Elsevier

DNA Repair

Volume 3, Issue 11, 2 November 2004, Pages 1409-1423
DNA Repair

Mini review
Mechanisms of DNA damage recognition and strand discrimination in human nucleotide excision repair

https://doi.org/10.1016/j.dnarep.2004.05.005Get rights and content

Abstract

Using only a limited repertoire of recognition subunits, the nucleotide excision repair (NER) system is able to detect a nearly infinite variety of bulky DNA lesions. This extraordinary substrate versatility has generally been ascribed to an indirect readout mechanism, whereby particular distortions of the double helix, induced by a damaged nucleotide, provide the molecular determinants not only for lesion recognition but also for subsequent verification or demarcation processes. Here, we discuss the evidence in support of a bipartite mechanism of substrate discrimination that is initiated by the detection of thermodynamically unstable base pairs followed by direct localization of the lesion through an enzymatic proofreading activity. This bipartite discrimination mechanism is part of a dynamic reaction cycle that confers high levels of selectivity to avoid futile repair events on undamaged DNA and also protect the intact complementary strand from inappropriate cleavage.

Introduction

The maintenance of DNA integrity is essential for the viability of cells and the health of organisms. However, the genome is under permanent attack from endogenous metabolic byproducts and environmental factors that alter the chemical structure of DNA and corrupt its nucleotide sequence. To cope with such genotoxic insults, all living organisms are endowed with a network of DNA repair systems that process multiple forms of DNA damage. Nucleotide excision repair (NER) is the only pathway by which mammalian cells remove bulky DNA adducts induced by UV light and electrophilic chemicals. It consists of a simple “cut and patch” system that operates by excision of a short single-stranded DNA fragment, followed by restoration of the duplex DNA structure through repair synthesis. This particular DNA repair mode was first discovered in Escherichia coli as a mechanism that eliminates UV radiation products from DNA [1], [2]. Subsequently, a functionally similar process for the excision of UV damage was identified among eukaryotes [3], [4]. Research in this area was stimulated by the finding that a genetic defect in the NER response causes the human hereditary disease xeroderma pigmentosum (XP), which is characterized by extreme photosensitivity and a 2000-fold increased incidence of sunlight-induced skin cancer. XP individuals also have a higher incidence of internal tumors and, in some cases, neurological abnormalities, probably reflecting the importance of NER in the repair of endogenous DNA damage [5]. Patients affected by this recessive disorder are classified into seven repair-deficient complementation groups designated XP-A through XP-G.

With the advent of targeted gene replacement in embryonic stem cells, several mouse models have been generated to analyze in detail the phenotypic consequences of a NER defect [6], [7], [8], [9], [10], [11]. These knock-out experiments lend support to the notion that the NER complex is an essential part of the cellular defense system that protects the genome against somatic mutations leading to cancer. In general, the lack of NER activity in mice not only recapitulates the predisposition to UV-induced skin cancer but also results in tumorigenesis after exposure to chemical carcinogens. For example, a homozygous XPA−/− mutant mouse, completely deficient in NER, is prone to tumors of the skin, lymphoid system and liver after treatment with polycyclic aromatic hydrocarbons [8], [10].

Section snippets

The human NER reaction

The NER mechanism involves (1) damage recognition and assembly of the incision complex, (2) dual DNA incision and damage excision, (3) DNA repair synthesis and ligation (Fig. 1). Most enzymatic steps that follow the recognition of a lesion in the human NER pathway are well characterized (see for example reference [12], [13]). A key NER intermediate is generated by transient unwinding of the duplex substrate, resulting in a partially open DNA structure with “Y-shaped” double to single strand

Subunits of the human NER machinery

All core components that carry out the NER reaction in both prokaryotic and eukaryotic organisms have been identified. Six polypeptides (UvrA, UvrB, UvrC and UvrD, DNA polymerase I, and DNA ligase) are necessary and sufficient for NER activity in prokaryotes [22]. The mammalian NER system displays a considerably higher degree of genetic complexity and its core subunits assemble into two large multi-enzyme machines [23], [24], [25], [26]. An initial complex performs the damage recognition and

Stepwise assembly of a recognition complex

It is becoming increasingly clear that, in mammalian cells, NER is executed by the sequential recruitment of repair proteins to the site of the DNA lesion, rather than by the action of a pre-assembled “repairosome” factory. It can be estimated that a functional “repairosome” may achieve a mass of nearly 3 MDa [31], but in vivo studies monitoring the movement of NER factors to sites of DNA repair are not compatible with such large machines and instead favor the nuclear translocation and stepwise

The role of DDB in damage recognition

Damaged DNA binding (DDB) is another factor with an affinity for damaged DNA. It stimulates excision of bulky UV lesions in human cells and, therefore, has been implicated in the damage recognition process. DDB is a heterodimer of p127 (DDB1) and p48 polypeptides (DDB2) whose small subunit is encoded by the XPE gene [45], [46]. Mutations in the 48 kDa subunit are found in all known cases of XP complementation group E [47], [48].

DDB appeared to be an initial damage sensor for several reasons.

Recognition of bulky lesions during transcription-coupled DNA repair

DNA repair is highly non-uniform in the context of mammalian chromosomes. While “global genome” repair (GGR) is active throughout the genome, regardless of whether any specific sequence is transcribed or not, living organisms have set up a more efficient “transcription-coupled” repair pathway (TCR) that removes DNA lesions only from the transcribed strand of active genes. For example, cyclobutane dimers are removed more rapidly from transcribed genes than from transcriptionally silent regions

Damage recognition in the GGR pathway

The GGR system should be highly specific for damaged DNA to avoid futile repair cycles among the 3 billion base pairs in the human genome and, at the same time, versatile in order to detect a broad spectrum of chemically unrelated lesions. The molecular mechanism of damage recognition has been a matter of much debate, in part raised by the fact that none of the individual core factors display a high enough specificity and versatility to function as a unique sensor of damaged substrates in the

The recruitment of XPG and XPF–ERCC1

Because XPG (but not XPF) associates with foci of DNA damage in XP-A cells [27], the 3′ endonuclease seems to be recruited ahead of XPF–ERCC1 (the 5′ endonuclease). Presumably through a stable interaction with TFIIH, XPG is already present in the NER complex before the arrival of the XPA subunit [137]. In reconstituted systems, however, XPA, RPA and XPG mutually stabilize their association with the NER substrate, suggesting that these three factors bind to DNA in a synergistic manner [18], [28]

A hypothesis for damage-specific and strand-selective DNA incision

How the human NER machinery recognizes many kinds of bulky DNA base adducts, and discriminates between these lesions and undamaged DNA (including the undamaged strand directly opposite the lesion), poses an experimental challenge that has not yet been fully resolved. Fig. 8 depicts a simplified model for DNA damage recognition in the human GGR pathway. Based on the evidence that has been reviewed here, we propose that XPC-hHR23B is the initial sensor for disrupted base pairs in the GGR pathway,

Acknowledgements

Research in the authors’ laboratory is supported by the Swiss National Science Foundation (grant 3100A0-101747).

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