Lecture Summaries


Introduction & Overview
The instructors and students will introduce themselves and we will discuss our scientific interests. The instructors will summarize the goals and expectations for the course. We will introduce the topic of DNA damage and repair, focusing on the major sources of DNA damage, the types of DNA damage that can occur, and some of the mechanisms by which cells can repair DNA damage. We will also introduce tools that can be used to search for scientific research papers, discuss how scientific publications are organized, and talk about how one evaluates the quality of a scientific research paper. Finally, a brief introduction to topics relevant to the papers to be read for week 2 will be provided.

Alkylating agents: How chemical warfare became medicine

The history of modern day chemotherapy is strongly intertwined with the chemical warfare agents used in World Wars I and II. As we will learn from the first paper, during the first World War medical examinations of people exposed to mustard gas and subsequent autopsy reports described signs of severely impaired bone marrow function and a loss of white blood cells, suggesting that exposure to mustard gas could inhibit the proliferation of specific cell types.

Interest in this phenomenon was renewed in 1943, when the German air raid in Bari, Italy on the SS John Harvey battleship exposed more than 1000 people—both US service men on the ship and civilians in the surrounding town—to its secret cargo containing mustard gas bombs. Medical examinations and autopsy reports reinforced the observations of impaired bone marrow function and loss of blood cells described in the first paper. This event also emphasized the risks associated with war gases such as mustard gas, and underscored the need to understand their mechanisms of action. By the 1940s, it was recognized that mustard gas and chemically related nitrogen mustards exerted the most severe effects on highly proliferative cells (those undergoing frequent cell division).

Therefore, scientists who were searching for chemicals that could be used to inhibit the growth of cancer hypothesized that diseases such as lymphoma, which is characterized by an inappropriate proliferation of blood cells, could be treated therapeutically with alkylating agents such as mustard gas. The second paper to be discussed presents one of the first clinical studies of the use of alkylating agents as chemotherapeutic drugs to treat patients with lymphomas, leukemias, and Hodgkin’s disease.

Direct Repair: Levels of _O_6-methylguanine-DNA methyltransferase (MGMT) as a potential predictor of response to alkylation-based chemotherapeutics

A DNA repair strategy that is often overlooked is direct reversal, whereby damaged DNA bases can be reverted back to the normal base without the requirement for multiple enzymatic steps that create intermediates or generate DNA strand breaks. The repair protein _O_6-methylguanine-DNA methyltransferase (MGMT) functions to directly repair alkyl adducts at the _O_6 position of guanine and to a lesser extent the _O_4 position of thymine. MGMT irreversibly transfers the alkyl group onto a cysteine residue in its active site in a “suicide” reaction, which causes a conformational change in the protein that targets it for ubiquitin-mediated degradation.

This week, we will explore how the levels of MGMT in a cell influence its response to alkylation-based chemotherapeutic agents and whether MGMT status can be used as a predictor of patient response. The first paper examines how modulation of MGMT expression in a mouse model alters the response to alkylation-induced damage at the whole-animal and tissue-specific levels. The second paper explores how MGMT expression in tumors of patients with glioblastoma multiforme influences the response to treatment with the mono-alkylating agent temozolomide, the toxicity of which is mediated by the formation of _O_6-methylguanine lesions in DNA.

Mismatch Repair: A guardian against replication errors

The mismatch repair (MMR) pathway primarily functions to increase the fidelity of DNA replication by correcting errors (such as mispaired bases and short insertions or deletions) that occur during DNA synthesis and escape proofreading, in which an incorrect base is recognized and excised by the exonuclease activity of the replicative polymerase so the correct base can be re-inserted. In the simplest terms, initiation of mismatch repair requires the recognition and binding of base/base mismatches or 1–2 nt insertions/deletions by MutSα (MSH2/MSH6 heterodimer). MutSα recognition serves to recruit components of the excision machinery, including MutLα (MLH1/PMS2 heterodimer), the processivity factor proliferating cell nuclear antigen, or PCNA, and EXO1 exonuclease which load onto the DNA at a preexisting nick and degrade it past the mispaired region. Repair synthesis of the degraded strand by a high-fidelity polymerase followed by DNA ligation completes the repair. Interestingly, the status of the MMR pathway plays a role in both cancer susceptibility and biological outcomes following chemotherapeutic treatments.

The first paper is an early study that derives a model for mismatch repair in yeast through analyzing the in vitro interactions among purified MSH2, MLH1 and PMS1 and DNA. The second paper explores the relationship between the status of the mismatch repair pathway in cells and sensitivity to the cytotoxic _O_6-methylguanine lesions induced by the prototypical alkylating agent MNNG or the alkylation-based chemotherapeutic agent TMZ. This study begins to explore the highly coordinated effort between the MMR and DNA damage response pathways to stimulate cell cycle checkpoints and repair or programmed cell death in response to alkylation-induced damage.

Mutations and Epigenetics: Multiple means by which inactivation of the mismatch repair pathway complicates the treatment of cancer

Predisposition to hereditary nonpolyposis colorectal cancer (HNPCC) has been associated with germline mutations in genes that encode proteins within the mismatch repair pathway. Such colorectal carcinomas display a mutator phenotype characterized by microsatellite instability (changes in the length of repetitive DNA sequences) and are resistant to alkylation-based chemotherapeutics.

From the first paper, we will see how the knowledge of mismatch repair mutations in yeast and genetic analysis were applied to discover several founder germline mutations in the human MutL homolog 1, hMLH1 that predisposed Finnish families to hereditary nonpolyposis colorectal cancer (HNPCC). Interestingly, not all microsatellite instability-positive colorectal carcinomas display genetic or epigenetic (e.g., silencing of the gene through promoter methylation) defects in the mismatch repair proteins. In recent years, it has been shown that cells mount a global response to damage, which extends beyond the transcriptional regulation and protein interactions of the core DNA repair, replication and cell cycle checkpoint proteins.

The second paper addresses how chromatin remodeling might contribute to mismatch repair efficiency by studying the interaction of MutSα with chromatin, specifically chromatin modifications on lysine 36 of histone 3 by the enzyme histone methyltransferase SETD2.

Base Excision Repair: Intermediate threat to genomic stability

Base excision repair (BER) is initiated by distinct glycosylases that recognize specific aberrant bases and cleave the glycosidic bond between the damaged base and the DNA backbone leaving an apurinic/apyrimidinic (AP) site. The phosphodiester backbone is then hydrolyzed by AP endonuclease (APE1) to create a single-strand break with 3’OH and 5’deoxyribose-5-phosphate (5’dRP) termini. DNA polymerase β functions to remove the 5’dRP with its lyase domain and reincorporate the missing nucleotide with its polymerase domain. Finally, the nick is resealed by either DNA ligase I or the XrccI/Ligase IIIα complex.

Importantly, if the entire multi-step BER is not completed, an accumulation of mutagenic and/or cytotoxic intermediates can arise which can potentially block transcription and replication or induce stress responses that can lead to apoptosis. Intriguingly, these intermediates might contribute to the onset of disease as well as to cytotoxic side effects of chemotherapeutics that function by inducing damage in both tumor and adjacent tissues.

The first paper uses mouse genetic experiments to address how expression of the Alkyladenine DNA glycosylase (Aag), which recognizes 7-methylguanine and 3-methyladenine lesions among others, can mediate alkylation-induced tissue damage and whole-animal lethality. The second paper utilizes a mutated version of the uracil-DNA glycosylase (UDG) to induce an excessive number of AP sites specifically in the mitochondrial genome to study how the accumulation of BER intermediates can contribute to behavioral defects, premature aging and neurodegenerative diseases similar to Parkinson’s and Alzheimer’s diseases.

Base Excision Repair: The GO system limits mutagenesis by oxidative damage

The major base lesion caused by oxidative damage, 8-oxoguanine (8-oxoG or GO lesion) is highly mutagenic because of its ability to pair with both cytosine and adenine. The “GO system” comprising OGG1 (MutM), MUTYH (MutY), and MTH1 (MutT), protects the cell against the potentially mutagenic and cytotoxic effects of oxidative damage, which can lead to carcinogenesis or degenerative disorders, respectively. The BER glycosylase OGG1 can excise 8-oxoG lesions from DNA when they are paired with cytosine, leaving an AP site that is further processed by endonucleases and repair synthesis. The glycosylase MUTYH excises the adenine incorrectly inserted opposite the 8-oxoG, allowing the repair polymerase lambda the opportunity to insert the correct C opposite the 8-oxoG and thus providing another opportunity for OGG1 to remove the oxidized lesion. Finally, MTH1 sanitizes the nucleotide pool of 8-oxoGTP by hydrolyzing oxidized purine nucleoside triphosphates to corresponding monophosphates so that an aberrant base cannot be incorporated into DNA.

Recent studies have identified mutations in MUTYH within colorectal carcinoma patients who do not display alterations in any of the critical mismatch repair genes, MSH2, MSH6, MLH1 or PMS1. The first paper investigates the biological and biochemical consequences of MUTYH variants in cells from patients with MUTYH-associated adenomatous polyposis (MAP) to address possible genotype/phenotype correlations and the pathogenicity of unclassified variants. The second paper addresses how cells stimulate distinct programmed cell death pathways when oxidative damage accumulates in either the nuclear or mitochondrial DNA because of modified levels of OGG1.

Field Trip to Blueprint Medicines

Blueprint Medicines is a bio-pharmaceutical company located in Cambridge. The vision of Blueprint Medicines is, “to improve the lives of patients by transforming subsets of cancer from acute diseases to manageable conditions.” We will visit Blueprint Medicines to tour its facilities and introduce students to the research strategies Blueprint Medicines uses in cancer research to systematically develop drugs that will personalize treatment and target specific genetic aberrations that drive the formation and progression of cancer.

While the majority of cancer drugs used in the clinic function by damaging DNA to prevent cell division or induce cell death, mutations in genes involved in DNA repair pathways can cause tumors to become resistant to these particular therapies. We will learn how Blueprint Medicines uses molecular signatures to develop drugs that are selective against cells with specific aberrations that cause cancer and mechanisms of resistance to drugs.

16,568 base pairs: Mitochondrial DNA repair mechanisms and the deadly consequences of failing to maintain the mitochondrial genome

While greater than 99% of our genetic information is stored in the nucleus, about 16 kilobases of DNA are found in the mitochondria. This DNA encodes gene products that are essential for human life, including proteins that are required for metabolic processes and RNA components of the translation machinery needed to produce mitochondrial proteins. Thus, maintenance of the mitochondrial genome is critically important for human health. A major threat to mitochondrial DNA comes from the metabolic process known as oxidative phosphorylation, which occurs exclusively in mitochondria. While this process generates ATP, the energy currency of the cell, it also releases chemically reactive oxygen species, which can damage DNA. To deal with this and other types of DNA damage, cells have evolved DNA repair mechanisms that operate in the mitochondria.

In the first paper, we will learn about how cells respond to mitochondrial DNA damage and how excessive mitochondrial DNA damage can lead to a loss of mitochondrial DNA and impaired oxidative phosphorylation. In the second paper, we will learn about a disease called Alpers’ syndrome, which can arise from mutations in the mitochondrial DNA polymerase and is characterized by a gradual loss of mitochondrial DNA, which ultimately leads to severe illness and death.

Extreme sun-sensitivity: Nucleotide excision repair defects in xeroderma pigmentosum patients

Anyone who has a sunburn knows firsthand about the consequences of exposing skin cells to ultraviolet radiation. Ultraviolet radiation in sunlight induces several types of DNA damage, including pyrimidine dimers and 6–4 photoproducts (light-induced molecular rearrangements of DNA bases). These types of DNA damage involve the formation of chemical bonds between two DNA bases. They constitute major structural disruptions that block both DNA polymerase and RNA polymerase and can induce mutations or cell death; peeling dead skin after a sunburn is the result of massive cell death triggered by exposure to UV light.

Cells are equipped with a DNA repair pathway known as nucleotide excision repair (NER), which deals with this type of DNA damage. Some individuals are born with the disease xeroderma pigmentosum (XP), which is caused by mutations in genes that are critical for efficient NER. XP is a rare disease that afflicts fewer than 1 in 20,000 live births and is characterized by premature aging. Because of the NER defect, people with XP are extremely sensitive to sun exposure, and have a nearly 2000-fold increased risk of skin cancer in sun-exposed skin. In the first paper, we will learn how cell lines derived from XP patients were used to deduce important details of the mechanism of NER. In the second paper, we will learn about single nucleotide polymorphisms (differences in the DNA sequences of people in the general population) in NER genes that predict disease susceptibility and therapeutic outcome.

Unwinding less: Depletion of Werner helicase activity as both a cause and a treatment of disease

Double-strand breaks are a particularly dangerous type of DNA damage, because in contrast to base mismatches, alkylation damage, and the bulky lesions induced by UV radiation, a template strand is not immediately available for restoring the correct sequence of the damaged DNA. Cells have evolved multiple pathways that act on double-strand breaks, including homologous recombination (HR), which uses a second DNA molecule as a template to accurately repair the broken DNA molecule, and non-homologous end joining (NHEJ), which is a more error-prone pathway that involves directly ligating (joining) broken DNA ends back together.

Double-strand break repair involves numerous proteins that play important roles in processing broken DNA ends; among them is a gene named WRN that encodes a DNA helicase that unwinds DNA and is involved in both HR and NHEJ pathways. People who have mutations that inactivate WRN suffer from a premature aging condition (known as Werner’s syndrome) and an increased risk of cancer. We will read a recent paper that reveals a new function for Werner syndrome protein in base excision repair. From a second paper we will learn how small-molecule inhibitors of the Werner syndrome protein were discovered and, perhaps counter-intuitively, could become useful drugs in the treatment of cancer.

Stem cells: DNA damage and differentiation, do they mix?

Stem cells are distinguished by their ability to differentiate into multiple different types of cells. This important biological role for stem cells raises the question: are they subject to any special standards with regard to maintaining the integrity of their genomes? The first paper we will discuss today explores this question in detail from the perspective of researchers who would like to use stem cells for regenerative medicine; DNA repair capacity in four pathways that we have discussed in previous weeks is compared in stem cells and non-stem cells.

A second intriguing question is whether there are particular consequences for human health when a DNA repair defect occurs in stem cells. In the case of the disease Fanconi anemia (FA), a defect in the repair pathway that deals with DNA inter-strand cross-links is associated with an inability to generate blood cells. Although the FA defect is manifest in all cells in affected individuals, the disproportionate effect on blood cells suggests that the repair defect is particularly harmful to hematopoetic stem cells (the progenitor cells that differentiate into blood cells). The second paper explores this topic at the molecular level by mechanistically investigating factors that control the DNA damage response in hematopoietic stem cells.

Students Oral Presentation Assignments
Students present 15-minute papers as described in the assignments section, and a final discussion evaluating the course is held

Course Info