Structural and functional conservation of the human homolog of the Schizosaccharomyces pombe rad2 gene, which is required for chromosome segregation and recovery from DNA damage

The rad2 mutant of Schizosaccharomyces pombe is sensitive to UV irradiation and deficient in the repair of UV damage. In addition, it has a very high degree of chromosome loss and/or nondisjunction. We have cloned the rad2 gene and have shown it to be a member of the Saccharomyces cerevisiae RAD2/S. pombe rad13/human XPG family. Using degenerate PCR, we have cloned the human homolog of the rad2 gene. Human cDNA has 55% amino acid sequence identity to the rad2 gene and is able to complement the UV sensitivity of the rad2 null mutant. We have thus isolated a novel human gene which is likely to be involved both in controlling the fidelity of chromosome segregation and in the repair of UV-induced DNA damage. Its involvement in two fundamental processes for maintaining chromosomal integrity suggests that it is likely to be an important component of cancer avoidance mechanisms

Role of Schizosaccharomyces pombe RecQ homolog recombination and checkpoint genes in UV Damage tolerance

The cellular responses to DNA damage are complex and include direct DNA repair pathways that remove the damage and indirect damage responses which allow cells to survive DNA damage that has not been, or cannot be, removed. We have identified the gene mutated in the rad12.502 strain as a Schizosaccharomyces pombe recQ homolog. The same gene (designated rqh1) is also mutated in the hus2.22 mutant. We show that Rqh1 is involved in a DNA damage survival mechanism which prevents cell death when UV-induced DNA damage cannot be removed. This pathway also requires the correct functioning of the recombination machinery and the six checkpoint tad gene products plus the Cds1 kinase. Our data suggest that Rqh1 operates during S phase as part of a mechanism which prevents DNA damage causing cell lethality. This process may involve the bypass of DNA damage sites by the replication fork. Finally, in contrast with the reported literature, we do not find that rqh1 (rad12) mutant cells are defective in UV dimer endonuclease activity

Cloning and characterisation of the rad9 DNA repair gene from Schizosaccharomyces pombe

The rad9.192 DNA repair mutant from the fission yeast, Schizosaccharomyces pombe, is sensitive to both UV and ionising radiation. The rad9 gene has been cloned by complementation of the gamma-ray sensitivity of the mutant cell line. A 4.3kb HindIII fragment was found to confer resistance to both types of radiation. The region of complementation was further localised to a 2.6kb HindIII-EcoRV fragment, which, by DNA sequence analysis, was found to contain sequences capable of coding for a 427 amino acid protein, if three introns were postulated to remove stop codons. The introns were confirmed by sequence analysis of cDNA clones and PCR products derived from cDNA. The product of transcription is a 1.6kb mRNA of low abundance. The putative rad9 protein shows no homology to any published sequence. A truncated protein is capable of complementing the radiation sensitivity of the rad9.192 mutant. Deletion of the gene is not lethal and the null allele has a similar phenotype to the rad9.192 mutant

Mouse Rad1 deletion enhances susceptibility for skin tumor development

Cells are constantly exposed to stresses from cellular metabolites as well as environmental genotoxins. DNA damage caused by these genotoxins can be efficiently fixed by DNA repair in cooperation with cell cycle checkpoints. Unrepaired DNA lesions can lead to cell death, gene mutation and cancer. The Rad1 protein, evolutionarily conserved from yeast to humans, exists in cells as monomer as well as a component in the 9-1-1 protein complex. Rad1 plays crucial roles in DNA repair and cell cycle checkpoint control, but its contribution to carcinogenesis is unknown. To address this question, we constructed mice with a deletion of Mrad1. Matings between heterozygous Mrad1 mutant mice produced Mrad1+/+ and Mrad1+/- but no Mrad1-/- progeny, suggesting the Mrad1 null is embryonic lethal. Mrad1+/- mice demonstrated no overt abnormalities up to one and half years of age. DMBA-TPA combinational treatment was used to induce tumors on mouse skin. Tumors were larger, more numerous, and appeared earlier on the skin of Mrad1+/- mice compared to Mrad1+/+ animals. Keratinocytes isolated from Mrad1+/- mice had significantly more spontaneous DNA double strand breaks, proliferated slower and had slightly enhanced spontaneous apoptosis than Mrad1+/+ control cells. These data suggest that Mrad1 is important for preventing tumor development, probably through maintaining genomic integrity. The effects of heterozygous deletion of Mrad1 on proliferation and apoptosis of keratinocytes is different from those resulted from Mrad9 heterozygous deletion (from our previous study), suggesting that Mrad1 also functions independent of Mrad9 besides its role in the Mrad9-Mrad1-Mhus1 complex in mouse cells

The S. pombe translation initiation factor eIF4G is sumoylated and associates with the SUMO protease Ulp2

SUMO is a small post-translational modifier, that is attached to lysine residues in target proteins. It acts by altering proteinprotein interactions, protein localisation and protein activity. SUMO chains can also act as substrates for ubiquitination, resulting in proteasome-mediated degradation of the target protein. SUMO is removed from target proteins by one of a number of specific proteases. The processes of sumoylation and desumoylation have well documented roles in DNA metabolism and in the maintenance of chromatin structure. To further analyse the role of this modification, we have purified protein complexes containing the S. pombe SUMO protease, Ulp2. These complexes contain proteins required for ribosome biogenesis, RNA stability and protein synthesis. Here we have focussed on two translation initiation factors that we identified as co-purifying with Ulp2, eIF4G and eIF3h. We demonstrate that eIF4G, but not eIF3h, is sumoylated. This modification is increased under conditions that produce cytoplasmic stress granules. Consistent with this we observe partial co-localisation of eIF4G and SUMO in stressed cells. Using HeLa cells, we demonstrate that human eIF4GI is also sumoylated; in vitro studies indicate that human eIF4GI is modified on K1368 and K1588, that are located in the C-terminal eIF4A- and Mnk-binding sites respectively

The Rad24 checkpoint protein of Saccharomyces cerevisiae: A complex problem

Rad24 functions in the DNA damage-dependent checkpoint pathway of Saccharomyces cerevisiae. Polyclonal antibodies were raised against Rad24 and other components of this pathway in order to investigate their biochemical functions. Studies demonstrated that Rad24, Mec3 or Rad17 did not interact with each other and did not appear to be modified after DNA damage treatment or during the cell cycle. Analysis of Rad24 in whole cell extracts demonstrated that its mass was considerably greater than its predicted molecular weight, suggesting that Rad24 is a component of a protein complex. A protocol was developed to purify the Rad24 complex to homogeneity. In addition to Rad24, the complex included polypeptides of 40kDa and 35kDa. The 40kDa species was found to contain Rfc2 and Rfc3 by mass spectrometry. Rfc2 and Rfc3 are subunits of Replication Factor C (RFC), a five subunit protein which is required for the loading of polymerases onto DNA during replication and repair. We hypothesised that other RFC subunits, all of which share sequence homologies with Rad24, might also be components of the Rad24 complex. Reciprocal co-immunoprecipitation studies were performed using extracts prepared from strains constructed containing epitope tagged RFC genes. These experiments showed that the small RFC proteins, Rfc2, Rfc3, Rfc4 and Rfc5 interact with Rad24, whereas the Rfc1 subunit does not. We suggest that this RFC-like Rad24 complex may function as a structure-specific sensor in the DNA damage checkpoint pathway. In order to address this hypothesis, conditional mutants of small Rfc subunits were tested for intact DNA damage responses outside of S phase. The biochemical activities of the Rad24 complex were investigated using a variety of techniques including gel mobility shift and ATP hydrolysis assays

Characterisation of the SUMO-like domains of Schizosaccharomyces pombe Rad60

The S. pombe Rad60 protein is required for the repair of DNA double strand breaks, recovery from replication arrest, and is essential for cell viability. It has two SUMO-like domains (SLDs) at its C-terminus, an SXS motif and three sequences that have been proposed to be SUMO-binding motifs (SBMs). SMB1 is located in the middle of the protein, SBM2 is in SLD1 and SBM3 is at the C-terminus of SLD2. We have probed the functions of the two SUMO-like domains, SLD1 and SLD2, and the putative SBMs. SLD1 is essential for viability, while SLD2 is not. rad60-SLD2Δ cells are sensitive to DNA damaging agents and hydroxyurea. Neither ubiquitin nor SUMO can replace SLD1 or SLD2. Cells in which either SBM1 or SBM2 has been mutated are viable and are wild type for response to MMS and HU. In contrast mutation of SBM3 results in significant sensitivity to MMS and HU. These results indicate that the lethality resulting from deletion of SLD1 is not due to loss of SBM2, but that mutation of SBM3 produces a more severe phenotype than does deletion of SLD2. Using chemical denaturation studies, FPLC and dynamic light scattering we show this is likely due to the destabilisation of SLD2. Thus we propose that the region corresponding to the putative SBM3 forms part of the hydrophobic core of SLD2 and is not a SUMO-interacting motif. Over-expression of Hus5, which is the SUMO conjugating enzyme and known to interact with Rad60, does not rescue rad60-SLD2Δ, implying that as well as having a role in the sumoylation process as previously described [1], Rad60 has a Hus5-independent function

Novel insights into the DNA interstrand cross-link repair in Schizosaccharomyces pombe: characterisation of Fan1 through standard and high-throughput genetic analysis

FAN1/MTMR15 (Fanconi anemia-associated nuclease 1 / Myotubularin-related protein 15) is a protein originally identified from a set of size-fractionated human brain cDNA libraries coding for large proteins in vitro (Nagase et al., 1999). FAN1 is widely conserved across eukaryotes, with the notable exception of S. cerevisiae (Smogorzewska et al., 2010; MacKay et al., 2010; Kratz et al., 2010; Liu et al., 2010; Shereda et al., 2010). Recent work has shown that FAN1 is a novel component of the Fanconi Anemia repair DNA pathway in higher eukaryotes (Smogorzewska et al., 2010; MacKay et al., 2010; Kratz et al., 2010; Yoshikiyo et al., 2010; Liu et al., 2010; Shereda et al., 2010). My work presents a biochemical and genetic characterisation of the FAN1 Schizosaccharomyces pombe ortholog Fan1. I show that, in contrast with the situation in higher eukaryotes, Fan1 in S. pombe does not strongly interact with components of the mismatch repair pathway. The disruption of fan1 causes a mild sensitivity to interstrand cross-linking agents, dramatically augmented by the concomitant deletion of the nuclease Pso2, suggesting a role for Fan1 in the resolution of DNA interstrand cross-links. Further genetic interactions are explored by use of an automated high-throughput screen, where a non-epistatic relationship is found with Pli1, a component of the SUMOylation pathway. Finally, I show that three conserved residues in the VRR_nuc nuclease domain are required for Fan1 activity in DNA repair. Taken together, the data presented points at a role for S. pombe Fan1 in the resolution of adducts created by DNA interstrand cross-linking agents

Early Steps in the DNA Base Excision Repair Pathway of a Fission Yeast Schizosaccharomyces pombe

DNA base excision repair (BER) accounts for maintaining genomic integrity by removing damaged bases that are generated endogenously or induced by genotoxic agents. In this paper, we describe the roles of enzymes functioning in the early steps of BER in fission yeast. Although BER is an evolutionarily conserved process, some unique features of the yeast repair pathway were revealed by genetic and biochemical approaches. AP sites generated by monofunctional DNA glycosylases are incised mainly by AP lyase activity of Nth1p, a sole bifunctional glycosylase in yeast, to leave a blocked 3′ end. The major AP endonuclease Apn2p functions predominantly in removing the 3′ block. Finally, a DNA polymerase fills the gap, and a DNA ligase seals the nick (Nth1p-dependent or short patch BER). Apn1p backs up Apn2p. In long patch BER, Rad2p endonuclease removes flap DNA containing a lesion after DNA synthesis. A UV-specific endonuclease Uve1p engages in an alternative pathway by nicking DNA on the 5′ side of oxidative damage. Nucleotide excision repair and homologous recombination are involved in repair of BER intermediates including the AP site and single-strand break with the 3′ block. Other enzymes working in 3′ end processing are also discussed

Nse2, a component of the Smc5-6 complex, is a SUMO ligase required for the response to DNA damage

The Schizosaccharomyces pombe SMC proteins Rad18 (Smc6) and Spr18 (Smc5) exist in a high-M(r) complex which also contains the non-SMC proteins Nse1, Nse2, Nse3, and Rad62. The Smc5-6 complex, which is essential for viability, is required for several aspects of DNA metabolism, including recombinational repair and maintenance of the DNA damage checkpoint. We have characterized Nse2 and show here that it is a SUMO ligase. Smc6 (Rad18) and Nse3, but not Smc5 (Spr18) or Nse1, are sumoylated in vitro in an Nse2-dependent manner, and Nse2 is itself autosumoylated, predominantly on the C-terminal part of the protein. Mutations of C195 and H197 in the Nse2 RING-finger-like motif abolish Nse2-dependent sumoylation. nse2.SA mutant cells, in which nse2.C195S-H197A is integrated as the sole copy of nse2, are viable, whereas the deletion of nse2 is lethal. Smc6 (Rad18) is sumoylated in vivo: the sumoylation level is increased upon exposure to DNA damage and is drastically reduced in the nse2.SA strain. Since nse2.SA cells are sensitive to DNA-damaging agents and to exposure to hydroxyurea, this implicates the Nse2-dependent sumoylation activity in DNA damage responses but not in the essential function of the Smc5-6 complex