Developing Single-Molecule TPM Experiments for Direct Observation of Successful RecA-Mediated Strand Exchange Reaction

RecA recombinases play a central role in homologous recombination. Once assembled on single-stranded (ss) DNA, RecA nucleoprotein filaments mediate the pairing of homologous DNA sequences and strand exchange processes. We have designed two experiments based on tethered particle motion (TPM) to investigate the fates of the invading and the outgoing strands during E. coli RecA-mediated pairing and strand exchange at the single-molecule level in the absence of force. TPM experiments measure the tethered bead Brownian motion indicative of the DNA tether length change resulting from RecA binding and dissociation. Experiments with beads labeled on either the invading strand or the outgoing strand showed that DNA pairing and strand exchange occurs successfully in the presence of either ATP or its non-hydrolyzable analog, ATPγS. The strand exchange rates and efficiencies are similar under both ATP and ATPγS conditions. In addition, the Brownian motion time-courses suggest that the strand exchange process progresses uni-directionally in the 5′-to-3′ fashion, using a synapse segment with a wide and continuous size distribution

Paleo-Immunology: Evidence Consistent with Insertion of a Primordial Herpes Virus-Like Element in the Origins of Acquired Immunity

BACKGROUND:The RAG encoded proteins, RAG-1 and RAG-2 regulate site-specific recombination events in somatic immune B- and T-lymphocytes to generate the acquired immune repertoire. Catalytic activities of the RAG proteins are related to the recombinase functions of a pre-existing mobile DNA element in the DDE recombinase/RNAse H family, sometimes termed the "RAG transposon". METHODOLOGY/PRINCIPAL FINDINGS:Novel to this work is the suggestion that the DDE recombinase responsible for the origins of acquired immunity was encoded by a primordial herpes virus, rather than a "RAG transposon." A subsequent "arms race" between immunity to herpes infection and the immune system obscured primary amino acid similarities between herpes and immune system proteins but preserved regulatory, structural and functional similarities between the respective recombinase proteins. In support of this hypothesis, evidence is reviewed from previous published data that a modern herpes virus protein family with properties of a viral recombinase is co-regulated with both RAG-1 and RAG-2 by closely linked cis-acting co-regulatory sequences. Structural and functional similarity is also reviewed between the putative herpes recombinase and both DDE site of the RAG-1 protein and another DDE/RNAse H family nuclease, the Argonaute protein component of RISC (RNA induced silencing complex). CONCLUSIONS/SIGNIFICANCE:A "co-regulatory" model of the origins of V(D)J recombination and the acquired immune system can account for the observed linked genomic structure of RAG-1 and RAG-2 in non-vertebrate organisms such as the sea urchin that lack an acquired immune system and V(D)J recombination. Initially the regulated expression of a viral recombinase in immune cells may have been positively selected by its ability to stimulate innate immunity to herpes virus infection rather than V(D)J recombination Unlike the "RAG-transposon" hypothesis, the proposed model can be readily tested by comparative functional analysis of herpes virus replication and V(D)J recombination

Single-molecule imaging of DNA pairing by RecA reveals a three-dimensional homology search

DNA breaks can be repaired with high fidelity by homologous recombination. A ubiquitous protein that is essential for this DNA template-directed repair is RecA. After resection of broken DNA to produce single-stranded DNA (ssDNA), RecA assembles on this ssDNA into a filament with the unique capacity to search and find DNA sequences in double-stranded DNA (dsDNA) that are homologous to the ssDNA. This homology search is vital to recombinational DNA repair, and results in homologous pairing and exchange of DNA strands. Homologous pairing involves DNA sequence-specific target location by the RecA-ssDNA complex. Despite decades of study, the mechanism of this enigmatic search process remains unknown. RecA is a DNA-dependent ATPase, but ATP hydrolysis is not required for DNA pairing and strand exchange, eliminating active search processes. Using dual optical trapping to manipulate DNA, and single-molecule fluorescence microscopy to image DNA pairing, we demonstrate that both the three-dimensional conformational state of the dsDNA target and the length of the homologous RecA-ssDNA filament have important roles in the homology search. We discovered that as the end-to-end distance of the target dsDNA molecule is increased, constraining the available three-dimensional (3D) conformations of the molecule, the rate of homologous pairing decreases. Conversely, when the length of the ssDNA in the nucleoprotein filament is increased, homology is found faster. We propose a model for the DNA homology search process termed 'intersegmental contact sampling', in which the intrinsic multivalent nature of the RecA nucleoprotein filament is used to search DNA sequence space within 3D domains of DNA, exploiting multiple weak contacts to rapidly search for homology. Our findings highlight the importance of the 3D conformational dynamics of DNA, reveal a previously unknown facet of the homology search, and provide insight into the mechanism of DNA target location by this member of a universal family of proteins

Reaction cycle of the yeast Isw2 chromatin remodeling complex

Members of the ISWI family of chromatin remodeling factors hydrolyze ATP to reposition nucleosomes along DNA. Here we show that the yeast Isw2 complex interacts with DNA in a nucleotide-dependent manner at physiological ionic strength. Isw2 efficiently binds DNA in the absence of nucleotides and in the presence of a nonhydrolyzable ATP analog. Conversely, ADP promotes the dissociation of Isw2 from DNA. In contrast, Isw2 remains bound to mononucleosomes through multiple cycles of ATP hydrolysis. Solution studies show that Isw2 undergoes nucleotide-dependent alterations in conformation not requiring ATP hydrolysis. Our results indicate that during an Isw2 remodeling reaction, hydrolysis of successive ATP molecules coincides with cycles of DNA binding, release, and rebinding involving elements of Isw2 distinct from those interacting with nucleosomes. We propose that progression of the DNA-binding site occurs while nucleosome core contacts are maintained and generates a force dissipated by disruption of histone–DNA interactions