Genetic alterations are central to the development of cancer. A single alteration—for example, one in a protein that controls cell proliferation, survival, or fate—can suffice to initiate the process of neoplastic transformation. Once the process starts, it invariably increases genomic instability, which in turn facilitates the emergence of additional alterations that promote malignant progression.
DNA Damage Response
Genetic alterations can readily arise from DNA damage caused by environmental or endogenous agents and also from the complex transactions during replication. To guard against these lesions, the cell has evolved a handful of specialized pathways that sense, signal, and repair them. Simple lesions, such as frequently occurring single-base modifications, can be sensed directly and repaired by direct reversal enzymes or by base excision. However, many single-strand lesions, such as adducts of the most potent environmental carcinogens, are chemically and structurally so diverse that they do not lend themselves to direct recognition.
We have been interested in understanding how just two nucleotide excision repair (NER) proteins, the xeroderma pigmentosum C (XPC) and XPE, suffice for the recognition of such diverse lesions. We found that XPC recognizes the lesion indirectly, by sensing the propensity of the DNA duplex to open up, with two complete base pairs flipped out and a large XPC β hairpin inserted through the duplex. Because inducing this open DNA conformation has a substantial energetic cost, XPC can recognize only lesions that substantially reduce the stability of the duplex. XPE, by contrast, inserts a smaller β turn through the DNA duplex and expels only the two damaged nucleotides, while the opposing bases remain stacked within the helix. Inducing this DNA conformation costs less, explaining XPE's heightened sensitivity for lesions that destabilize the duplex minimally. To achieve this, however, XPE recognizes lesion-specific features, such as a precompressed phosphodiester backbone characteristic of intrastrand crosslinks. Consequently, it can sense only a limited set of lesions with these features, which nevertheless tend to be the ones that evade XPC.
Lesions affecting both DNA strands, such as DNA double-strand breaks (DSBs) and interstrand crosslinks (ICLs), present additional challenges, as the missing information has to be copied from the sister chromatid by homologous recombination (HR). These lesions elicit a signaling response characterized by cell cycle arrest, chromatin changes, and activation of HR. Our research in this area focuses on the ATM and ATR checkpoint kinases that sense these events and initiate the DNA damage response; the end resection pathway that processes DSBs to single-stranded (ss) DNA, which is the substrate for the RAD51 recombinase; and the mechanism of HR. In earlier work, we looked at why HR requires BRCA2. We found that BRCA2 binds to the resected DSB, recruits RAD51, and facilitates the nucleation of RAD51-ssDNA filament formation, the rate-limiting step. The RAD51-ssDNA filament then binds to donor double-stranded (ds) DNA, searches for homology, and catalyzes strand exchange, the key reaction in recombination.
To address the mechanism of strand exchange, we determined a series of crystal structures of minifilaments of RecA (the prokaryotic RAD51 homolog), bound to the ssDNA substrate or to the strand-exchanged heteroduplex product. We do not yet know the structure of RecA-ssDNA-dsDNA, in part because of its transient nature, but we have a rough model based on mutational data. Movie 1, based on an interpolation of three structures and one model, illustrates key mechanistic aspects of HR. In the presynaptic structure, RecA holds the ssDNA in a conformation that resembles the dsDNA product, with the edges of the bases exposed for homology sampling by the incoming DNA. In the synaptic model, the donor dsDNA is melted locally by stretching and unstacking, freeing one of its strands for homology sampling through Watson-Crick pairing. In the postsynaptic structure where complementary strands have exchanged, the new heteroduplex is stable and the reaction is irreversible. On ATP hydrolysis, a heteroduplex is released if strand exchange was successful; otherwise, the filament re-forms and samples homology with the donor duplex at a new register.
Repair of ICLs requires, in addition to HR, the Fanconi anemia (FA) pathway of at least 13 proteins. The FA pathway is activated when a replication fork encounters an ICL. ATR then phosphorylates and activates the downstream FANCI-FANCD2 (ID) complex, leading to its monoubiquitination by a seven-protein FA complex and the recruitment of at least two nucleases that excise the ICL. Our research in this area is aimed at understanding the function and activation of the ID complex, and the mechanism of ICL excision. The structure of the ~300 kDa ID complex revealed two large basic grooves, one on each protein, that could accommodate dsDNA, and adjacent narrower basic grooves that could accommodate ssDNA. We confirmed these DNA-binding activities with in vitro biochemistry and by determining a low-resolution structure of a FANCI-dsDNA-ssDNA complex. This led to the model that the complex recognizes DNA structures arising from the collapse of two replication forks converging onto an ICL (Figure 1). This model is being addressed in current work aimed at reconstituting ID activation by phosporylation and ubiquitination.
The mTOR Growth Regulatory Pathway
The mammalian target of rapamycin (mTOR) protein kinase is a master regulator of cell growth, and its deregulation is a common event in cancer. The mTOR pathway senses a wide range of environmental cues, including nutrient, energy, and oxygen levels, as well as growth factors. In response, it regulates many growth-related processes, such as overall the rate of protein synthesis, metabolic output, survival, and cytoskeletal organization. These functions are mediated by two mTOR complexes, mTORC1 and mTORC2, that differ in their upstream inputs. Distinct subunits in each complex account for these functional differences, and we have been interested in understanding how they control the kinase activity. A related focus has been the mechanism of inhibition by rapamycin, which is mTORC1 specific and was thought to act allosterically.
We have determined the structure of a truncated mTOR containing the phosphatidylinositol 3-kinase-related protein kinase (PIKK) domain and the helical solenoid region shared by other PIKK family members. The kinase-active site and catalytic mechanism turned out to be remarkably conserved with canonical protein kinases. In the complex with the constitutive mammalian lethal with SEC13 protein 8 (mLST8) subunit, the mTOR kinase has a fully active conformation, raising the question of how the kinase activity is regulated. Part of the answer is the location of the active site, which is highly recessed because of the FKBP12-rapamycin-binding (FRB) domain and an inhibitory helix protruding from the catalytic cleft (Figure 2). Hyperactivating mutations map to the structural framework that holds these elements in place, indicating the kinase is controlled by restricted access. Through in vitro biochemistry, we found that the FRB domain acts as a gatekeeper, with its rapamycin-binding site interacting with privileged substrates to grant them access to the restricted active site. Essentially, FKBP12-rapamycin inhibits both directly, by blocking a substrate recruitment site, and indirectly, by further restricting active-site access.
This work was supported in part by grants from the National Institutes of Health.
As of April 15, 2014