Review
DNA–protein crosslinks: their induction, repair, and biological consequences

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Abstract

The covalent crosslinking of proteins to DNA presents a major physical challenge to the DNA metabolic machinery. DNA–protein crosslinks (DPCs) are induced by a variety of endogenous and exogenous agents (including, paradoxically, agents that are known to cause cancer as well as agents that are used to treat cancer), and yet they have not received as much attention as other types of DNA damage. This review summarizes the current state of knowledge of DPCs in terms of their induction, structures, biological consequences and possible mechanisms of repair. DPCs can be formed through several different chemistries, which is likely to affect the stability and repair of these lesions, as well as their biological consequences. The considerable discrepancy in the DPC literature reflects both the varying chemistries of this heterogeneous group of lesions and the fact that a number of different methods have been used for their analysis. In particular, research in this area has long been hampered by the inability to chemically define these lesions in intact cells and tissues. However, the emergence of proteomics as a tool for identifying specific proteins that become crosslinked to DNA has heralded a new era in our ability to study these lesions. Although there are still many unanswered questions, the identification of specific proteins crosslinked to DNA should facilitate our understanding of the down-stream effects of these lesions.

Introduction

The purpose of this review is to summarize our current understanding of the mechanisms of induction and repair, as well as the biological consequences, of the types of DNA lesion known as DNA–protein crosslinks (DPCs). A DPC is created when a protein becomes covalently bound to DNA. Such events occur following exposure of cells to a variety of cytotoxic, mutagenic, and carcinogenic agents, including ultraviolet light and ionizing radiation (IR), metals and metalloids such as chromium, nickel and arsenic, various aldehydes, and some important chemotherapeutic drugs including cisplatin, melphalan, and mitomycin C. Humans are continuously exposed to DPC-inducing agents present in environmental pollutants such as cigarette smoke and automotive and diesel exhaust, industrial chemicals and foodstuffs, as well as physiological metabolites, such as products of lipid peroxidation. Understanding the biology of these lesions is complicated by several factors. For example, different agents induce DPCs by different mechanisms (Fig. 1). Proteins can become crosslinked to DNA directly through oxidative free radical mechanisms or they can be crosslinked indirectly through a chemical or drug linker or through coordination with a metal atom. A subtype of these crosslinking mechanisms involves a sulfhydryl linkage to the amino acid. This results in numerous types of DPCs that are chemically distinct and whose formation is influenced by factors such as cellular metabolism, cell-cycle phase, and temperature. It is likely that these different types of crosslinks will be more or less susceptible to various mechanisms of reversal (e.g., hydrolysis) and enzyme-catalyzed repair, given their different chemical structures and physical conformations. They may also have different cellular consequences.

The timing of this review coincides with the emergence of proteomics as a tool for studying biological complexes involving unknown proteins, so that the identification and quantification of specific proteins that become crosslinked to DNA is now possible without the necessity for presumption. This approach has been recently highlighted because of its success in identifying proteins involved in complex cellular structures such as the spliceosome [1] and lipid rafts [2]. Such studies have highlighted an important issue that may have compromised earlier studies of this type, namely that of protein abundance and solubility under a given set of assay conditions, which may greatly influence the proteins that are identified to the exclusion of others. These issues may have contributed to discrepancies among earlier studies.

Two classes of DPC, the attachment of topoisomerases to DNA and the association of DNA and protein caused by hyperthermia, have been reviewed recently [3], [4], and will not be discussed in depth in this review.

Section snippets

Detection of DPCs

Early studies of DPCs tended to focus on the issue of whether cellular protein became associated with DNA and quantifying these DPCs following exposure of a test system to a given genotoxic agent. Existing techniques for the quantitation of DPCs differ in their detection limit/sensitivity level and associated problems. DPC induction can be measured using the comet assay because the crosslinking of proteins to DNA retards the migration of DNA fragments, resulting in a reduced tail moment [5], [6]

Formaldehyde-induced DPCs

Formaldehyde is a widely studied DPC-inducing agent, and the crosslinking of proteins to DNA by formaldehyde is used for the investigation of DNA–protein interactions in a technique called chromatin immunoprecipitation (ChIP). To perform ChIP, cells are treated with formaldehyde resulting in the covalent crosslinking of proteins to the DNA sequences with which they are associated. The DNA is then fragmented and the protein–DNA complex is isolated by immunoprecipitation with an antibody to the

Radiation-induced DPCs in cells

Exposure of cells to IR results in the generation of many localized ROS within a short distance of each other and of the DNA (Fig. 3). Many of these, including the extremely reactive hydroxyl radical (radical dotOH), will be generated at high levels within small discrete regions known as spurs, blobs, and short tracks [30]. When these ionization-dense regions overlap a DNA molecule, this can result in what are variously referred to as “locally multiply damaged sites” or “clustered lesions”, because each

Stability of DPCs in vitro

Different types of DPCs appear to have very different chemical stability. Aldehyde-induced DPCs are reversed by spontaneous hydrolysis and are also reversible by incubation at elevated temperatures (discussed in [24]). Acetaldehyde-induced DPCs are hydrolytically unstable, and in in vitro experiments only ∼25% of these DPCs remained after 8 h at 37 °C [61], [62]. By comparison, malondialdehyde-induced DPCs formed in vitro using purified DNA and histone protein had a much longer half-life of 13.4

Biological consequences of DPCs

The covalent crosslinking of proteins to DNA is expected to interrupt DNA metabolic processes such as replication, repair, recombination, transcription, chromatin remodeling, etc. Indeed, the effect of agents that cause DPCs on DNA replication has been widely investigated ([63], [64], [65] and others). DPCs are expected to act as bulky helix-distorting adducts and would therefore be likely to physically block the progression of replication or transcription complexes and/or prevent access of

Proteins involved in DPCs

Determining which proteins become crosslinked to DNA by these various genotoxic agents and how they are bound may help to unravel the biological consequences of DPCs as well as the mechanisms of their repair. A number of investigators have tried to identify proteins that can become crosslinked to DNA using in vitro systems with purified proteins and DNA or by isolating DPCs from cells exposed to various DNA-damaging agents. Several proteins have been shown to be amenable to crosslinking in

Crosslinking of DNA to the nuclear matrix

The nuclear matrix is a three-dimensional network that is necessary for DNA organization and nuclear structure and function. This framework consists of the nuclear membrane with the nuclear lamina and pore proteins, the internal network of ribonuclear proteins, and nucleolar proteins [134]. The nuclear matrix contains anchoring sites for the DNA called matrix attachment regions (MARs) and the DNA is organized into loops of 50–200 kbp between these anchor sites. Loop domain anchoring allows for

Enzymatic repair of DPCs

Studies on some types of cellular DPCs indicate that these lesions can be longer-lived than other types of damage and persist through several DNA-replication cycles [144], [145] and are only partially repaired [146], which may result in permanent DNA alterations and have serious consequences for replication, transcription, and repair processes [147]. A significant background level of accumulated DPCs has been reported in some types of mammalian cells [44], [59], and in mice this frequency

Conclusions

Because DPCs have received less attention than other types of DNA damage, their biological consequences and mechanisms of repair are not well understood. In part, this is because DPC-inducing agents inevitably induce other types of DNA and protein damage. Possible biochemical consequences of the covalent crosslinking of proteins to DNA are blockage of replication, transcription and recombination. Evidence is mounting that DPCs contribute to the cytotoxic, mutagenic, and carcinogenic effects of

Acknowledgements

This article was made possible through support from the Alberta Cancer Board Pilot Project #R-465 and Research Initiative Program Grant #RI-202 (to D.M.), and by grants from the Canadian Institutes of Health Research #MT-14056 (to D.M.) and the National Cancer Institute of Canada #013104 with funds from the Canadian Cancer Society (to M.W.).

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