DNA Repair, Genetic Instability, and Cancer

The following sections are included:

• Preface

• Contributors

• Contents

Mechanisms have evolved to help protect our genetic material from endogenous and exogenous damage that are vital for an organism's survival. This ability to recognize damaged DNA and simultaneously regulate cell cycle progression and DNA repair is critical for genomic stability, and defects in these pathways are hallmarks of cancer. The DNA damage response pathway is a multi-component signal transduction network that consists of a multitude of proteins whose functions are still under investigation. This review will focus on the current findings in the field including how the DNA damage response influences cancer predisposition and formation.

Base damage or hydrolytic decay originating from endogenous processes or caused by environmental compounds constantly occurs in genomic DNA. Base excision repair (BER) is the major repair mechanism that removes oxidized, alkylated, deaminated bases as well as apurinic/apyrimidinic (AP) sites. BER is initiated by specific and versatile DNA glycosylases, many of which have redundant functions. The AP site generated by a DNA glycosylase is processed by a stepwise process that involves AP endonuclease, AP lyase, DNA polymerase and DNA ligase. Mammalian BER can operate via distinct subpathways. Both the biochemistry and structural biology of the BER components have progressed rapidly over the last decade. Recent biochemical studies are also beginning to identify an intricate network of protein-protein interactions that may play an important role in mediating concerted steps in BER and in coordinating BER with other DNA metabolic pathways. Gene knockout animal models of the majority of the BER enzymes have been generated or attempted, which provided significant insights into their biological role in the protection of genomic stability. The importance of BER in humans is shown by the findings that defects in the BER enzymes can result in increased risk for cancer, neurodegenerative and other disorders. This is best illustrated by the recent discovery that biallelic germline mutations in DNA glycosylase MYH lead to increased risk for colorectal cancer.

One of the major challenges for the earliest forms of life was to protect the cellular DNA from the highly mutagenic solar ultraviolet and from exposure to environmental mutagens. The nucleotide excision repair (NER) pathway is responsible for the removal of a large variety of lesions, including those induced by ultraviolet light and bulky adduct-forming chemicals. The NER mechanism consists of two major steps. The incision step involves introduction of dual incisions flanking the DNA lesion and subsequent removal of the damage-containing oligonucleotide. The next repair synthesis step restores the damaged site by filling in the gap created by the incision step. Humans with NER deficiencies suffer from the DNA repair syndrome, xeroderma pigmentosum (XP) with manifestations of both acute sunlight sensitivity and profoundly elevated occurrence of skin cancer. XP is a classical example of the direct relationship between genomic instability and cancer development. This chapter highlights the molecular mechanism of NER and its impact on human cancers.

DNA mismatch repair (MMR) is an important genome caretaker system. It ensures genomic stability by correcting mismatches generated during DNA replication and recombination, suppressing homeologous recombination, and triggering apoptosis of cells with severe DNA damage. Protein components required for these reactions are highly conserved through evolution, and MutS-like and MutL-like proteins in mammalian cells are key players responsible for the initiation steps of both the strand-specific mismatch correction and the MMR-dependent apoptotic signaling. The inactivation of the genes encoding these activities leads to genome-wide instability, particularly in simple repetitive sequences, and the predisposition to certain types of cancer, including hereditary non-polyposis colorectal cancer.

Homologous recombination (HR) is highly relevant to cancer intervention for two major reasons. First, defects or deregulation in HR systems are major contributing factors in tumorigenesis. HR is important for the accurate repair of DNA double-strand breaks and DNA interstrand crosslinks, and it restarts stalled or collapsed replication forks. During these processes, HR restores the gross structure of DNA with minimum probability of mutation. HR is also tightly co-ordinated with mitosis and cell cycle checkpoint regulation, to ensure that cells with excessive DNA damage have minimum chance of being propagated. Thus, HR plays pivotal roles in maintaining genome stability and preventing tumorigenesis. Although HR is generally an error-free DNA repair process, hyper-recombination, hypo-recombination and altered HR outcomes can all lead to genomic instability. Thus HR can be mutagenic and contribute to genomic instability when it is not properly regulated. Second, the status of HR in cancer cells may be used as a marker for tailoring individual cancer therapy and improving prognosis evaluation. Current studies are focused on the stepwise DNA structural changes that occur during HR, and on the identification and characterization of proteins involved in HR. There are two categories of HR proteins: those with enzymatic activity that directly promote the chemical reactions during HR, and accessory proteins that regulate HR enzymes and co-ordinate HR reactions with other important cellular processes. It is important to fully identify and characterize the proteins of both categories as they may serve as targets for cancer therapy, or as markers for diagnosis, prognosis and treatment monitoring. In addition, a detailed picture of HR protein structure and function may provide new opportunities for cancer intervention through improvements in our understanding of cellular responses to therapeutic DNA damage.

Of the various types of DNA damage that can occur within the mammalian cell nucleus, the DNA double-strand break (DSB) is perhaps the most dangerous. DSBs are induced by ionizing radiation, chemotherapeutic drugs, as well as by the byproducts of cellular metabolism. Understanding exactly how a mammalian cell responds to and repairs a DSB is very important because, on one hand, DSBs cause cancer while, on the other hand, DSBs are induced by chemotherapeutic agents to treat the disease. Of the two main mechanisms by which cells can repair DSBs, NHEJ (non-homologous end joining) and HR (homologous repair), NHEJ is the predominant repair pathway in mammalian cells. In this chapter we describe the main steps in the NHEJ pathway of repair highlighting major recent discoveries, and also provide a perspective on the link between defective NHEJ and human disease.

Fanconi anemia (FA) is a rare genetic disorder characterized by congenital abnormalities, bone marrow failure, chromosomal instability and cancer predisposition. The disease has often been considered as a model for studying the repair of interstrand DNA crosslinks (ICLs) because the hallmark feature of FA is cellular hypersensitivity to drugs that cause ICLs. To date, 11 FA genes have been discovered, and their products function in the same DNA damage response network as the breast cancer susceptibility gene products, BRCA1 and BRCA2. One FA protein, FANCD1, is identical to BRCA2, whereas another one, FANCJ is the same as BRIP1, a DNA helicase interacting with BRCA1. A key component of this network is a multisubunit complex, termed FA core complex, which contains at least eight FA proteins, including a ubiquitin ligase (FANCL) and a DNA translocase (FANCM), and is required for the monoubiquitination of FANCD2 in response to DNA damage. Studies of FA have provided novel insights into the mechanisms of genome maintenance and DNA repair.

A fundamental question in DNA repair is how a lesion, when embedded in millions to billions of normal base pairs, is detected. Extensive structural and functional studies reveal atomic details of DNA repair protein and nucleic acid interactions. This chapter summarizes seemingly diverse mechanisms for lesion recognition and presents a general strategy utilized by various DNA repair pathways.

In addition to the numerous DNA repair pathways, living organisms also possess other mechanisms to avoid the severe consequences of DNA damage that stalls replication fork progression. Eukaryotic cells from yeast to humans have a pathway known as DNA post-replication repair (PRR) or DNA damage tolerance (DDT), which employs one of two means to bypass the replication-blocking lesion instead of removing it. One method is translesion DNA synthesis (TLS), which utilizes a panel of specialized DNA polymerases. The fidelity of these TLS polymerases varies and is largely dependent on the lesions encountered. With the exception of Polŋ bypass of UV-induced thymine dimers, TLS is generally regarded as error-prone. An alternative means of lesion bypass is highly accurate and is believed to involve template switching or sister chromatid exchange. Pioneer studies in budding yeast reveal that DDT is regulated by the covalent modifications of PCNA, a trimeric sliding-clamp processivity factor for replication. The Rad6-Rad18 complex monoubiquitylates PCNA at the K164 residue and this monoubiquitylated PCNA recruit TLS polymerases. The Rad5-Ubc13-Mms2 complex polyubiquitylates PCNA at the same residue via a noncanonical K63 chain that promotes error-free DDT.

The DNA of an organism suffers from external environmental stresses as well as internal insults and errors resulting from replication and recombination. There are multiple DNA repair pathways to maintain the integrity of the genome. DNA nucleases play a crucial role in mismatch repair, nucleotide excision repair, base excision repair and double-strand break repair. This chapter summarizes the role of nucleases in DNA repair, replication and recombination. One of such nucleases, flap endonuclease-1 (FEN-1), is discussed in detail as a multifunctional and structure-specific nuclease involved in several nucleic acid processing pathways, including RNA primer removal, long patch base excision repair, and the resolution of di- and tri-nucleotide repeat secondary structures and stalled DNA replication forks. The multiple functions of FEN-1 are regulated via several means, including the formation of complexes with different protein partners, nuclear localization in response to cell cycle or DNA damage, and post-translational modifications. Its functional deficiency is predicted to cause genetic diseases, including Huntington's disease, myotonic dystrophy and cancers.

Genome sequence information from human as well as model organisms together with the advancement in high-throughput technology have transformed traditional biology researches into a new era of large-scale systems biology. Systems biology aims to provide a system-level understanding of biology which includes genome organization, epigenome, transcriptome and proteome. Information from these “-omics” researches can be used to build biological and mathematical models to describe biological phenomena and to give predictive values. In this review, the author provides an overview of the recent progress made in genomics and epigenomics, including a summary of some of the recent studies in this field carried out by the author and colleagues.

The role of DNA repair in the etiology of cancer has been well illustrated in several hereditary syndromes, in which an inherited defect in DNA repair is associated with an extraordinarily high incidence of cancer. However, such an association between an inherited DNA repair defect and the risk of cancer has not been apparent in the general population. In the past 10 years, there has been a growing body of literature that addressed this important research question at the population level. Several assays that measure DNA repair phenotypes (e.g. transfection-based host-cell reactivation, expression of genes and proteins, and cytogenetics, etc.) have been applied to population-based studies. It has been demonstrated that a suboptimal DNA repair capacity for removing DNA damage induced by ultraviolet light and benzo[a]pyrene diol epoxide, two-well known environmental carcinogens, is associated with an increased risk of skin cancer (e.g. melanoma and non-melanoma skin cancers) and tobacco-related cancers (e.g. lung and head and neck cancers), respectively. More recently, the completion of the Human Genome Project and the initiation of the Environmental Genome Project have led to the discovery of numerous single nucleotide polymorphisms in DNA repair genes, followed by a wave of association studies, for cancer in particular. Published data appear to have established a genetic basis for a suboptimal repair phenotype in the general population and provide a rationale for future genetic screening for at-risk populations who can be targeted for the primary prevention of cancer.

The following sections are included:

• INDEX