Crystal structure of a DNA decamer containing a cis-syn thymine dimer

Abstract

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...

Electrically monitoring DNA repair by photolyase

“…The intensity of the redox signal corresponds to a surface coverage of Ϸ1 pmol͞cm 2 , markedly lower than the expected density of DNA on the surface, 12 pmol͞cm 2 , as measured previously by radioactive tagging (10). The presence of a TϽϾT within a DNA duplex leads to a kink in the DNA (26,27), and such disruptions in base stacking are also expected to be associated with an attenuation in signal from DNA-bound redox probes. Indeed, using methylene blue in an assay for CT efficiency (9), we observe a decrease in the methylene blue redox signal on electrodes modified with TϽϾT DNA relative to those modified with undamaged DNA.…”

Rotational Position of a 5-Methylcytosine-containing Cyclobutane Pyrimidine Dimer in a Nucleosome Greatly Affects Its Deamination Rate

“…This preference is also seen for bent DNA that is not in contact with a protein (34) and has been attributed to the greater degree of rotational freedom in the phosphate backbone, making it easier to for adjacent pyrimidines to adopt a photoreactive conformation (35,36). When DNA containing randomly distributed CPDs is assembled into nucleosomes, the CPDs also favor positions away from the surface (37), which is consistent with the 30°b end that they make toward the major grove of DNA (38). Despite the distortion of DNA caused by CPDs, nucleosome core particles containing CPDs in different rotational settings can be readily prepared and isolated (39 -41).…”

“…The same would be expected to hold true for the CPDs as the 3Ј-pyrimidine of a CPD has been found to adopt roughly the same position as it does in the undamaged DNA (38). Thus, the C4 position of the m C of both the inside and the outside CPDs would appear to be in a similar, unobstructed environment, suggesting that factors other than steric interference by the histone proteins must play a role in inhibiting or enhancing deamination of the CPDs.…”

Inhibition of Nucleotide Excision Repair by High Mobility Group Protein HMGA1

“…The structural hallmark of AT-rich DNA that is recognized by HMGA1 proteins is a narrow minor groove, where alternating dAdT, poly(dT), and dAdAdTdT sequences produce minor grooves that are almost 2 Å narrower than that of B-DNA (48). In contrast, DNA containing a cyclobutane thymine dimer is bent by ϳ30 o and, compared with B-form DNA, the CPD lesion itself exhibits an extreme widening of both minor and major groves and has a severe change in base pairing geometry on its 5Ј-side (19). Such structural differences in DNA might explain any slight variations in binding affinity of HMGA1 for UV photoproducts.…”

“…Among a variety of possible UV-induced photoproducts, cyclobutane pyrimidine dimers (CPDs) are the most abundant, stable forms (17) and, if unrepaired, are known to cause mutations and skin cancer (2,18). CPDs alter DNA structure from normal B-form, causing severe bending of the helical axis and disruption of Watson-Crick base pairs at the lesion sites (19). Additionally, these lesion-induced distortions can obstruct progression of both RNA and DNA polymerases (20,21).…”