Editorial pubs.acs.org/CR
Introduction: Genome Modifying Mechanisms
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subfamily serve as key effectors in the innate immune system, targeting foreign DNA elements for hypermutation via cytosine deamination. After introducing these canonical immune functions in their review, Bhagwat and colleagues address the increasing evidence that purposeful DNA deamination can go awry. The authors highlight genomic studies that show the hallmarks of AID/APOBEC activity in oncogenic translocations or mutational landscapes, and discuss the mechanisms involved in deamination. These insights further illustrate the benefits that can arise from accessing a dynamic genome, while also revealing the risks incumbent in enzymatic genome modification. Beyond a role in the ongoing evolutionary arms race, tailoring DNA bases with chemical modifications provides a mechanism for expanding the coding potential of the genome. DNA cytosine methylation is the best known modification in mammalian genomes and can function to muffle portions of the genome to help shape development or cell identity. The scope of epigenetic DNA modifications expanded yet further with the discovery that pyrimidine bases, including 5-methylcytosine, can be oxidized by Fe(II)/α-ketoglutarate dependent dioxygenase enzymes. Oxidized bases are subject to further alterations such as glucosylation, and the realm of epigenetically relevant methylation has recently been extended to encompass adenine methylation in DNA. Base-flipping is a common theme that runs through many enzymes that chemically tailor DNA bases. The extrusion of a nucleobase out of the DNA helix allows for interrogation of the base identity and permits chemical modifications at otherwise inaccessible sites. One such class of DNA modifying enzymes that typically works through base flipping are DNA glycosylases. These enzymes provide an interesting example of the competing demands of stability and adaptability that are at play within the genome. When viewed through the prism of genomic fidelity, DNA glycosylases are part of the base excision “repair” pathway, functioning to excise lesions within the genome. However, these enzymes also play critical roles outside of repair that allow for greater diversity to be accessed in the genome. DNA glycosylases function following deamination by AID to promote antibody diversification, and they can act on 5methylcytosine or its oxidized derivatives as part of a dynamic cycle of DNA demethylation. In their review, Drohat and colleagues describe how such “moonlighting” functions of DNA glycosylases, acting on bases that are not lesions, occur at a mechanistic level. The authors explore the functional consequences of DNA glycosylases acting to promote DNA dynamics and help to reaffirm a view of the genome where both fidelity and adaptability are central. While modifications at the nucleotide level are one class of DNA modifying processes, the other major class involves movement of larger segments of DNA. Retroviral DNA integrases, such as HIV integrase, provide an interesting
enomes must fulfill two competing demands: stability and adaptability. On the one hand, faithful maintenance of the genome is essential, since this blueprint encodes the core functions of life. Genomic stability is achieved through a variety of mechanisms, including high-fidelity replicative polymerases and robust DNA repair processes. On the other hand, adaptability is also critical, as all organisms require the capacity to respond to changing environments. Indeed, purposeful, rather than pathological, DNA alterations underlie a wide range of biological phenomena, from host−pathogen interactions to embryonic development and differentiation. The dynamic genome is the focus of this issue, particularly the DNA modifying enzymes that purposefully modify the DNA code. The chemical transactions catalyzed by these enzymes can alter the nature of individual nucleobases embedded in DNA, promote rearrangements of large stretches of DNA, or integrate foreign DNA. Recent developments have uncovered new modifications to canonical bases and have shed light on the molecular mechanisms at work within the active sites of various DNA modifying enzymes. Importantly, an improved knowledge of the natural mechanisms by which genomes change has offered the ability to synthetically tailor genomes to probe new biology. The significance of DNA modifications is evident even in the most ancient evolutionary battles between bacteria and bacteriophages. Bacteria use specific DNA modifications, such as adenine methylation in specific sequence contexts, to distinguish self from nonself, and their restriction enzymes allow for defense against foreign DNA. Bacteriophages respond by utilizing even more elaborate alterations to overcome the associated restriction mechanisms. In their review, Weigele and Raleigh highlight the tremendous diversity of nucleobase modifications that exist in bacteriophage genomes. These modifications can be introduced by perturbing the free nucleotide pools or by manipulating the bases already embedded within DNA. Their review delves into the scope of enzymes that introduce these marks into DNA, as well as the enzymes that can recognize and restrict modified DNA. Many of these DNA modifications were discovered around the inception of modern molecular biology; however, only now, in the era of genomics, are the candidate pathways and enzymes being exposed. The theme of DNA modifications playing a central role in self versus nonself recognition persists in higher organisms. Nowhere is the dynamic genome more evident than in the mammalian immune system. In adaptive immunity, recombination of large blocks of DNA by the RAG1 and RAG2 enzymes yields one layer of variety in B- and T-cell lineages. Further rounds of adaptation are required to mature antibody responses in B-cells through somatic hypermutation and class switch recombination. These reactions are initiated by the purposeful deamination of cytosine bases by activation-induced deaminase (AID), which generates uracil bases within DNA at the immunoglobulin locus. AID belongs to a broader family of AID/APOBEC DNA deaminases. Members of the APOBEC3 © 2016 American Chemical Society
Special Issue: Genome Modifying Mechanisms Published: October 26, 2016 12653
DOI: 10.1021/acs.chemrev.6b00584 Chem. Rev. 2016, 116, 12653−12654
Chemical Reviews
Editorial
AUTHOR INFORMATION
intersection of the themes of genomic stability and variety, which both become relevant when genomes interact. Following reverse transcription, the viral integrase enzyme catalyzes insertion of the viral genome into actively transcribed regions of the host genome. Cherepanov and colleagues build upon recent solution of retroviral integrase−DNA structures to describe the mechanisms underlying DNA processing and strand transfer. As the integration process is critical to the viral life cycle, these structures have also helped to reveal the mechanisms of action of anti-HIV integrase inhibitors, provide insight into viral resistance mutations, and set the stage for next-generation drug design. Studying genome modifying mechanisms not only provides insight into the processes that regulate genome dynamics, but also offers the opportunity to harness their power. Recently, efforts focused on zinc finger proteins, TALEs, and the CRISPR/Cas system provide compelling evidence for the fact that mechanistic insights into DNA modifying enzymes can be rapidly translated to yield powerful synthetic genome editing tools. From a historical perspective, transposons have proven to be particularly versatile and valuable genome editing tools, and recent mechanistic developments help to refine and expand their impact on biology. Hickman and Dyda describe the transposase enzymes that can catalyze the movement of DNA transposons from one genomic location to another, with a particular focus on the DD(E/D) family of transposases. Highlighted applications include the use of these transposons or their variants to perform genome-wide insertional mutagenesis studies or to introduce transgenes into genomes. While integrases and transposases tend to mobilize DNA to nonspecific target sites, site-specific recombination processes are exemplified by the tyrosine site-specific recombinases (TSSRs) reviewed by Buchholz and colleagues. These enzymes exchange DNA strands between specific, typically palindromic, sequences via an active site tyrosine−DNA intermediate. As with nucleobase modifications, there is a great diversity of TSSRs at the bacteria−bacteriophage interface, with interesting questions arising about how these systems have been tailored for specific functions in prokaryotic organisms. Insights into the molecular requirements for specific family members, such as Cre, have permitted these natural genome modifying enzymes or designed variants to become stalwarts in the genome editing toolbox. Furthermore, revolutionary new approaches, such as the Brainbow approach for lineage tracing, rely upon T-SSRs. Genome modifying mechanisms continue to draw significant interest because studies on the associated enzymes achieve dual purposes. For one, understanding the diversity of catalyzed reactions provides new insights into the dynamic nature of the genome. On the other hand, revealing the mechanisms involved in natural DNA modification offers multidisciplinary new tools to introduce, perturb, or modify genes. Together, this issue highlights the biochemistry of DNA modifying enzymes and provides key illustrative examples of the role these enzymes play in shaping and reshaping genomes.
Notes
Views expressed in this editorial are those of the authors and not necessarily the views of the ACS. Biographies
Rahul M. Kohli received his B.S. degree from the University of Michigan in 1994, where he was introduced to enzymology under Professor Vincent Massey. He went on to obtain M.D. and Ph.D. degrees from Harvard Medical School in 2004, completing his graduate studies under Professor Christopher T. Walsh. After completing a residency in internal medicine at the University of Pennsylvania, Kohli pursued postdoctoral training in infectious diseases and biochemistry at Johns Hopkins University, studying under Professor James T. Stivers. He took on his current position as an Assistant Professor of Medicine, and of Biochemistry & Biophysics, at the University of Pennsylvania’s Perelman School of Medicine in 2010. His research interests are focused on DNA modifying enzymes and pathways that contribute to immunity, epigenetics, and acquired antibiotic resistance.
Gregory D. Van Duyne was born in Auburn, New York, and graduated from Cornell University with a B.A. in Chemistry in 1983. He studied the structures of bioactive natural products at Cornell with Jon Clardy, receiving his Ph.D. degree in chemistry in 1988. Van Duyne was a Research Associate at Cornell from 1988 to 1991 and then joined Paul Sigler’s laboratory at Yale as a Postdoctoral Fellow, where he pursued research on the structures of regulatory protein−DNA complexes. Van Duyne joined the faculty of the University of Pennsylvania School of Medicine in 1995 and from 2000 to 2012, he was an Investigator of the Howard Hughes Medical Institute. He is currently the Jacob Gershon-Cohen Professor of Medical Science and investigates the mechanisms of action and biomedical applications of bacteriophage and retroviral integrases.
Rahul M. Kohli
University of Pennsylvania
Gregory D. Van Duyne
University of Pennsylvania 12654
DOI: 10.1021/acs.chemrev.6b00584 Chem. Rev. 2016, 116, 12653−12654
