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...

Human replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein (subunits of 70, 32, and 14 kDa) that is required for cellular DNA metabolism. RPA has been reported to interact specifically with damaged double-stranded DNA and to participate in multiple steps of nucleotide excision repair (NER) including the damage recognition step. We have examined the mechanism of RPA binding to both single-stranded and double-stranded DNA (ssDNA and dsDNA, respectively) containing damage. We show that the affinity of RPA for damaged dsDNA correlated with disruption of the double helix by the damaged bases and required RPAs ssDNA-binding activity. We conclude that RPA is recognizing single-stranded character caused by the damaged nucleotides. We also show that RPA binds specifically to damaged ssDNA. The specificity of binding varies with the type of damage with RPA having up to a 60-fold preference for a pyrimidine(6-4)pyrimidone photoproduct. We show that this specific binding was absolutely dependent on the zinc-finger domain in the C-terminus of the 70-kDa subunit. The affinity of RPA for damaged ssDNA was 5 orders of magnitude higher than that of the damage recognition protein XPA (xeroderma pigmentosum group A protein). These findings suggest that RPA probably binds to both damaged and undamaged strands in the NER excision complex. RPA binding may be important for efficient excision of damaged DNA in NER.

A fast method to detect and sequence photomodified oligodeoxynucleotides (ODNs) by exonuclease digestion and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is reported. Upon treatment of modified ODNs with both phosphodiesterase I and phosphodiesterase II, the digestion stops at the sites of photomodification. Post-source decay (PSD) of MALDI-produced ions from two enzymatic digestion end products distinguishes isomers such as 5'-d(T[cis-syn]TAAGC) and 5'-d(CGAAT[cis-syn]T), which have symmetrical or identical compositions at the 3' and 5' ends, respectively. Studies have also been done to follow the kinetics for enzyme degradation of photomodified ODNs. The calculated rate constants from a mathematical treatment of the time-dependent MALDI data clearly show that the enzymatic digestion rate slows as the enzyme approaches the modified site.