Calf thymus DNA polymerase epsilon readily uses short, synthetic oligonucleotides as substrates for both polymerase and exonuclease activity. These substrates were used to examine the mechanism of inhibition by aphidicolin. Aphidicolin competes with each of the four dNTPs for binding to a pol epsilon.DNA complex. Importantly, aphidicolin binds equally well regardless of the identity of the next template base to be replicated (Ki approximately 0.6 microM). Hydrolysis of synthetic templates of defined sequence by the 3'-->5' exonuclease was examined. pol epsilon preferred to hydrolyze single-stranded DNA 3-fold better than double-stranded DNA (Vmax/KM), while under Vmax conditions single-stranded DNA was hydrolyzed 100-fold faster than double-stranded DNA. Aphidicolin did not inhibit exonuclease activity on single-stranded DNA; however, activity on double-stranded DNA was partially inhibited. Formation of an E.[template.primer].aphidicolin ternary complex inhibits exonuclease activity. However, even under conditions where the polymerase site is completely blocked by a template-primer, the exonuclease retains significant activity.

Machado-Joseph disease, an autosomal dominant multisystem motor degeneration, has been described mainly in people of Portuguese descent. Our report documents the presence of Machado-Joseph disease in the Chinese population, based on the specific molecular marker of a CAG repeat array in the 3' end of the MJD gene. We screened 21 Chinese families with dominant spinocerebellar ataxia. The results showed that Machado-Joseph disease with CAG expansion accounted for 52% of families with autosomal dominant cerebellar ataxia in this series. The clinical characteristics, besides the well-documented cerebellar ataxia, dysarthria, nystagmus, corticospinal dysfunctions, a variable degree of facial muscle fasciculation, and proprioceptive loss, included loss of optokinetic nystagmus and autonomic nervous system dysfunction. The CAG repeat number in the MJD gene ranged from 14 to 39 among normal alleles, and from 63 to 81 among MJD alleles. There was a strong inverse correlation (gamma = -0.77) between number of CAG repeats and age at symptom onset, accounting for 60% of the variance of age at onset. A strong clinical anticipation of age at onset existed in successive generations. Mild instabilities of expanded CAG repeat numbers during meiotic transmission occurred, with no significant difference according to the gender of the transmitting parent. Finally, brain metabolism in Machado-Joseph disease, studied with positron emission tomography, was characterized by significant progressive regional hypometabolism in the occipital cortex, as well as the cerebellar hemispheres, vermis, and brainstem.