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Werner's syndrome (WS) is an autosomal recessive disorder in humans characterized by the premature development of a partial array of age-associated pathologies. WRN, the gene defective in WS, encodes a 1432 amino acid protein (hWRN) with intrinsic 3'-->5' DNA helicase activity. We recently showed that hWRN is also a 3'-->5' exonuclease. Here, we further characterize the hWRN exonuclease. hWRN efficiently degraded the 3' recessed strands of double-stranded DNA or a DNA-RNA heteroduplex. It had little or no activity on blunt-ended DNA, DNA with a 3' protruding strand, or single-stranded DNA. The hWRN exonuclease efficiently removed a mismatched nucleotide at a 3' recessed terminus, and was capable of initiating DNA degradation from a 12-nt gap, or a nick. We further show that the mouse WRN (mWRN) is also a 3'-->5' exonuclease, with substrate specificity similar to that of hWRN. Finally, we show that hWRN forms a trimer and interacts with the proliferating cell nuclear antigen in vitro. These findings provide new data on the biochemical activities of WRN that may help elucidate its role(s) in DNA metabolism.

A rapid method for mapping exposed cytosine residues in 5'-[32P]-labeled RNA molecules is suggested. The exposed cytosines (C's) are converted into uracyls (U's) by bisulphite treatment at pH 5.8 in the presence of Mg2+, followed by complete modification of the residual (non-exposed) C's by a methoxyamine and bisulphite mixture at pH 5.0. The control RNA is modified only by methoxyamine and bisulphite without the preliminary C?U conversion. The location of the exposed C's is determined by comparing the products of partial T1, T2, A and U2 ribonuclease digestions of the C ? U converted and control RNAs after slab gel polyacrylamide electrophoresis and autoradiography. The method has been applied for mapping exposed cytosine bases in tRNATrp (yeast) which have been found in the anticodon loop and at the 3'-end of the molecule. In tRNATrp (beef liver), in addition to the same exposed bases, C in the diHU-loop is exposed. The data obtained are in full agreement with that is known about exposed C's for other tRNAs.