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  • br UVC induced DNA damage a

    2020-01-20


    UVC-induced DNA damage: a model to study DNA damage tolerance The UV components of sunlight that reaches the Earth surface are one of the most carcinogenic agents humans are exposed to, and the leading cause of skin cancer [4,12]. UV light causes different types of DNA damage through indirect and direct modes. UV rays can be absorbed by chromophores present in skin cells, leading to the generation of reactive species of oxygen MRT68921 sale (ROS) that can damage the DNA by oxidizing bases [13]. UV light can also be directly absorbed by the DNA molecule, leading to the dimerization of nucleotides, in particular adjacent pyrimidines [13,14], which results in strong distortions of the DNA double helix, able to arrest the progression of the replicative polymerases (Fig. 1). The electromagnetic spectrum of UV radiation is typically sub-divided in three ranges based on the wavelengths: UVA (315–400 nm), UVB (280–315 nm) and UVC (100–280 nm). Because UVC radiation is the most reactive of the UV spectrum, it is completely absorbed by the ozone layer but also maximally absorbed by the DNA molecule. For this reason, exposure to UVC radiation generates the highest frequency of pyrimidine dimers relative to ROS-induced DNA damage compared to UVA [15,16] and has been extensively used to study how distortive DNA damage, in particular pyrimidine dimers, are repaired or tolerated. The most frequent pyrimidine dimers generated upon exposure to UVC radiation are cys-sin cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproducts (6-4PP) [16,17] (Fig. 1). Although CPDs and 6-4PPs are simultaneously formed upon UVC irradiation, these two types of damage differ in several aspects. First, CPDs, and in particular TT-CPDs, are the most abundant UV lesions. 6-4PPs are the second most frequent UV lesions, but they only represent 25–30% of the amount of CPDs [18]. Also, due to their biochemical structure, 6-4PPs induce a major deformation on the DNA double helix compared to CPDs. In fact, a CPD leads to a distortion from 7 to 30° compared to the normal conformation of DNA, while a 6-4PP generates distortion of about 44° of the double helix, leading to loss of MRT68921 sale pairing [19]. These structural differences define how each lesion is repaired and tolerated by TLS.
    DNA damage tolerance by translesion DNA synthesis (TLS) polymerases In human cells, 6-4PPs are completely removed within 3–6 h after exposure to UV radiation, while about 50% of CPDs still persist 24 h later [[20], [21], [22]]. An important outcome of this is that most cells progressing though the S phase (DNA synthesis) of the cell cycle will encounter CPD lesions before their complete removal. Strikingly, cells from XP patients, defective in NER, progressing through the S-phase will have to deal with both CPDs and 6-4PPs. Bulky lesions on DNA physically block the progression of the replicative polymerases (pols) Pol ε and Pol δ, leading to so-called replication stress [23,24]. While the replicative polymerases are stalled by intra-strand crosslinks, such as UV-induced pyrimidine dimers, the progression of the CMG helicase (composed of CDC45 protein, MCM2-7 complex and the GINS complex) is not perturbed by this type of damage. As a result, the replicative helicase continues to unwind the parental DNA, while the replicative Pol is stalled. This uncoupling of the helicase and the polymerase leads to the formation of stretches of ssDNA [25,26]. ssDNA regions are rapidly coated by the ssDNA-binding protein RPA (replication protein A) and activate the ATR/Chk1 pathway, culminating in the maintenance of replication fork integrity, stabilization and restart, as well as cell survival [27,28]. In case of impaired preservation of fork integrity and/or prolonged fork stalling, arrested forks can break, leading to double-strand breaks (DSBs), which can result in chromosomal rearrangements or cell death [1,23]. In order to avoid that fate, cells have evolved two universal strategies to deal with DNA replication-blocking lesions: homology-dependent repair (HDR) mechanisms and translesion DNA synthesis (TLS) [29,30]. Briefly, HDR, also known as DNA damage avoidance, relies on the identical sister chromatid to serve as a template for DNA replication at the site of the damage [29,31]. It is noteworthy that the generation of DSBs at stalled replication forks may also represent the starting point of the particular HDR pathway called Break-Induced Replication [24,28,29]. Although HDR mechanisms, and in particular homologous recombination (HR), are important mechanisms for lesion bypass in eukaryotes as yeast, in mammalian cells the replication of a template containing lesions is mainly performed without strand transfer by recombination [32]. Interestingly, recent studies have shown that proteins involved in the repair of DSB by HR such as Brca1 and Brca2 (breast cancer susceptibility genes 1 and 2) are involved in replication fork protection independently of their role in HR [33,34]. In fact, a key protein in HR, Rad51, was shown to be crucial for fork reversal, a replication fork remodeling that promotes template switch through the regression of the fork and the subsequent annealing of the two nascent strands [24,35,36]. Alternatively, damaged DNA can be directly replicated by specialized polymerases, called TLS polymerases. A study revealed that both TLS and HDR act to tolerate DNA damage in mammalian cells but the preference and efficiency of one mechanism above the other depends on the nature of the DNA lesion [37]. According to the purposes of this review, TLS will be described and discussed in more details below.