Spontaneous DNA synapsis by forming noncanonical intermolecular structures

We report the spontaneous formation of DNA-DNA junctions in solution in the absence of proteins visualised using atomic force microscopy. The synapsis position fits with potential G-quadruplex (G4) sites. In contrast to the Holliday structure, these conjugates have affinity for G4 antibodies. Molecular modelling was used to elucidate the possible G4/IM-synaptic complex structures. Our results indicate a new role of the intermolecular noncanonical structures in chromatin architecture and genomic rearrangement.

Control of the serine integrase reaction: roles of the coiled-coil and helix E regions in DNA site synapsis and recombination

Bacteriophage serine integrases catalyze highly specific recombination reactions between defined DNA segments called att sites. These reactions are reversible depending upon the presence of a second phage-encoded directionality factor. The bipartite C-terminal DNA binding region of integrases includes a recombinase domain (RD) connected to a zinc-binding domain (ZD), which contains a long flexible coiled-coil (CC) motif that extends away from the bound DNA. We directly show that the identities of the phage A118 integrase att sites are specified by the DNA spacing between the RD and ZD DNA recognition determinants, which in turn, directs the relative trajectories of the CC motifs on each subunit of the att -bound integrase dimer. Recombination between compatible dimer-bound att sites requires minimal length CC motifs and 14 residues surrounding the tip where pairing of CC motifs between synapsing dimers occurs. Our alanine-scanning data suggests that molecular interactions between CC motif tips may differ in integrative ( attP x attB ) and excisive ( attL x attR ) recombination reactions. We identify mutations in 5 residues within the integrase oligomerization helix that control the remodeling of dimers into tetramers during synaptic complex formation. Whereas most of these gain-of-function mutants still require the CC motifs for synapsis, one mutant efficiently, but indiscriminantly, forms synaptic complexes without the CC motifs. However, the CC motifs are still required for recombination, suggesting a function for the CC motifs after initial assembly of the integrase synaptic tetramer. Importance The robust and exquisitely-regulated site-specific recombination reactions promoted by serine integrases are integral to the life cycle of temperate bacteriophage, and in the case of the A118 prophage, are an important virulence factor by Listeria monocytogenes . The properties of these recombinases have led to their repurposing into tools for genetic engineering and synthetic biology. In this report, we identify determinants regulating synaptic complex formation between correct DNA sites, including the DNA architecture responsible for specifying the identity of recombination sites, features of the unique coiled-coil structure on the integrase that are required to initiate synapsis, and amino acid residues on the integrase oligomerization helix that control the remodeling of synapsing dimers into a tetramer active for DNA strand exchange.

Cryo-EM structure of the Rous sarcoma virus octameric cleaved synaptic complex intasome

Despite conserved catalytic integration mechanisms, retroviral intasomes composed of integrase (IN) and viral DNA possess diverse structures with variable numbers of IN subunits. To investigate intasome assembly mechanisms, we employed the Rous sarcoma virus (RSV) IN dimer that assembles a precursor tetrameric structure in transit to the mature octameric intasome. We determined the structure of RSV octameric intasome stabilized by a HIV-1 IN strand transfer inhibitor using single particle cryo-electron microscopy. The structure revealed significant flexibility of the two non-catalytic distal IN dimers along with previously unrecognized movement of the conserved intasome core, suggesting ordered conformational transitions between intermediates that may be important to capture the target DNA. Single amino acid substitutions within the IN C-terminal domain affected intasome assembly and function in vitro and infectivity of pseudotyped RSV virions. Unexpectedly, 17 C-terminal amino acids of IN were dispensable for virus infection despite regulating the transition of the tetrameric intasome to the octameric form in vitro. We speculate that this region may regulate the binding of highly flexible distal IN dimers to the intasome core to form the octameric complex. Our studies reveal key steps in the assembly of RSV intasomes.

RAD51AP1 mediates RAD51 activity through nucleosome interaction

RAD51 Associated Protein 1 (RAD51AP1) is a key protein in the homologous recombination DNA repair pathway (HR). Loss of RAD51AP1 leads to defective HR, genome instability and telomere erosion. RAD51AP1 physically interacts with the RAD51 recombinase and promotes RAD51-mediated capture of the donor DNA, synaptic complex assembly and displacement-loop formation when tested with synthetic, nucleosome-free DNA substratesin vitro. In cells, however, DNA is packaged into chromatin, posing an additional barrier to the complexities of the HR reaction. How RAD51AP1 functions as an HR activator in the context of chromatin has remained unclear.In this study, we show that RAD51AP1 binds to Nucleosome Core Particles (NCPs). We identified a C-terminal region in RAD51AP1 and its previously mapped DNA binding domain as critical for mediating the association between RAD51AP1 and both the NCP and the histone octamer. We show that RAD51AP1 is capable of promoting duplex DNA capture and initiating joint-molecule formation with the NCP and chromatinized template DNA, respectively. Together, our results suggest that RAD51AP1directlyassists the RAD51-mediated search of donor DNA in chromatin. We present a model, in which RAD51AP1 anchors the DNA template through affinity for its nucleosomes to the RAD51-ssDNA nucleoprotein filament.

XLF acts as a flexible connector during non-homologous end joining

Non-homologous end joining (NHEJ) is the predominant pathway that repairs DNA double-strand breaks in vertebrates. During NHEJ DNA ends are held together by a multi-protein synaptic complex until they are ligated. Here, we use Xenopus laevis egg extract to investigate the role of the intrinsically disordered C-terminal tail of the XRCC4-like factor (XLF), a critical factor in end synapsis. We demonstrate that the XLF tail along with the Ku-binding motif (KBM) at the extreme C-terminus are required for end joining. Although the underlying sequence of the tail can be varied, a minimal tail length is required for NHEJ. Single-molecule FRET experiments that observe end synapsis in real-time show that this defect is due to a failure to closely align DNA ends. Our data supports a model in which a single C-terminal tail tethers XLF to Ku, while allowing XLF to form interactions with XRCC4 that enable synaptic complex formation.

Coping with DNA Double-Strand Breaks via ATM Signaling Pathway in Bovine Oocytes

As a common injury almost all cells face, DNA damage in oocytes—especially double-strand breaks (DSBs), which occur naturally during the first meiosis phase (meiosis I) due to synaptic complex separation—affects the fertilization ability of oocytes, instead of causing cancer (as in somatic cells). The mechanism of oocytes to effectively repair DSB damage has not yet been clearly studied, especially considering medically induced DSBs superimposed on naturally occurring DSBs in meiosis I. It was found that maturation rates decreased or increased, respectively corresponding with overexpression or interference of p21 in bovine oocytes. At the same time, the maturation rate of bovine oocytes decreased with a gradual increase in Zeocin dose, and the p21 expression in those immature oocytes changed significantly with the gradual increase in Zeocin dose (same as increased DSB intensity). Same as p21, the variation trend of ATM expression was consistent with the gradual increase in Zeocin dose. Furthermore, the oocytes demonstrated tolerance to DSBs during meiosis I, while the maturation rates decreased when the damage exceeded a certain threshold; according to which, it may be that ATM regulates the p53–p21 pathway to affect the completion of meiosis. In addition, nonhomologous recombination and cumulus cells are potentially involved in the process by which oocytes respond to DSB damage.

XLF acts as a flexible connector during non-homologous end joining

Non-homologous end joining (NHEJ) is the predominant pathway that repairs DNA double strand breaks in vertebrates. During NHEJ DNA ends are held together by a multi-protein synaptic complex until they are ligated. Here we investigate the role of the intrinsically disordered C-terminal tail of XLF, a critical factor in end synapsis. We demonstrate that the XLF tail along with the Ku binding motif (KBM) at the extreme C-terminus are required for end joining. While the underlying sequence of the tail can be varied, a minimal tail length is required for NHEJ. Single-molecule FRET experiments that observe end synapsis in real-time show that this defect is due to a failure to closely align DNA ends. Our data supports a model in which a single C-terminal tail tethers XLF to Ku while allowing XLF to form interactions with XRCC4 that enable synaptic complex formation.

Non-homologous end joining minimizes errors by coordinating DNA processing with ligation

Genome stability requires efficient and faithful repair of DNA double-strand breaks (DSBs). The predominant DSB repair pathway in human cells is non-homologous end-joining (NHEJ), which directly ligates DNA ends1–5. Broken DNA ends at DSBs are chemically diverse, and many are not compatible for direct ligation by the NHEJ-associated DNA Ligase IV (Lig4). To solve this problem, NHEJ end-processing enzymes including polymerases and nucleases modify ends until they are ligatable. How cells regulate end processing to minimize unnecessary genomic alterations6 during repair of pathological DSBs remains unknown. Using a biochemical system that recapitulates key features of cellular NHEJ, we previously demonstrated that DNA ends are initially tethered at a distance, followed by Lig4-mediated formation of a “short-range synaptic complex” in which DNA ends are closely aligned for ligation7. Here, we show that a wide variety of end-processing activities all depend on formation of the short-range complex. Moreover, using real-time single molecule imaging, we find that end processing occurs within the short-range complex. Confining end processing to the Lig4-dependent short-range synaptic complex promotes immediate ligation of compatible ends and ensures that incompatible ends are ligated as soon as they become compatible, thereby minimizing end processing. Our results elucidate how NHEJ exploits end processing to achieve versatility while minimizing errors that cause genome instability.