1.6 DNA lesions
1.6.1 Overview
Endo-‐ and exogenous agents constantly damage DNA. For instance, exposure to UV radiation, alkylating agents and oxidative species leads to the formation of abasic sites, pyrimidine dimers, alkylated adducts and oxidative lesion products. To maintain the genomic integrity and reduce the mutagenic potential cells allocate with multiple repair pathways and specialized enzymes. However, several health statistics could show that DNA lesions can be highly mutagenic and sometimes carcinogenic e.g. in Europe in 2000 ∼35 000 new cases of UV radiation damage-‐induced skin cancer were diagnosed (68). Further, the tobacco-‐
derived nitrosamine NNK is associated with lung cancer resulting in ∼334 800 deaths in Europe in 2006 (69). Therefore the biological prevalence of the DNA lesions and their chemical structures need to be determined. The main aspect here are (i) identification and quantification of DNA lesions in model systems and in vivo, (ii) to assess influences of lesions on physical properties of DNA e.g. thermal stability, and (iii) to elucidate the impact of the lesions on DNA function e.g. enzyme-‐mediated processes such as replication.
Within the last decade years specialized DNA polymerases, responsible for translesion DNA synthesis (TLS), were identified and characterized. W. Yang and R. Woodgate published a clear summary of this class of enzymes emphasizing the relationship of the bypass properties and the structural features (29). In brief, many of the TLS enzymes are member of the Y-‐family of DNA polymerases exhibiting universal features to manage bypass of a variety of DNA lesions. In a simplified model TLS polymerases can be categorized into two classes. The first class of enzyme is highly specialized and responsible for bypassing a certain DNA lesion e.g. the human pol η is able to bypass thymine-‐thymine cyclobutane dimer with high efficiency. Interestingly, patients showing mutations or defects in the human pol η gene suffer from sunlight-‐sensitive and cancer-‐prone Xeroderma pigmentosum variant (XP-‐V) syndrome (70, 71). The second class of enzymes is the all-‐rounder and has the ability to accommodate different DNA lesions e.g.
the archaeal Dpo4 DNA polymerase from the Y-‐family. A series of structural studies show this low fidelity polymerase bound to damaged substrates such as oxidative damage (72, 73), UV cross-‐linking (74),
benzo-‐[a]pyrene diol epoxide adduct (BPDE) (75), and abasic site lesions (76). However, efficient catalysis is mainly observed in case of an abasic site lesion(76, 77).
1.6.2 Abasic site
The most common DNA damage under physiological conditions are abasic sites resulting mainly from spontaneous hydrolysis of the N-‐glycosidic bond between the sugar moiety and the nucleobase in DNA (78). Abasic sites also occur as intermediates during excision repair of damaged nucleotides (79) or can be manifested in several chemical structures such as C4’-‐oxidized abasic site (C4-‐
AP) after treatment of DNA with antitumor antibiotics like bleomycin (80, 81). The abasic site L (2’-‐
deoxyribonolacetone) results from one-‐electron nucleotide oxidation (82, 83). In general, it has been estimated that 10000 abasic sites are formed in human cell per day (78,
84, 85). Guanine and adenine nucleobases are cleaved most efficiently resulting in the abasic sugar moiety (AP, Figure 9A). To investigate the biochemical impact of AP a stabilized tetrahydrofuran analog is used as a model.
Since the genetic information gets lost by the cleavage of the nucleobase, abasic sites bear a high mutagenic potential (85-‐87). To face this problem nature offers a whole arsenal of enzymes and possible pathways. In most cases, the lesion is removed by DNA repair systems using the sister strand to guide for incorporation of the right nucleotide. However, undetected lesions or those, formed during S phase, pose a challenge to DNA polymerases and block replication (26, 88). Additionally, it was found that the mutagenic potential of these lesions in translesion synthesis is more pronounced in animal compared with bacterial cells presumably because of higher translesion synthesis in eukaryotes (87, 89, 90).
A set of studies concerning the behavior of DNA polymerases, belonging to different families, showed that there are multiple mechanisms to overcome an abasic site. Most translesion DNA polymerases from family X and Y follow various loop out mechanisms (76, 77, 91-‐94). Thereby, the nucleotide selection is influenced by the following upstream templating bases resulting in deletions and complex mutation spectra. Recently, an amino acid templating mechanism was found for the “error-‐free” bypass of an abasic site by the yeast Rev1 DNA polymerase belonging to the family Y (95). Since guanine is cleaved most efficiently (85), the preference of Rev1 for dCMP incorporation opposite an abasic site represents the
“best-‐guess”.
In contrast, in vitro and in vivo studies of the replicative DNA polymerases from family A (including human DNA polymerases γ and θ) and B (including human DNA polymerases α, ε and δ) in the presence of the stabilized tetrahydrofuran abasic site analog F (Figure 9D) have shown that purines, in particular adenosine, and to a lesser extent guanosine, are most frequently incorporated opposite the lesion. The strong preference for adenosine incorporation opposite an abasic site has been termed ‘A-‐rule’ (89, 91, Figure 9 Structures of different forms of abasic DNA lesions.
96-‐104). The apparent selectivity for incorporation of purines ultimately results in transversion on this assumption numerous of non-‐natural nucleotide analogs were studied regarding their behavior in the presence of an abasic site. If the induced fit model is taken as a selection criteria opposite abasic sites, a non-‐natural nucleotide analog with nearly identical size to the Watson-‐Crick base pair, should show the highest incorporation efficiency. By steric examination Matray and Kool identified the pyrene nucleoside triphosphate (dPTP) as a perfect match in the absence of a templating base (Figure 10) (47). Indeed, they could show that the pyrene modified nucleotide is incorporated by DNA polymerase I from E. coli with higher efficiency than any other natural nucleotide, demonstrating that a simple steric model is sufficient for efficient incorporation. Further the fluorescent nucleobase analog is used to identify and sequence abasic site lesions in DNA. Studies of several nucleotide
analogs identified 5-‐nitro-‐1-‐indoyl-‐nucleotide (dNITP) as the ‘specific partner’ opposite an abasic site, since dNIMP is incorporated with increased efficiency by RB69 DNA polymerase, a α-‐like DNA polymerase, compared to dPMP (106). The structure of RB69 DNA polymerase capturing an artificial 5-‐nitro-‐1-‐indoyl-‐nucleotide (dNITP) opposite an abasic site in the active site of the enzyme elucidated that a
processing an adenosine opposite an abasic site lesion failed so far (97). The structure from RB69 DNA polymerase in presence of a guanosine opposite an abasic is still not sufficient to explain the ‘A-‐rule’.
Further the assignment of the behavior from TLS DNA polymerases to explain the preference of adenosine is unsatisfactory, given that these families use different, sequence-‐depending mechanisms that might compete with the A-‐rule when bypassing abasic sites. Therefore, due to missing structures of members from these sequence families, the selection criteria for adenosine opposite an abasic site remains to be elucidated.