Mini reviewA model for initial DNA lesion recognition by NER and MMR based on local conformational flexibility
Introduction
Initial recognition of a lesion against the background of normal DNA is the crucial step in DNA repair. However, the mechanisms by which repair proteins recognize damaged and mismatched DNA are not well understood. The three main classifications of repair pathways are base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (MMR). Although many BER systems recognize very specific forms of damage [1], the NER system must recognize a wide array of structurally dissimilar covalently modified bases ranging from UV-induced pyrimidine dimers to bulky covalent adducts [2], [3], [4]. Similarly, the MMR system recognizes a range of mismatched base pairs and short insertion/deletion loops, despite the diversity of substrates and structural effects on DNA that this presents [5]. Additionally, MMR recognizes many lesions that were previously thought to be repaired only by NER [6], [7], [8], [9]. Since neither the NER nor MMR systems recognize single substrates with obvious chemical features on which a classic “lock and key” recognition motif can be based, recognition likely operates through detection of more subtle characteristics of damaged and mismatched DNA. In contrast, BER pathways appear to recognize very specific chemical features of the damaged bases with little cross reactivity. A further complication is that normal DNA itself is structurally diverse on the local level, due to differences in base sequence [10]. DNA also undergoes sequence dependent conformational rearrangements on a variety of time scales [11], [12], [13], [14], [15], [16]. These rearrangements represent interchange between energetically similar conformational states, the number and relative energies of which determine the local conformational flexibility for a DNA molecule [17], [18]. Experimental observations suggest that covalent damage and mismatched base pairs alter this energy landscape, allowing normally unpopulated conformational states to become accessible (Fig. 1). These observations suggest that detection of DNA lesions and discrimination among NER and MMR pathways may depend on the differences in conformational flexibility between normal, covalently damaged, and mismatched DNA.
Thermodynamic destabilization of the double helix caused by covalent damage and base mismatches is a commonly proposed mechanism by which repair pathways with broad specificity such as NER and MMR recognize their substrates [19], [20]. The term thermodynamic destabilization considered in this model refers to changes in the enthalpic and entropic contributions to the free energy of local strand separation in the context of the DNA double helix. These changes in the free energy are most often observed as changes in the melting behavior of lesion-containing DNA relative to the parent DNA molecule. More negative values of free energy are associated with higher melting temperatures and thus a more thermodynamically stable double helix (Table 1) [21], [22].
Many experimental observations suggest that recognition of damaged DNA by NER relies on more than altered base pairing thermodynamics. Certain types of damage recognized by NER, such as alflatoxin B1 (AFB1-APY) [23], [24] and psoralen (HMT) adducts [25], actually thermodynamically stabilize the DNA helix relative to their undamaged parent sequences. There appears to be a trend in mammalian NER systems to recognize thermodynamically destabilizing lesions more efficiently than stabilizing lesions [19]. However, undamaged DNA sequences with thermodynamic stabilities intermediate between those of DNA molecules containing stabilizing and destabilizing lesions are ignored by NER proteins. Additionally, some types of damage that have been observed to thermodynamically destabilize DNA, such as etheno adducts [26], are not substrates for NER or MMR and are instead repaired by BER pathways that have more tightly defined substrate requirements. These observations suggest that initial recognition of the lesion by NER relies on some additional feature of damaged DNA, other than exclusively the detection of thermodynamic destabilization of the DNA double helix.
Likewise, there are compelling arguments and experimental data that suggest factors other than the local thermodynamic destabilization of base pairing caused by mismatches are the primary determinant of MMR lesion recognition. The thermodynamic stability of DNA, including mismatched DNA, is highly sequence dependent [22]. Using the nearest-neighbor model to calculate the local thermodynamic stability of double-stranded DNA [21], the least stabilizing trimer sequence in Watson–Crick DNA is TAT/ATA, with a ΔG°37 contribution to helix stability of −1.33 kcal/mol. Several other Watson–Crick trimers are also observed to have ΔG°37 contributions above –2 kcal/mol. The G-G mismatch-containing trimer sequence GGC/CGG has a ΔG°37 contribution of −2.22 kcal/mol [27], making it more thermodynamically stabilizing than many normal DNA trimers. However, the G-G mismatch is among the most efficiently recognized by MMR [28]. The recognition of diverse lesions by repair proteins is not closely correlated overall with their thermodynamic stability [29]. As is the case for NER, the recognition of mismatched DNA by MMR clearly relies on more than detection of the local thermodynamic destabilization of the DNA double helix. These observations suggest that an alternative mechanism for recognition of these lesions by both NER and MMR is some form of indirect readout of DNA conformational features induced by covalent damage or base mismatches.
Indirect readout is a mechanism by which proteins bind tightly to specific DNA sequences by detecting DNA conformational features that are affected by sequence context and other factors, rather than direct interaction with functional groups of particular bases [30], [31], [32], [33], [34], [35]. For example, the energy required to kink DNA and the conformational equilibria of backbone phosphate groups in response to protein binding are influenced by the DNA base sequence. In some cases, bases that are not directly contacted by the protein or are outside its binding footprint can have significant effects on protein binding. One of the most compelling mechanisms proposed for indirect readout is the probing for flexibility of a DNA substrate that allows it to fit into the binding site of a probing protein [36]. In order for specific recognition to occur, the energy cost for deforming the DNA must be low enough for the DNA to be tightly bound to the protein as it scans along the double helix. Thus, DNA that is intrinsically more flexible in ways complementary to modes of deformation by protein binding will make a better substrate for the protein. DNA that is already deformed due to sequence or damage in ways similar to the protein-bound conformation (preorganization) also favors protein binding. The protein complex XPC–HR23B is believed to initially recognize covalently damaged DNA in mammalian systems. These proteins form a complex with damaged DNA and deforms the DNA at lesion sites by bending of the double helix [37]. This observation suggests that increased flexibility of the damaged DNA at or near the lesion site is vital to damage recognition by the complex. The multiprotein factor XPA–RPA, another damage recognition component of the mammalian NER system, has also been shown to bind damaged DNA through an indirect readout mechanism [38].
Initial recognition of base mismatches by MMR appears to operate by a similar, albeit functionally distinct, mechanism to that of NER. The MutS homodimer in prokaryotes and the homologous MutSα heterodimer in eukaryotes are the protein components of MMR responsible for initial recognition of base mismatches and short insertion/deletion loops in DNA. The co-crystal structures of MutS bound to DNA containing a G-T mismatch [39] and another of MutS bound to DNA containing a thymidine insertion [40] suggest that mismatch recognition by MMR involves probing the DNA for deformability and structural flexibility.
Section snippets
An alternative model for DNA lesion recognition
In order to accommodate these reported observations, we propose a model in which damage- and mismatch-induced increases in local flexibility and associated conformational heterogeneity relative to normal DNA are the principal determinants by which NER and MMR proteins can recognize and bind to a lesion. The thermodynamics of local strand separation are less important to initial lesion recognition in this model, as NER and MMR recognize substrates with wide ranges of base pairing thermodynamic
Dynamics in NER substrates
The literature contains many examples of NMR-based studies of the structure and dynamics of DNA containing lesions that are NER substrates. One such lesion is caused by metabolic activation of the ubiquitous carcinogen benzo[a]pyrene, present in tobacco smoke and automobile exhaust [52], [53]. Endogenous enzymes activate this hydrocarbon to its carcinogenic form as a diol epoxide [54] (Fig. 2a), which then reacts with DNA to produce a variety of covalent adducts [55]. Many of these lesions have
Dynamics in MMR substrates
The MutS protein of MMR responsible for recognition of base mismatches and small insertion/deletion loops also recognizes many of the lesions discussed above as NER substrates [6], [7], [8], [9], [77]. It is unclear at present why these substrate specificities overlap. Studies of the structure and internal dynamics of MutS substrates that are not NER substrates have shed some light on this issue. Early NMR studies of G-T, G-A, C-A, and C-T mismatch-containing DNA showed increased exchange with
Substrate discrimination by NER versus MMR
In the eukaryotic NER system, DNA damage is first detected by the XPC–HR23B complex in global genome repair or by a stalled RNA polymerase complex in transcription-coupled repair [2], [3], [4]. A study using scanning force microscopy revealed that the XPC–HR23B complex distorts bound DNA, suggesting that the flexibility of the DNA is crucial for efficient binding and damage recognition [37]. It appears that strand separation is also a critical component of recognition by this complex [37].
Conclusions
Understanding how DNA repair pathways such as NER and MMR recognize non-standard DNA against the background of sequence dependent variations in the conformational flexibility of normal DNA provides insight into the molecular basis of carcinogenesis. Through the use of NMR spectroscopy and complementary computational methods, many aspects of DNA structure and internal dynamics have been elucidated. DNA damage and base mismatches have been observed in a number of cases to induce increased
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2022, Biomedicine and PharmacotherapyCitation Excerpt :This may show that cornelian cherry phenolics protect dsDNA against oxidation not only a priori but also after the oxidation occurs. The destabilization of double helix at singular locus may help NER and BER mechanisms delete the oxidated form of guanosine [13,33]. Results of the interaction study, content of individual phenolics in tested cultivars of C. mas and interaction energies between them and B2 and the base-pairs helped us construct PCA model of the potential genoprotective effect (Fig. 4).
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2018, DNA RepairCitation Excerpt :Complementary base stacking is a main factor in molecule intrinsic rigidity of the DNA [55]. A “single stranded character” region of double stranded DNA may arise as a result of bulky DNA damage formation and subsequent loss of complementary base stacking [56,57]. It should be also noted, that non modified DNA is not a static molecule.