Cell Cycle Checkpoint Function Research Articles

The human XRCC9 gene corrects chromosomal instability and mutagen sensitivities in CHO UV40 cells.

The Chinese hamster ovary (CHO) mutant UV40 cell line is hypersensitive to UV and ionizing radiation, simple alkylating agents, and DNA cross-linking agents. The mutant cells also have a high level of spontaneous chromosomal aberrations and 3-fold elevated sister chromatid exchange. We cloned and sequenced a human cDNA, designated XRCC9, that partially corrected the hypersensitivity of UV40 to mitomycin C, cisplatin, ethyl methanesulfonate, UV, and gamma-radiation. The spontaneous chromosomal aberrations in XRCC9 cDNA transformants were almost fully corrected whereas sister chromatid exchanges were unchanged. The XRCC9 genomic sequence was cloned and mapped to chromosome 9p13. The translated XRCC9 sequence of 622 amino acids has no similarity with known proteins. The 2.5-kb XRCC9 mRNA seen in the parental cells was undetectable in UV40 cells. The mRNA levels in testis were up to 10-fold higher compared with other human tissues and up to 100-fold higher compared with other baboon tissues. XRCC9 is a candidate tumor suppressor gene that might operate in a postreplication repair or a cell cycle checkpoint function.

Dpb11, which interacts with DNA polymerase II(epsilon) in Saccharomyces cerevisiae, has a dual role in S-phase progression and at a cell cycle checkpoint.

DPB11, a gene that suppresses mutations in two essential subunits of Saccharomyces cerevisiae DNA polymerase II(epsilon) encoded by POL2 and DPB2, was isolated on a multicopy plasmid. The nucleotide sequence of the DPB11 gene revealed an open reading frame predicting an 87-kDa protein. This protein is homologous to the Schizosaccharomyces pombe rad4+/cut5+ gene product that has a cell cycle checkpoint function. Disruption of DPB11 is lethal, indicating that DPB11 is essential for cell proliferation. In thermosensitive dpb11-1 mutant cells, S-phase progression is defective at the nonpermissive temperature, followed by cell division with unequal chromosomal segregation accompanied by loss of viability.dpb11-1 is synthetic lethal with any one of the dpb2-1, pol2-11, and pol2-18 mutations at all temperatures. Moreover, dpb11 cells are sensitive to hydroxyurea, methyl methanesulfonate, and UV irradiation. These results strongly suggest that Dpb11 is a part of the DNA polymerase II complex during chromosomal DNA replication and also acts in a checkpoint pathway during the S phase of the cell cycle to sense stalled DNA replication.

Cell cycle checkpoints and DNA repair preserve the stability of the human genome.

Chemical carcinogenesis in the regenerating rat liver is cell-cycle-dependent. Proliferating hepatocytes were maximally susceptible to initiation by a single dose of benzo[a]pyrene diolepoxide I when at the G1/S border. Hepatocytes in early G1 or late S/G2/M were less susceptible and non-proliferating G0 hepatocytes were resistant to initiation. Radiation clastogenesis in proliferating human fibroblasts also is cell-cycle-dependent. Ultraviolet radiation (UV) induced maximal frequencies of chromosomal aberrations in synchronized cells that were at the G1/S border. Cells in early G1 or G2 were significantly less sensitive. For both initiation of chemical carcinogenesis and UV-clastogenesis, it appears that replication of damaged DNA is required and DNA repair before replication reduces cellular risk. If DNA repair is protective, cell cycle checkpoints which delay DNA replication and mitosis should augment this protective influence by providing more time for repair. The contribution of cell cycle checkpoint function to DNA repair during cell cycle-dependent clastogenesis was studied using ataxia telangiectasia (AT) fibroblasts. The AT cells displayed a defect in the coupling of DNA damage to checkpoints which control the G1/S and G2/M transitions and the rate of replicon initiation in S phase cells. UV-clastogenesis in AT cells was cell-cycle-dependent with irradiation at the G1/S boundary inducing 3-times more aberrations than treatment in G0 at the time of release into the cell cycle. Thus, DNA excision repair during the pre-replicative G1 phase was protective even in cells with defective checkpoint function. However, following irradiation at the G1/S border, AT cells displayed about 6-fold increased levels of UV-induced chromosome aberrations in comparison to normal human fibroblasts that were treated at this time. These observations indicate that secondary and tertiary DNA lesions that are produced during replication of UV-damaged DNA (replicative gaps and double-strand breaks) also depend on checkpoint function for repair. The replicon initiation and G2-delay checkpoints that operate after initiation of S phase appear to play a major role in protection against UV-clastogenesis.