation or camptothecin, an inhibitor of topoisomerase I, suggesting that Pnk1 plays an important role in the repair of DNA strand breaks produced by these agents. The pnk1 deletion mutants were not hypersensitive to ethyl methanesulfonate, methyl methanesulfonate, or 4-nitroquinoline N-oxide. Expression of human PNKP in pnk1delta cells restores resistance to gamma-radiation or camptothecin, suggesting that the functions of yeast Pnk1 and human PNKP have been conserved.