Homologous recombination is a multi-step DNA processing reaction. We consider this process in the context of DNA repair where homologous recombination is required to underpin replication and repair DNA double-strand breaks. Precision and control of this essential, but potentially dangerous, DNA rearrangement is achieved by requiring specific sequential interactions among the proteins and DNA molecules involved. This can be described as a process of sequential assembly and disassembly of functional complexes, which is in addition controlled in time and space in the nucleus. We address the central defining event of homologous recombination, DNA strand exchange catalyzed by RAD51. Mammalian cells require BRCA2 to deliver RAD51 to sites of damage and assemble a functional nucleoprotein complex on single- stranded DNA. These functions clearly involve dynamic interactions between BRCA2, RAD51 and DNA among other partners. In order to define the fundamental processes that drive these required events we are quantifying the diffusive behavior of BRCA2 and associated partners in live cells by single particle tracking. This revealed that nuclear BRCA2 is multimeric and diffuses together with RAD51 (1). We have expanded this to define changes in diffusive behavior in response to DNA damage agents and the role of specific BRCA2 domains. In concert using super resolution microscopy we have revealed that the arrangement of proteins at sites of DNA damage. Although BRCA2 and RAD51 diffusing together they are not associated at the sites of DNA damage but organized separately (2). Our analysis of purified BRCA2 protein by scanning force microscopy (SFM) identified three interesting architectural aspects; BRCA2 is often multimeric, parts of the protein convert easily between extended and globular forms and interaction with RAD51 induces a dramatic reorganization (2). We are dissecting BRCA2 to identify which regions of the protein are responsible for the conformational flexibility we observe with SFM imaging. One aim is to link these conformational changes to known BRAC2 variants thereby removing some uncertainty concerning their functional significance. These combined results begin to define dynamic movement through the nucleus and structural rearrangement of BRCA2, RAD51 and their associated complexes that are essential elements of precision and control in homologous recombination DNA repair.
Many cellular membrane fission reactions are driven by ESCRT pathways, which culminate in remodeling and disassembly of ESCRT-III polymers by the AAA ATPase Vps4. HIV-1 and many other viruses recruit an ESCRT pathway in order to bud from cells. Recent advances in understanding of the budding machinery will be summarized, with special emphasis on HIV and findings from our 3.2 Å resolution cryo-EM structure of the active Vps4 hexamer in complex with its cofactor Vta1, ADP·BeFx, and an ESCRT-III substrate peptide. Five Vps4 subunits form a helix, with interfaces between the first four of these subunits apparently bound to ADP.BeFx (ATP) and the interface between the fourth and fifth subunit bound to ADP, as if it is just commencing dissociation from the helix. The final Vps4 subunit completes a notched-washer configuration as if transitioning between the ends of the helix. The ESCRT-III peptide binds in an extended (beta-strand) conformation against the five helical subunits. Two classes of side chain binding pockets are formed primarily by Vps4 pore loop 1 residues, with four copies of each pocket propagating along the highly solvated pore through the Vps4 hexamer. The pockets accommodate a wide range of residues, while main chain hydrogen bonds help dictate substrate-binding orientation. The structure supports a ‘conveyor belt’ model of translocation in which ATP binding allows a Vps4 subunit to join the growing end of the helix and engage the substrate, while hydrolysis and release promotes helix disassembly and substrate disengagement at the lagging end. In this manner Vps4 may disassemble ESCRT-III to reveal a metastable membrane configuration that resolves by fission and virus budding. This model likely applies to other ESCRT pathways and may be generally applicable to multiple other protein-translocating AAA ATPases.
CRISPR-Cas systems in prokaryotes provide an adaptive immunity against invading nucleic acids. CRISPR-Cas systems are very diverse and are categorized into five main types depending on the number and arrangement of Cas genes. Each Type is specified by the so-called signature protein, which is conserved in the particular Type, for example, Cas9 in Type II and Cas10 in Type III. In the Type II systems Cas9-guide RNA complex alone provides immunity against invading DNA. Cas9 protein guided by crRNA binds to the target sequence and Cas9 protein cuts both DNA strands. The initiation of the target site binding by the Cas9 critically depends on a short sequence motif called PAM located in the vicinity of the target sequence complimentary to crRNA. In the gene editing experiments PAM sequence requirement may limit target site selection if genome specific target sites are desired. Cas9 orthologues with distinct PAM specificities may help expand the sequence space targeted by Cas9. To explore the space of Cas9 orthologs, we established a phylogeny-guided bioinformatics approach coupled with a rapid biochemical screen that allowed to identify new Cas9 variants. In contrast to Cas9 that acts as a stand-alone protein that cuts invading DNA, the Type III CRISPR-Cas system provide immunity against invading nucleic acids through the coordinated degradation of transcriptionally-active DNA and its transcripts. Three catalytic domains are required to mount an immune response. Ribonuclease domain guided by crRNA recognizes viral RNA transcript and initiates its degradation. The deoxyribonuclease domain of Cas10 becomes activated upon target RNA binding and launches simultaneous degradation of DNA template. We recently showed that target RNA binding also triggers Cas10 Palm domain dependent synthesis of cyclic oligoadenylates (cOA) from ATP. We further showed that cOAs act as signaling molecules that couple Type III immunity and Csm6 ribonuclease thereby demonstrating a novel cyclic oligonucleotide-based signaling pathway in prokaryotic antiviral defense systems.