SummaryDNA damage can stall the DNA replication machinery, leading to genomic instability. Thus, numerous mechanisms exist to complete genome duplication in the absence of a pristine DNA template, but identification of the enzymes involved remains incomplete. Here, we establish that Primase-Polymerase (PrimPol; CCDC111), an archaeal-eukaryotic primase (AEP) in eukaryotic cells, is involved in chromosomal DNA replication. PrimPol is required for replication fork progression on ultraviolet (UV) light-damaged DNA templates, possibly mediated by its ability to catalyze translesion synthesis (TLS) of these lesions. This PrimPol UV lesion bypass pathway is not epistatic with the Pol η-dependent pathway and, as a consequence, protects xeroderma pigmentosum variant (XP-V) patient cells from UV-induced cytotoxicity. In addition, we establish that PrimPol is also required for efficient replication fork progression during an unperturbed S phase. These and other findings indicate that PrimPol is an important player in replication fork progression in eukaryotic cells.

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.SUMO is a small ubiquitin-like protein that is covalently attached to target proteins. In yeasts and lower eukaryotes, SUMO is encoded by a single gene, while in higher eukaryotes there are three isoforms, SUMO-1, SUMO-2, and SUMO-3. The attachment of SUMO to target proteins is similar to the process of ubiquitination: SUMO is produced as a precursor protein which is processed to the mature form by SUMO proteases, revealing a diglycine motif. SUMO is subsequently activated by the formation of a thioester bond with a cysteine residue on the SUMO E1-like activator enzyme, a heterodimer known as SAE. SUMO is then passed to an E2-like SUMO conjugator, with which it also forms a thioester bond at a cysteine residue. SUMO ligases have been identified in several organisms. However, whereas E3 ligases are required for the attachment of ubiquitin to targets both in vitro and in vivo, the requirement for SUMO ligases for the attachment of SUMO to targets appears to be less stringent in vitro, and possibly also in vivo. This would be consistent with reports that several SUMO target proteins interact directly with the E2-like SUMO conjugator (e.g., see reference 4).Two classes of SUMO ligases have been identified. Proteins in the first category contain C3HC4-like RING domains, while proteins in the second category do not. Members of the first category include the Saccharomyces cerevisiae proteins Siz1 and Siz2 (16) and the mammalian PIAS family of proteins (20,32,38). Members of the second category include the RanBP2 and Pc proteins (18, 33). In S. cerevisiae (budding yeast), the SIZ1 and SIZ2 genes are not essential for viability, and null mutants do not show the severe cell and nuclear morphologies (16) that are observed with mutants that are defective in other components of the sumoylation system (17, 39). It remains...

The xeroderma pigmentosum group D (XPD) protein has a dual function, both in nucleotide excision repair of DNA damage and in basal transcription. Mutations in the XPD gene can result in three distinct clinical phenotypes, XP, trichothiodystrophy (TTD), and XP with Cockayne syndrome. To determine if the clinical phenotypes of XP and TTD can be attributed to the sites of the mutations, we have identified the mutations in a large group of TTD and XP-D patients. Most sites of mutations differed between XP and TTD, but there are three sites at which the same mutation is found in XP and TTD patients. Since the corresponding patients were all compound heterozygotes with different mutations in the two alleles, the alleles were tested separately in a yeast complementation assay. The mutations which are found in both XP and TTD patients behaved as null alleles, suggesting that the disease phenotype was determined by the other allele. If we eliminate the null mutations, the remaining mutagenic pattern is consistent with the site of the mutation determining the phenotype.

A number of cell-cycle checkpoint genes have been shown to play important roles in meiosis. We have characterized the human and mouse counterpart of the Schizosaccharomyces pombe Rad3 protein, named Atr (for ataxia-telangiectasia-and rad3-related), and the protein that is mutated in ataxia-telangiectasia, Atm. We demonstrate that ATR mRNA and protein are expressed in human and mouse testis. More detailed analysis of specific cells in seminiferous tubules shows localization of Atr to the nuclei of cells in the process of meiosis I. Using immunoprecipitation and immunoblot analysis, we show that Atr and Atm proteins are-300 and 350 kD relative molecular mass, respectively, and further demonstrate that both proteins have associated protein kinase activity. Further, we demonstrate that Atr and Atm interact directly with meiotic chromosomes and show complementary localization patterns on synapsing chromosomes. Atr is found at sites along unpaired or asynapsed chromosomal axes, whereas Atm is found along synapsed chromosomal axes. This is the first demonstration of a nuclear association of Atr and Atm proteins with meiotic chromosomes and suggests a direct role for these proteins in recognizing and responding to DNA strand interruptions that occur during meiotic recombination. [ Mitotic cells guard against genetic instability and increase their survival following DNA damage both by direct DNA repair mechanisms and by delaying progression through the cell cycle. Depending on the position of the cell within the cell cycle at the time of irradiation, DNA damage in mammalian cells can effect specific checkpoints that prevent passage from G 1 to S phase, progression through S phase, or passage from G 2 into mitosis. These checkpoints are thought to prevent dele-terious events such as replication of damaged DNA or the segregation of fragmented chromosomes during mi-tosis (Hartwell and Kastan 1994) and therefore assure the accurate transmission of the genetic material.