The base excision repair: mechanisms and its relevance for cancer susceptibility
Introduction
Although several DNA damaging agents are present as environmental contaminants, the majority of DNA damage has an endogenous origin and it is mainly due to: (i) oxidation of DNA by reactive oxygen species (ROS) which are generated in the cells by normal aerobic metabolism; (ii) spontaneous deamination of DNA bases, and (iii) methylation of DNA bases by S-adenosylmethionine. The majority of these lesions is blocking lesions or constitutes a pause for the progression of replicative DNA polymerases (Pols) thus leading to cytotoxicity. Some of them, after bypass, show highly miscoding properties (for example, O6-methylguanine and 8-oxoguanine can mispair with thymine and adenine leading to GC > AT and GC > TA mutations, respectively). With the exception of O6-MeGua, which is directly repaired by damage reversal by O6-MeGua-methyltransferase (MGMT), the base excision repair (BER) is the main mechanism to repair DNA alkylation and oxidative damage. This repair pathway can be subdivided in five steps: (i) base removal by a specific DNA glycosylase; (ii) incision at the resulting abasic site by an AP-endonuclease; (iii) processing of the produced blocked termini; (iv) filling in of the gap; and (v) resealing of the damaged DNA strand. The first step is characterised by the action of a DNA glycosylase, which, after recognition of the specific modified base, cleaves the N-glycosidic bond giving rise to an apurinic/apyrimidinic (AP) site. This lesion, which can also be generated spontaneously or by radiation and chemicals, is subsequently acted upon an AP-endonuclease or an AP-lyase to generate a single-strand break (ssb). After the cleavage of the phosphodiester bond, the BER mechanism can proceed through two different sub-pathways: the short-patch BER and the long-patch BER (Fig. 1 ). They are differentiated by the repair gap size and the enzymes involved. The first sub-pathway is characterised by the resynthesis of a single nucleotide at the lesion site by DNA polymerase β (Pol β). The long-patch BER involves the resynthesis of a longer oligonucleotide spanning two to seven nucleotides in length by either Pol β or the replicative Pols δ/ε. The displacement of the lesion-containing strand requires the action of additional players, namely a flap-endonuclease, FEN1, which recognises and cleaves at the base of the flap structure and PCNA that is the loading clamp for the replicative Pols. In the following sections, the mechanisms of BER and the protein factors involved (with the exception of factors specifically involved in repair of ssb) will be described. The recent progress that has been made in assigning cellular functions to the different protein factors will be summarised.
Section snippets
Base removal
The first step of BER is catalysed by DNA glycosylases which hydrolyse the N-glycosylic bond between the target base and the sugar moiety thus releasing the free damaged base and giving rise to an AP site. Most of DNA glycosylases have a low molecular mass (from 20 to 40 kDa), do not require cofactors and show broad substrate specificity. These enzymes have been classified as monofunctional and bifunctional DNA glycosylases. The monofunctional DNA glycosylases operate by a base flipping
AP-endonuclease
The release of the base catalysed by damage-specific DNA glycosylases creates an AP site in the DNA, which is incised by an AP-endonuclease. The major human AP-endonuclease (APE1, HAP1, Apex), which is homologous to E. coli Xth protein, was independently discovered as an AP-endonuclease [85], [86] and a redox-regulator of the DNA binding domain of several transcription factors [87]. APE1 plays a central role in BER. It is implicated in both short- and long-patch repair pathways. APE1 nicks the
Flap-endonuclease 1
The incision of the AP site by APE1 generates a dRP moiety, which must be removed to allow BER completion. During short-patch BER, the associated deoxyribose 5′-phosphatase (dRPase) activity of Pol β (see below) removes the dRP. Alternatively the long-patch BER may remove the dRP residue along with several downstream nucleotides by flap-endonuclease I (FEN1) that, along with DNA ligase I, interacts with PCNA to complete repair synthesis [108], [109]. FEN1 is a multifunctional,
DNA polymerases β and δ/ε
Several lines of evidence indicate that different Pols are alternatively engaged in the filling-in process of BER. At the moment the best characterised BER Pols are Pol β and Pol δ/ε. The role of Pol β in BER is well known [125], mainly in the short-patch pathway, as demonstrated by in vitro assays with both human cell-free extracts [126] and purified human proteins [95]. Pol β is a polypeptide of 39 kDa and it is constituted by two domains connected by a protease sensitive hinge region: the
DNA ligase I
DNA ligase I has been identified as a component of a high molecular weight replication complex [177], [178] and shown to be specifically required for the efficient joining of Okazaki fragments when DNA replication is reconstituted from highly purified components [179]. Moreover, using affinity chromatography with either Pol β antibody or Pol β as the ligand, a multiprotein complex that catalysed the repair of a uracil-containing DNA substrate was partially purified from a bovine testis nuclear
The complexity of BER
One specific feature of BER is its versatility and apparent redundancy. DNA glycosylases present a broad substrate specificity and other BER enzymes like APE1 and Pol β are able to catalyse more than one repair step. On the other hand, there is an apparent redundancy at the level of enzymes required to accomplish specific BER steps, for example, the resynthesis and ligation steps. To increase the complexity of this network, proteins involved in other repair mechanisms enter into the scenario by
Acknowledgements
This work is dedicated to the memory of Tamara Basic-Zaninovic. We will never forget her enthusiasm for science and joy of life. This work was partially supported by the Associazione Italiana Ricerca sul Cancro (A.I.R.C.) and by Compagnia di San Paolo, Turin, Italy (project coordinator: Dr. G. Frosina).
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