Requirement of proliferating cell nuclear antigen in RAD6-dependent postreplicational DNA repair.

The proliferating cell nuclear antigen (PCNA) acts as a processivity factor for replicative DNA polymerases and is essential for DNA replication. In vitro studies have suggested a role for PCNA-in the repair synthesis step of nucleotide excision repair, and PCNA interacts with the cyclin-dependent kinase inhibitor p21. However, because of the lack of genetic evidence, it is not clear which of the DNA repair processes are in fact affected by PCNA in vivo. Here, we describe a PCNA mutation, pol30-46, that confers ultraviolet (UV) sensitivity but has no effect on growth or cell cycle progression, and the mutant pcna interacts normally with DNA polymerase delta and epsilon. Genetic studies indicate that the pol30-46 mutation is specifically defective in RAD6-dependent postreplicational repair of UV damaged DNA, and this mutation impairs the error-free mode of bypass repair. These results implicate a role for PCNA as an intermediary between DNA replication and postreplicational DNA repair.

Fibroblast growth factor (FGF) homologous factors: new members of the FGF family implicated in nervous system development.

Four new members of the fibroblast growth factor (FGF) family, referred to as fibroblast growth factor homologous factors (FHFs), have been identified by a combination of random cDNA sequencing, data base searches, and degenerate PCR. Pairwise comparisons between the four FHFs show between 58% and 71% amino acid sequence identity, but each FHF shows less than 30% identity when compared with other FGFs. Like FGF-1 (acidic FGF) and FGF-2 (basic FGF), the FHFs lack a classical signal sequence and contain clusters of basic residues that can act as nuclear localization signals. In transiently transfected 293 cells FHF-1 accumulates in the nucleus and is not secreted. Each FHF is expressed in the developing and adult nervous systems, suggesting a role for this branch of the FGF family in nervous system development and function.

The redox/DNA repair protein, Ref-1, is essential for early embryonic development in mice.

The DNA-binding activity of AP-1 proteins is modulated, in vitro, by a posttranslational mechanism involving reduction oxidation. This mode of regulation has been proposed to control both the transcriptional activity and the oncogenic potential of Fos and Jun. Previous studies revealed that reduction of oxidized Fos and Jun by a cellular protein, Ref-1, stimulates sequence-specific AP-1 DNA-binding activity. Ref-1, a bifunctional protein, is also capable of initiating the repair of apurinic/apyrymidinic sites in damaged DNA. The relationship between the redox and DNA repair activities of Ref-1 is intriguing; both activities have been suggested to play an important role in the cellular response to oxidative stress. To investigate the physiological function of Ref-1, we used a gene targeting strategy to generate mice lacking a functional ref-1 gene. We report here that heterozygous mutant mice develop into adulthood without any apparent abnormalities. In contrast, homozygous mutant mice, lacking a functional ref-1 gene, die during embryonic development. Detailed analysis indicates that death occurs following blastocyst formation, shortly after the time of implantation. Degeneration of the mutant embryos is clearly evident at embryonic day 5.5. These findings demonstrate that Ref-1 is essential for early embryonic development.

APC mutations in colorectal tumors with mismatch repair deficiency.

We have investigated the influence of genetic instability [replication error (RER) phenotype] on APC (adenomatous polyposis coli), a gene thought to initiate colorectal tumorigenesis. The prevalence of APC mutations was similar in RER and non-RER tumors, indicating that both tumor types share this step in neoplastic transformation. However, in a total of 101 sequenced mutations, we noted a substantial excess of APC frameshift mutations in the RER cases (70% in RER tumors versus 47% in non-RER tumors, P < 0.04). These frameshifts were characteristic of mutations arising in cells deficient in DNA mismatch repair, with a predilection for mononucleotide repeats in the RER tumors (P < 0.0002), particularly (A)n tracts (P < 0.00007). These findings suggest that the genetic instability that is reflected by the RER phenotype precedes, and is responsible for, APC mutation in RER large bowel tumors and have important implications for understanding the very earliest stages of neoplasia in patients with tumors deficient in mismatch repair.

Ultraviolet-induced movement of the human DNA repair protein, Xeroderma pigmentosum type G, in the nucleus.

Xeroderma pigmentosum type G (XPG) is a human genetic disease exhibiting extreme sensitivity to sunlight. XPG patients are defective XPG endonuclease, which is an enzyme essential for DNA repair of the major kinds of solar ultraviolet (UV)-induced DNA damages. Here we describe a novel dynamics of this protein within the cell nucleus after UV irradiation of human cells. Using confocal microscopy, we have localized the immunofluorescent, antigenic signal of XPG protein to foci throughout the cell nucleus. Our biochemical studies also established that XPG protein forms a tight association with nuclear structure(s). In human skin fibroblast cells, the number of XPG foci decreased within 2 h after UV irradiation, whereas total nuclear XPG fluorescence intensity remained constant, suggesting redistribution of XPG from a limited number of nuclear foci to the nucleus overall. Within 8 h after UV, most XPG antigenic signal was found as foci. Using beta-galactosidase-XPG fusion constructs (beta-gal-XPG) transfected into HeLa cells, we have identified a single region of XPG that is evidently responsible both for foci formation and for the UV dynamic response. The fusion protein carrying the C terminus of XPG (amino acids 1146-1185) localized beta-gal specific antigenic signal to foci and to the nucleolus regions. After UV irradiation, antigenic beta-gal translocated reversibly from the subnuclear structures to the whole nucleus with kinetics very similar to the movements of XPG protein. These findings lead us to propose a model in which distribution of XPG protein may regulate the rate of DNA repair within transcriptionally active and inactive compartments of the cell nucleus.

Lack of the DNA repair protein O6-methylguanine-DNA methyltransferase in histologically normal brain adjacent to primary human brain tumors.

Exposure to exogenous alkylating agents, particularly N-nitroso compounds, has been associated with increased incidence of primary human brain tumors, while intrinsic risk factors are currently unknown. The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) is a major defense against the carcinogenicity of N-nitroso compounds and other alkylators. We report here that in 55% (64/117) of cases, histologically normal brain tissue adjacent to primary human brain tumors lacked detectable MGMT activity [methyl excision repair-defective (Mer-) status]. The incidence of Mer- status in normal brain tissue from brain tumor patients was age-dependent, increasing from 21% in children 0.25-19 years of age to 75% in adults over 50. In contrast, Mer- status was found in 12% (5/43) of normal brain specimens from patients operated for conditions other than primary brain tumors and was not age-dependent. The 4.6-fold elevation in incidence of Mer- status in brain tumor patients is highly significant (chi2 = 24; p < or = 0.001). MGMT activity was independent of age in the lymphocytes of brain tumor patients and was present in lymphocytes from six of nine tumor patients whose normal brain specimen was Mer-. DNA polymerase beta, apurinic/apyrimidinic endonuclease, and lactate dehydrogenase activities were present in all specimens tested, including Mer- specimens from brain tumor patients. Our data are consistent with a model of carcinogenesis in human brain in which epigenetically regulated lack of MGMT is a predisposing factor and alkylation-related mutagenesis is a driving force.

Characterization of promoters and stable transfection by homologous and nonhomologous recombination in Plasmodium falciparum.

Genetic studies of the protozoan parasite Plasmodium falciparum have been severely limited by the inability to introduce or modify genes. In this paper we describe a system of stable transfection of P. falciparum using a Toxoplasma gondii dihydrofolate reductase-thymidylate synthase gene, modified to confer resistance to pyrimethamine, as a selectable marker. This gene was placed under the transcriptional control of the P. falciparum calmodulin gene flanking sequences. Transfected parasites generally maintained plasmids episomally while under selection; however, parasite clones containing integrated forms of the plasmid were obtained. Integration occurred by both homologous and nonhomologous recombination. In addition to the flanking sequence of the P. falciparum calmodulin gene, the 5' sequences of the P. falciparum and P. chabaudi dihydrofolate reductase-thymidylate synthase genes were also shown to be transcriptionally active in P. falciparum. The minimal 5' sequence that possessed significant transcriptional activity was determined for each gene and short sequences containing important transcriptional control elements were identified. These sequences will provide considerable flexibility in the future construction of plasmid vectors to be used for the expression of foreign genes or for the deletion or modification of P. falciparum genes of interest.

Molecular cloning of a rat chromosome putative recombinogenic sequence homologous to the hepatitis B virus encapsidation signal.

Previously, we reported that a 61-bp subgenomic HBV DNA sequence (designated as 15AB, nt 1855-1915) is a hot spot for genomic recombination and that a cellular protein binding to 15AB may be the putative recombinogenic protein. In the present study, we established the existence of a 15AB-like sequence in human and rat chromosomal DNA by Southern blot analysis. The 15AB-like sequence isolated from the rat chromosome demonstrated a 80.9% identity with 5'-CCAAGCTGTGCCTTGGGTGGC-3', at 1872-1892 of the hepatitis B virus genome, thought to be the essential region for recombination. Interestingly, this 15AB-like sequence also contained the pentanucleotide motifs GCTGG and CCAGC as an inverted repeat, part of the chi known hot spot for recombination in Escherichia coli. Importantly, a portion of the 15AB-like sequence is homologous (82.1%, 23/28 bp) to break point clusters of the human promyelocytic leukemia (PML) gene, characterized by a translocation [t(15;17)], and to rearranged mouse DNA for the immunoglobulin kappa light chain. Moreover, 15AB and 15AB-like sequences have striking homologies (12/15 = 80.0% and 13/15 = 86.7%, respectively) to the consensus sequence for topoisomerase II. Our present results suggest that this 15AB-like sequence in the rat genome might be a recombinogenic candidate triggering genomic instability in carcinogenesis.

Targeted disruption of the Rad51 gene leads to lethality in embryonic mice.

The mouse Rad51 gene is a mammalian homologue of the Escherichia coli recA and yeast RAD51 genes, both of which are involved in homologous recombination and DNA repair. To elucidate the physiological role of RAD51 protein, the gene was targeted in embryonic stem (ES) cells. Mice heterozygous for the Rad51 null mutation were intercrossed and their offspring were genotyped. There were no homozygous (Rad51-/-) pups among 148 neonates examined but a few Rad51-/- embryos were identified when examined during the early stages of embryonic development. Doubly knocked-out ES cells were not detected under conditions of selective growth. These results are interpreted to mean that RAD51 protein plays an essential role in the proliferation of cell. The homozygous Rad51 null mutation can be categorized in cell-autonomous defects. Pre-implantational lethal mutations that disrupt basic molecular functions will thus interfere with cell viability.