Various 5'O-N-protected deoxynucleoside-3'-O-beta-cyanoethyl-N,N-dialkylamino-/N- morpholinophosphoramidites were prepared from beta-cyanoethyl monochlorophosphoramidites of N,N-dimethylamine, N,N-diisopropylamine and N-morpholine. These active deoxynucleoside phosphates have successfully been used for oligodeoxynucleotide synthesis on controlled pore glass as polymer support and are very suitable for automated DNA-synthesis due to their stability in solution. The intermediate dichloro-beta- cyanoethoxyphosphine can easily be prepared free from any PC1(3) contamination. The active monomers obtained from beta-cyanoethyl monochloro N,N- diisopropylaminophosphoramidites are favoured. Cleavage of the oligonucleotide chain from the polymer support, N-deacylation and deprotection of beta-cyanoethyl group from the phosphate triester moiety can be performed in one step with concentrated aqueous ammonia. Mixed oligodeoxynucleotides are characterized by the sequencing method of Maxam and Gilbert.

It is well known that exposure to UV induces DNA damage, which is the first step in mutagenesis and a major cause of skin cancer. Among a variety of photoproducts, cyclobutane-type pyrimidine photodimers (CPD) are the most abundant primary lesion. Despite its biological importance, the precise relationship between the structure and properties of DNA containing CPD has remained to be elucidated. Here, we report the free (unbound) crystal structure of duplex DNA containing a CPD lesion at a resolution of 2.0 Å. Our crystal structure shows that the overall helical axis bends Ϸ30°t oward the major groove and unwinds Ϸ9°, in remarkable agreement with some previous theoretical and experimental studies. There are also significant differences in local structure compared with standard B-DNA, including pinching of the minor groove at the 3 side of the CPD lesion, a severe change of the base pair parameter in the 5 side, and serious widening of both minor and major groves both 3 and 5 of the CPD. Overall, the structure of the damaged DNA differs from undamaged DNA to an extent that DNA repair proteins may recognize this conformation, and the various components of the replicational and transcriptional machinery may be interfered with due to the perturbed local and global structure. The cis-syn pyrimidine dimer (cyclobutane-type pyrimidine photodimer, CPD) is the major photoproduct induced by UV light present in sunlight (1) and is one of the principal causes of skin cancer (2). Evidence for the formation of the thymine dimer in DNA was first obtained Ͼ40 years ago (3, 4) and a few years later for cytosine-containing CPDs (5). Because of the mutagenic and protein-DNA-disrupting properties of CPDs, many organisms have evolved enzymes to specifically recognize and repair cis-syn dimers, such as Escherichia coli photolyase (6, 7), T4 endoV (8), as well as general repair enzymes such as E. coli uvrABC (9) and human excinuclease (10). Additionally, dimers are efficiently repaired in transcription-coupled repair by virtue of their ability to block synthesis by RNA polymerases (11,12). CPDs also block DNA replication (13) and are efficiently bypassed in a nonmutagenic manner by DNA damage bypass polymerases such as E. coli pol V (14) and the recently discovered yeast (15) and human polymerase (16,17). There is also evidence that the mismatch repair system can recognize mismatches opposite thymine dimers (18). Understanding the mechanism by which these proteins recognize and process CPDs will be greatly aided by a structure for CPD-containing DNA.The efficiency of damage repair is likely to depend on the extent to which those changes alter the structure of the DNA, hence making it recognizable for the repair enzymes involved. It has been suggested that the binding affinities of the repair enzymes for the CPD-containing DNA depend on the degree of DNA unwinding or kinking caused by those lesions (19,20). The first evidence that CPD formation causes large alterations in the structure of DNA is offered by circular dichroism studies and...

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A matrix screen is presented with optimized conditions for the crystallization of nucleic acid fragments. The screen consists of 24 conditions only and utilizes minimal amounts of sample. It has been tested on a series of DNA and RNA oligomers, and yielded diffraction-quality crystals of many specimens including drug-DNA complexes. Preliminary X-ray analysis of the crystals is reported.