MRNIP is a replication fork protection factor.

The remodeling of stalled replication forks to form four-way DNA junctions is an important component of the replication stress response. Nascent DNA at the regressed arms of these reversed forks is protected by RAD51 and the tumor suppressors BRCA1/2, and when this function is compromised, stalled forks undergo pathological MRE11-dependent degradation, leading to chromosomal instability. However, the mechanisms regulating MRE11 functions at reversed forks are currently unclear. Here, we identify the MRE11-binding protein MRNIP as a novel fork protection factor that directly binds to MRE11 and specifically represses its exonuclease activity. The loss of MRNIP results in impaired replication fork progression, MRE11 exonuclease-dependent degradation of reversed forks, persistence of underreplicated genomic regions, chemosensitivity, and chromosome instability. Our findings identify MRNIP as a novel regulator of MRE11 at reversed forks and provide evidence that regulation of specific MRE11 nuclease activities ensures protection of nascent DNA and thereby genome integrity.

A systems comparison of once- versus twice-daily milking of pastured dairy cows.

The objective of this study was to compare the effect of milking frequency (once vs. twice-daily milking) and breed (Holstein-Friesians vs. Jerseys) on milk and milk solids (MS; milk fat + milk protein), yield per cow, milk composition, somatic cell count and lactation length; cow body weight, body condition score, and reproductive performance over a 4-yr period. Total cow numbers in each herd were 30, 35, 36, and 42 for Holstein-Friesians milked once or twice daily, and Jerseys milked once or twice daily, respectively. Forty hectares of pasture were subdivided into 4 smaller pastures of 10 ha each. Stocking rates for the once-daily herds were 16.7% greater than the twice-daily herd in their respective breed. An increased stocking rate was chosen to achieve equal milk and MS per ha from the 2 milking frequencies. Annual milk, fat, protein, and lactose yields per cow were less for once-daily than for twice-daily milking. Interactions were detected between milking frequency and breed for annual milk, fat, protein, and lactose yields per cow, because Jerseys were relatively less affected by once-daily than by twice-daily milking than Holstein-Friesians. Holstein-Friesian cows milked once daily produced 31.2% less milk and 29.4% less MS per cow than their twice-daily counterparts. In contrast, Jersey cows milked once daily produced 22.1% less milk and 19.9% less MS per cow than their twice-daily counterparts. Milk per ha was 17.7 and 9% less for the once-daily Holstein-Friesians and once-daily Jersey herds, respectively, compared with their twice-daily counterparts, because the greater stocking rate for the once-daily herds did not fully compensate for the milk loss per cow. Milking once daily increased somatic cell count throughout the year in both breeds. Cows milked once daily conceived 3 d earlier, took 5 d less from calving to conception, and needed 11% fewer controlled internal drug release devices than those milked twice daily. Milking once daily is a viable milking option for New Zealand farmers who are prepared to trade-off loss of MS income for increased time to accomplish other non-milking activities.

Low-dose hyper-radiosensitivity: a consequence of ineffective cell cycle arrest of radiation-damaged G2-phase cells.

This review highlights the phenomenon of low-dose hyper- radiosensitivity (HRS), an effect in which cells die from excessive sensitivity to small single doses of ionizing radiation but become more resistant (per unit dose) to larger single doses. Established and new data pertaining to HRS are discussed with respect to its possible underlying molecular mechanisms. To explain HRS, a three-component model is proposed that consists of damage recognition, signal transduction and damage repair. The foundation of the model is a rapidly occurring dose-dependent pre-mitotic cell cycle checkpoint that is specific to cells irradiated in the G2phase. This checkpoint exhibits a dose expression profile that is identical to the cell survival pattern that characterizes HRS and is probably the key control element of low-dose radiosensitivity. This premise is strengthened by the recent observation coupling low- dose radiosensitivity of G2-phase cells directly to HRS. The putative role of known damage response factors such as ATM, PARP, H2AX, 53BP1 and HDAC4 is also included within the framework of the HRS model.

Expression of the DNA-PK binding protein E4-34K fails to confer radiation sensitivity to mammalian cells.

PURPOSE: The adenovirus E4orf6 34 kDa protein (E4-34k) is known to disrupt V(D)J recombination as a result of its interaction with the catalytic subunit of cellular DNA-dependent protein kinase (DNA-PK(cs)), a major participant in the repair of DNA double-strand breaks (DSB). Previous studies have shown that cells with disrupted DSB repair and V(D)J recombination due to attenuation of DNA-PK(cs) activity exhibit a radiation-sensitive phenotype. It is not known at present whether the E4-34k protein can also modify cellular response to ionizing radiation. In an attempt to develop a novel gene therapy strategy to modify cellular radiation response, we sought to determine if expression of the adenovirus E4-34k protein resulted in sensitization to clinically relevant doses of ionizing radiation. MATERIALS AND METHODS: In order to minimize potential bias resulting from selection procedures, we performed clonogenic survival assays on DU 145 prostate cancer cells, RKO colorectal cancer cells and 293 kidney cells following transient transfection of E4-34k- and/or E1B-55k-expressing plasmids. Western blots and immunohistochemical analyses were used to demonstrate E4-34k expression within transfected cells. FACS sorting was carried out to enrich cells transfected with a plasmid that expresses both E4-34k and enhanced green fluorescent protein. RESULTS: It is shown that E4-34k expression does not affect cellular radiosensitivity of transiently transfected populations of either DU 145 prostate or RKO colon cancer cell lines. Similarly, the radiosensitivity of human embryonic kidney 293 cells, which constitutively express the E1B-55k protein, was also unaffected. The radiosensitivity of DU 145 cells co-transfected with E4-34k- and E1-55K-expressing plasmids was unchanged, suggesting that the adenovirus E1B-55k protein does not augment any effects E4-34k might have on DNA-PK(cs) activity. CONCLUSIONS: The lack of radiosensitization by E4-34k expression is quite intriguing as it is known that E4-34k interaction with DNA-PK(cs) causes disruption of V(D)J recombination, a process dependent on DSB rejoining. These data suggest that for future studies, preferential targeting of DNA-PK(cs) DSB activity will be required to influence cellular radiosensitivity.

Development of a novel rapid assay to assess the fidelity of DNA double-strand-break repair in human tumour cells.

Cellular survival following ionising radiation-mediated damage is primarily a function of the ability to successfully detect and repair DNA double-strand breaks (DSBs). Previous studies have demonstrated that radiosensitivity, determined as a reduction in colony forming ability in vitro, may be related to the incorrect repair (misrepair) of DSBs. The novel rapid dual fluorescence (RDF) assay is a plasmid-based reporter system that rapidly assesses the correct rejoining of a restriction-enzyme produced DSBs within transfected cells. We have utilised this novel assay to determine the fidelity of DSB repair in the prostate tumour cell line LNCaP, the bladder tumour cell line MGH-U1 and a radiosensitive subclone S40b. The two bladder cell lines have been shown in previous studies to differ in their ability to correctly repair plasmids containing a single DSB. Using the RDF assay we found that a substantial portion of LNCaP cells [80.4 +/- 5.3(standard error)%] failed to reconstitute reporter gene expression; however, there was little difference in this measure of DSB repair fidelity between the two bladder cell lines (48.3 +/- 3.5% for MGH-U1; 39.9 +/- 8.2% for S40b). The RDF assay has potential to be developed to study the relationship between DSB repair fidelity and radiosensitivity as well as the mechanisms associated with this type of repair defect.

Ribozyme minigene-mediated RAD51 down-regulation increases radiosensitivity of human prostate cancer cells.

The strand transferase RAD51 is a component of the homologous recombination repair pathway. To examine the contribution of RAD51 to the genotoxic effects of ionising radiation, we have used a novel ribozyme strategy. A reporter gene vector was constructed so that expression of an inserted synthetic double-stranded ribozyme-encoding oligonucleotide would be under the control of the cytomegalovirus immediate-early gene enhancer/promoter system. The prostate tumour cell line LNCaP was transfected with this vector or a control vector, and a neomycin resistance gene on the vector was used to create geneticin-resistant stable cell lines. Three stable cell lines were shown by western blot analysis to have significant down-regulation of RAD51 to 20-50% of the levels expressed in control cell lines. All three cell lines had a similar increased sensitivity to gamma-irradiation by 70 and 40%, respectively, compared to normal and empty vector-transfected cells, corresponding to dose-modifying factors of approximately 2.0 and 1.5 in the mid-range of the dose-response curves. The amount of RAD51 protein in transfected cell lines was shown to strongly correlate with the alpha parameter obtained from fitted survival curves. These results highlight the importance of RAD51 in cellular responses to radiation and are the first to indicate the potential use of RAD51-targeted ribozyme minigenes in tumour radiosensitisation.