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RESEARCH BRIEFSStructure Suggests How DNA Repair Enzyme Spots TroubleA crystal structure by HMS researchers showing a versatile human repair enzyme bound to a damaged DNA base gives an initial view into the method of this fix-it molecule. The enzyme, 3-methyladenine DNA glycosylase (AAG), works on damaged bases that are chemically and structurally diverse, leaving the myriad intact bases in the genome alone. Determining how AAG recognizes such a variety of substrates is a key to understanding how it can distinguish damaged from normal bases.
This new crystal structure reveals DNA repair enzyme 3-methyladenine DNA glycosylase (AAG) bound to a damaged base. The base (black) is flipped out of the DNA double helix (blue) and into AAG's active site (orange and green). Courtesy of Albert Lau A structure published previously by Tom Ellenberger, associate professor of biological chemistry and molecular pharmacology; Albert Lau, a graduate and research fellow from the department; and colleagues suggested that AAG flipped bases out of the DNA helix and into its active site. This structure did not contain a damaged base and so it only hinted at how AAG cleaves damaged bases but not undamaged bases. In the Dec. 5 PNAS, Ellenberger, Lau, and colleagues show their X-ray crystallography snapshot of AAG in action with a damaged base. This structure suggests that AAG distinguishes damaged from undamaged bases using a combination of factors including hydrogen bonding and a large active site that snugly fits the damaged base. The researchers also tested whether mutating amino acids in AAG that were predicted from the crystal structure to be important for AAG function affected AAG's ability to repair DNA in vivo. The in vivo results correlated well with their hypotheses, providing evidence that "the crystal structure reflects physiologic conformation," said Lau. In the future, he says, it will be important to solve the structure of AAG with other types of damaged bases to determine if the insights provided by this structure hold true with AAG's other substrates. Although helpful to the normal cell, AAG is detrimental when it undoes the intentional damage of chemotherapeutic agents. Understanding how AAG recognizes its substrates may lead to the development of AAG inhibitors, thus allowing particular chemotherapeutic agents to work more effectively. —Heather Ettinger Key Acid Bond May Activate Cell Death ProteinA research team led by Stanley Korsmeyer, the Sidney Farber professor of pathology at the Dana–Farber Cancer Institute, has found the chemical mechanism that activates a molecule involved in programmed cell death, a study detailed in the Dec. 1 Science. Korsmeyer's team has been studying BID, part of a family of proteins dubbed BCL-2 that help regulate the process of apoptosis. One of the ways that apoptosis unfolds is by causing the dysfunction of cell organelles like the mitochondria. BID responds to a death signal in the cell cytosol by undergoing a conformational change that enables it to target and infiltrate the mitochondrial membrane and activate one of its family members in the mitochondria, BAK. BID is activated by the cell death protein caspase-8, which splits it in two. The larger piece is subsequently found at the mitochondrial membrane and is able to integrate into it, but how this process occurs was hazy. Korsmeyer's team discovered that the two fragments of BID do not immediately separate as was thought, but rather travel to the mitochondria together as a complex. The team found that the separation of the two fragments is not itself responsible for BID's activation. When caspase-8 cleaves BID, it exposes a site on one of the fragments that bonds to myristic acid in the process of myristoylation, which can help cytosolic proteins attach to a membrane. Myristoylation helps explain how BID gets activated and is able to target the mitochondria. "It's like a switch," said Solly Weiler, a research associate in Korsmeyer's lab and one of the paper's first authors. Proteins generally undergo myristoylation as they are translated by RNA, and this study may be the earliest example of how a eukaryotic protein can be myristoylated after translation. The paper shows that "myristoylation can be used as a signal transduction mechanism," Weiler said, and may apply to other types of proteins. Added Phosphoryl Groups Bring Axons Greater BreadthIn order to rapidly relay signals through the conduction of action potentials, neuronal axons must have a substantial diameter. During axon radial growth, which is regulated by myelinating oligodendroglia, neurofilaments accumulate within the expanding region in a process that is not well understood. Researchers at HMS now contribute new information that identifies a particular phosphorylation event as crucial to this accumulation. The work is published in the Nov. 27 Journal of Cell Biology. Past research has suggested that either interneurofilament spacing or subunit composition might account for the accumulation of neurofilaments and the radial expansion of axons. Subunits consist of heavy, medium, and light neuronal intermediate filaments. Using several mouse models, Ralph Nixon, former HMS associate professor of psychiatry and currently at New York University School of Medicine, and his colleagues found that subunits assemble within an axon much earlier than the occurrence of radial expansion. Their experiments, including those performed by Ivelisse Sanchez, lead author of the paper and an HMS instructor in cell biology, show that the determining event may instead be the phosphorylation of an amino acid motif of the sequence Lysine-Serine-Proline (KSP) on heavy and medium neurofilament subunits. In fact, mice with defects in myelination were deficient in the KSP phosphorylation of neurofilaments. And neurons deficient in heavy subunits maintained their axonal diameter by compensating for the critical KSP epitope on medium neurofilament subunits. The group found that this particular phosphorylation event is the only change that coincides with axon expansion during myelination and occurs selectively within regions of the axon that undergo neurofilament accumulation. Chromosome Remodeler Plays Role in Diversifying Immune SystemIn order to establish the diversity of the immune system, immunoglobulin and T cell receptor genes undergo a complex series of recombinatory steps necessary for the expression of the correct receptors in the appropriate cells. Yet the regulation of this so-called V(D)J cleavage and rearrangement is unclear. Research at Massachusetts General Hospital now explains how chromatin modification can act to mediate the accessibility of DNA to these V(D)J processes. Normally, the chromatin structure of chromosomes provides significant impediments to binding proteins because nucleosomes, the repeating subunits of chromatin made up of DNA coiled around histones, can render DNA sequences inaccessible. But Marjorie Oettinger, an HMS associate professor of genetics at MGH, and her team have found that chromatin remodeling by a protein complex called human SWI/SNF in addition to histone acetylation can act to increase the accessibility of nucleosomal DNA to V(D)J cleavage. The SWI/SNF chromatin-remodeling complex is a known player in transcriptional activation and repression, but its role in V(D)J rearrangement reported in the November Molecular Cell is a novel finding. The work draws on studies showing that the human SWI/SNF protein complex is capable of remodeling nucleosomal DNA in the presence of ATP. In addition, data show that acetylation of the core histones on DNA can render chromatin more accessible. Oettinger and her group applied these findings to their own studies, showing that these two mechanisms, acetylation and human SWI/SNF remodeling, can act in concert on an individual nucleosome to achieve levels of V(D)J cleavage approaching those observed on naked DNA. —This and brief above by Tracy Hampton |
