Circadian oscillation of nucleotide excision repair in mammalian brain

Abstract

The circadian clock regulates the daily rhythms in the physiology and behavior of many organisms, including mice and humans. These cyclical changes at molecular and macroscopic levels affect the organism's response to environmental stimuli such as light and food intake and the toxicity and efficacy of chemo- and radiotherapeutic agents. In this work, we investigated the circadian behavior of the nucleotide excision repair capacity in the mouse cerebrum to gain some insight into the optimal circadian time for favorable therapeutic response with minimal side effects in cancer treatment with chemotherapeutic drugs that produce bulky adducts in DNA. We find that nucleotide excision repair activity in the mouse cortex is highest in the afternoon/evening hours and is at its lowest in the night/early morning hours. The circadian oscillation of the repair capacity is caused at least in part by the circadian oscillation of the xeroderma pigmentosum A DNA damage recognition protein.

Fig. 1.

Day and night nucleotide excision repair activity in the mouse brain. (A) Diagram of the excision assay used to measure repair capacity. A 140-bp duplex containing a centrally located (6-4) photoproduct (triangle) adjacent to a ³²P label (circle) is incubated with CFE, which removes the damage by dual incisions in the form of 24- to 32-nt-long oligomers. (B) Excision activities in cerebral CFEs of two mice killed at ZT06 (1300) and two killed at ZT18 (0100). Each lane represents brain extract from one mouse. (C) Quantitative analysis of the excision activity as a function of two circadian (ZT) times. The averages and standard deviations were calculated from three independent excision assays for each mouse (n = 6 for both ZT06 and ZT18). (D) Kinetics of excision activity of CFE, from ZT06 and ZT18 brains. Kinetic assays were conducted with two of the brain extracts used in B. Ten femtomoles of substrates were loaded as a control (lane 1). (E) Quantitative analysis of data in D.

Fig. 2.

Circadian oscillation of nucleotide excision repair activity in the mouse brain. (A) Excision assays with mice cerebrum extracts prepared from brains harvested at the indicated ZT and CT times. Ten femtomoles of substrates were loaded as a control. (B) Quantitative analysis of the excision activity as a function of ZT or CT. For ZT, each data point represents the average of excision activity from the cerebrums of three mice, and the bars represent SE. For CT, the data points represent the average of duplicate experiments conducted with extracts from a single mouse for each data point. Bars indicate SD.

Fig. 3.

Effect of the circadian clock on day and night expression patterns of clock, excision repair, and checkpoint proteins. (A–C) Extracts from brains harvested at ZT18 (night) and ZT06 (day) were analyzed by immunoblotting. (A) Clock proteins. Clock shows no oscillation, whereas Cry1 and Per2 oscillate antiphase with Bmal1. (B) Nucleotide excision repair proteins. XPA is highly expressed during the day and down-regulated at night. The other excision repair proteins do not exhibit a measurable variation between ZT18 and ZT06. (C) Checkpoint proteins. None of the checkpoint/cell cycle proteins tested exhibits a measurable difference between day (ZT06) and night (ZT18) values. (D) Circadian oscillation of XPA. Brains were harvested from mice under either LD or DD conditions, and the levels of XPA over a circadian period were analyzed by immunoblotting along with the four core clock proteins as a reference. Note the zenith values for XPA at afternoon/evening hours under both ZT and CT conditions are in phase with Bmal1 and antiphase with Cry1 and Per2 as expected for a Clock·Bmal1-controlled protein. Clock that does not oscillate is used as a loading control for a nonoscillating protein. (E) Quantitative analysis of XPA oscillation. Each data point represents XPA value from the cerebrum of one mouse, and the values are expressed relative to that of XPA at CT14, which was the highest.

Fig. 4.

Complementation of excision repair-defective nighttime brain extract with XPA protein. (A) Specificity of XPA complementation. To the ZT06 brain extract XPA or XPC was added at the indicated amounts in the reaction mixtures of the excision assays. Only the part of the gels containing the excision products is shown. The assay with the ZT06 extract is included as positive control. (B) Quantification of data shown in A. (C) Comparison of the effect of XPA on night- and daytime cerebrum extracts excision activities. ZT06 and ZT18 extracts were supplemented with the indicated amounts of XPA, and the excision assays were carried out for 60 min. (D) Quantitative analysis of data in C.