Yanbin Zhang - US grants

DNA interstrand crosslinks (ICLs) can be induced endogenously (e.g. lipid peroxidation) and by environmental agents (e.g. cigarette smoking, automobile exhausts, and pollution). The ICL damage tethers both strands of DNA duplex and blocks essential DNA metabolic functions such as replication. It remains poorly understood how the ICL damage is repaired in humans. ICLs are also the primary toxic lesions induced by many bi-functional chemotherapeutic drugs to kill cancerous cells. Cells develop resistance to such agents through up-regulating their repair capacity of ICLs, thereby compromising the therapeutic efficacy. Fanconi anemia (FA, FANC) is a hereditary disorder characterized by bone marrow failure, developmental defects, predisposition to cancers, hypersensitivity to crosslinking agents, indicating involvement of FA proteins in repair and tolerance of ICLs. At least 13 FANC genes have been identified thus far. Eight (FANC-A, B, C, E, F, G, L, and M) of the thirteen FANC gene products are found in a protein complex, termed as the FA core complex. Based on the preliminary data and previous observations, we hypothesize that FANCM participates in the incision step of ICL repair, and the FA core complex is involved in the regulation of incision endonuclease activities for precise and efficient incision of ICLs. The overall goal of this proposal is to delineate the mechanism of the dual incisions on both sides of ICL damage (unhooking) and to determine how the FA core complex helps maintaining stability of replication forks and contributes to the ICL unhooking when the DNA replication fork encounters an ICL. We will characterize the enzymatic properties of FANCM, identify the endonucleases that carry out the ICL incision, and test whether they collaborate with each other for successful ICL unhooking. By employing RNA interference and cDNA complementation analyses, we will verify the in vitro discoveries in human fibroblast cells through monitoring the ICL incision-induced production of DNA double strand breaks. We will test how components of the FA core complex are involved in maintaining stability of replication forks and in regulating activities of the ICL incision endonucleases. We will purify all components of the FA core complex, evaluate their DNA damage recognition activity, profile their physical and functional interactions with the incision endonucleases, and delineate the regulatory mechanism of damage incision in a biochemically defined in vitro system. We will also determine whether the FA core complex recruits and regulates the endonucleases in human cells through RNA interference and confocal microscopy. Understanding the mechanism of the ICL recognition and incision will not only contribute to the overall clarification of the ICL repair process, but also provide a novel basis for interventional strategies. For example, developing inhibitors of the ICL repair would lead to future translational research for chemosensitizers to overcome the clinically observed drug resistance in cancer patients.

DESCRIPTION (provided by applicant): Nucleoside reverse transcriptase (RT) inhibitors continue to be an important component of therapy against human immunodeficiency virus type 1 (HIV-1). These compounds are phosphorylated by cellular enzymes and are incorporated into DNA by HIV-1 RT leading to chain termination during viral DNA synthesis. The ability of mutants of HIV-1 RT to remove chain-terminating nucleotides from nascent DNA chains (excision activity) is an important mechanism of drug resistance, and mutants with elevated excision activity are often selected during therapy. We propose to develop an analytical approach to determine what influences excision activity in infected cells that will help us understand what drives mutant selection in vivo. Aim 1 is to use purified wild type and mutant HIV-1 recombinant proteins to evaluate the roles of RNA cleavage by the ribonuclease H activity of RT and template fragment dissociation in regulating excision rescue of blocked DNA chains. Aim 2 is to determine the effect of HIV nucleocapsid proteins on the release of secondary ribonuclease H cleavage fragments and excision of chain terminators. Aim 3 is to develop methods to measure intracellular excision activity and to use lentivirus vectors packaged with wild type or mutant RTs to evaluate factors that regulate excision in infected cells. The use of the excision reaction of HIV RT as a therapeutic target is limited by our lack of understanding of the intracellular reaction, including factors that influence timing and rate of the reaction, stability of the enzyme-substrate complexes that carry out secondary ribonuclease H cleavage and chain terminator excision, contribution of viral and cellular factors to these reactions, and accessibility of the viral replication machinery to acceptor substrates and inhibitory molecules. These questions will be addressed by the proposed studies. PUBLIC HEALTH RELEVANCE: Developoment of new therapies against HIV will continue to be necessary because of the rapid mutation rate of this virus that facilitates selection of drug resistance and because of the need to continue therapy in each patient over many years. Research is proposed to characterize intracellular processes that influence the ability of HIV to escape from a class of drugs that blocks viral DNA synthesis, which will provide insight into the potency of these drugs in different tissues and metabolic conditions, the tissue sites where selection of resistance mutants takes place, and how therapeutic strategies can be changed to optimize drug efficacy and avoid selection of resistant mutants.

Mismatch Repair (MMR) of errors during DNA replication is essential for human life. Deficiencies in MMR cause a mutator phenotype and is the underlying cause of hereditary non-polyposis colorectal cancer. Helicase activity facilitates mismatch removal and is required for bacterial MMR. Our preliminary results show that certain members of the human RecQ family of DNA helicases dramatically stimulate DNA excision in a mismatch-specific manner. This suggests that human DNA helicases participate in MMR in ways that have not been previously appreciated. The present proposal will test the hypothesis that mammalian helicases play an essential role in MMR by regulating the mismatch removal step. In this proposal, we describe a systematic approach to define the role of human RecQ helicases in MMR. Using our in vitro reconstitution system we will explore the mechanisms of MMR stimulation. Furthermore, we will extend the studies to cell-based experimental systems through genetic experiments, confocal microscopy, and functional tests. Aim 1 is to determine physical and functional interactions between RecQ helicases and MMR proteins and their role in MMR. Aim 2 is to study role of RecQ helicases in MMR through genetic complementation and cell-based analyses. Unveiling the biological role of RecQ helicases in mismatch repair will not only open a new level of mechanistic understanding of the human MMR pathway and its involvement in cellular response to environmental toxins such as chromium, it may also lead to development of new diagnostic markers and more effective therapeutic strategies for treating cancer and other diseases associated with MMR deficiency.

Interstrand crosslinks (ICLs) are deleterious DNA damages in which both strands are covalently bound together, generating a stalled replication state cytotoxic to cells. Defective repair of ICLs is associated with Fanconi Anemia syndrome (FA) and predisposition to cancer. At least seventeen FA genes have been identified up to date. This proposal focuses on a specific FA protein, FANCA, a member of the FA core complex that is mutated in 64% of the entire FA patient population and does not have a clear function. Our preliminary study shows that FANCA destabilizes (unwinds) DNA helix and recognizes DNA interstrand crosslink damage (ICL) in a replication fork. Intriguingly, FANCA anneals single-strand DNA and catalyzes strand exchange as well. Furthermore, FANCA synergistically interacts with Rad51, the major recombinase in double strand break (DSB) repair. Based on these data, we hypothesize that FANCA directly participates in repair of interstrand crosslinks through its DNA-destabilizing (`unwinding') activity and facilitates subsequent repair of double strand DNA breaks by catalyzing strand annealing and exchange. In order to delineate the role of FANCA in DNA repair, we will use a biochemically defined in vitro system, a cell-based DSB repair study system, and a living cell imaging system to accomplish four aims: 1) Delineate the molecular mechanism of how FANCA catalyzes DNA `unwinding', single-strand annealing, and strand exchange. 2) Determine the role of FANCA in recognition and incision of DNA interstrand crosslinks. 3) Study the biological role of FANCA in double strand break repair. 4) Examine the physiological role of the DNA `unwinding', single- strand annealing, and strand exchange activities of FANCA in the etiology of Fanconi anemia. Understanding the role of FANCA in DNA repair will not only contribute to the overall clarification of the ICL repair process, but also provide novel insights into the etiology of Fanconi anemia and its associated cancer.

Abstract One of the most predominant hallmarks driving cancer development is genome instability. It creates genome- wide diversity that enables cells to acquire additional capabilities required for cancer development and progression. Most of the ~400 genes known to be mutated and implicated in cancer development are a direct result of increased genome instability. Therefore, understanding the molecular mechanisms of genome instability in cancer cells is imperative for the development of novel treatment strategies. Fanconi Anemia (FA) is a hereditary disorder caused by mutations in at least 22 genes and clinically characterized by bone marrow failure and predisposition to cancer. This proposal focuses on FANCA, a gene that is mutated in ~64% of the entire FA patient population. During the preliminary studies, we found that FANCA promotes error-prone DNA repair that drives genome instability; its expression is upregulated in many cancer types, and the expression level is strongly associated with breast cancer progression and inversely correlates with cancer patient survival. Intriguingly, FANCA recruitment to double strand breaks and DNA damage sites requires active transcription in a KillerRed live cell analysis. More importantly, knockout of FANCA in a triple negative breast cancer cell MDA-MB-231 initiates cell cycle arrest and cellular senescence and abolishes breast cancer formation in mice. Based on these preliminary data, we hypothesize that high expression of FANCA in cancer cells promotes error-prone repair, genome instability, and cell cycle progression. To delineate the role of FANCA in genome instability and cancer development, we will use a biochemically defined in vitro system, a transcription-coupled DSB repair reporter system, a KillerRed live cell imaging system, a xenograft mouse model, and genome-wide instability analysis to accomplish three aims: Aim 1 is to determine the molecular mechanism of how FANCA contributes to R-loop-mediated genome instability; Aim 2 is to study the role of FANCA in DSB-mediated genome instability and how FANCA is regulated; Aim 3 is to determine the relationship between FANCA-mediated genome instability and cell cycle progression. Completion of this proposal will define a novel role for FANCA in genome instability. This work will also elucidate the significance of FANCA as a unique, rationale-driven target for cancer treatment. The outcome of this proposal will expand treatment strategies for cancer patients with elevated FANCA expression and genome instability.