Many clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-based genome editing technologies take advantage of Cas nucleases to induce DNA ...double-strand breaks (DSBs) at desired locations within a genome. Further processing of the DSBs by the cellular DSB repair machinery is then necessary to introduce desired mutations, sequence insertions, or gene deletions. Thus, the accuracy and efficiency of genome editing are influenced by the cellular DSB repair pathways. DSBs are themselves highly genotoxic lesions and as such cells have evolved multiple mechanisms for their repair. These repair pathways include homologous recombination (HR), classical nonhomologous end joining (cNHEJ), microhomology-mediated end joining (MMEJ) and single-strand annealing (SSA). In this review, we briefly highlight CRISPR-Cas9 and then describe the mechanisms of DSB repair. Finally, we summarize recent findings of factors that can influence the choice of DNA repair pathway in response to Cas9-induced DSBs.
Clustered regularly interspaced short palindromic repeats (CRISPR)- CRISPR-associated protein 9 (Cas9)-mediated genome editing offers a powerful approach as a potential therapy for monogenic human genetic diseases.Precise template-free base deletions can be achieved through microhomology-mediated end joining (MMEJ) repair and depend on local target site sequence.The DNA repair pathway choice in CRISPR-Cas9 induced-double-strand breaks (DSBs) is regulated by several key factors including the cell cycle, target site sequence and chromatin structure, and the identity of the donor DNA template.Homology-directed repair (HDR)-related DNA repair pathways in response to CRISPR-Cas9 induced-DSBs in mammalian cells are complicated and relatively inefficient. Different DNA repair pathways might be used to repair each end at a DSB resulting in the potential for asymmetric repair.
The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA-DNA base-pairing to target foreign DNA in bacteria. ...Cas9-guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9-RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9-RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9-RNA. Competition assays provide evidence that DNA strand separation and RNA-DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate scission of double-stranded DNA.
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DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Replication protein A (RPA) is a ubiquitous eukaryotic single-stranded DNA (ssDNA) binding protein necessary for all aspects of DNA metabolism involving an ssDNA intermediate, including DNA ...replication, repair, recombination, DNA damage response and checkpoint activation, and telomere maintenance. The role of RPA in most of these reactions is to protect the ssDNA until it can be delivered to downstream enzymes. Therefore a crucial feature of RPA is that it must bind very tightly to ssDNA, but must also be easily displaced from ssDNA to allow other proteins to gain access to the substrate. Here we use total internal reflection fluorescence microscopy and nanofabricated DNA curtains to visualize the behavior of Saccharomyces cerevisiae RPA on individual strands of ssDNA in real-time. Our results show that RPA remains bound to ssDNA for long periods of time when free protein is absent from solution. In contrast, RPA rapidly dissociates from ssDNA when free RPA is present in solution allowing rapid exchange between the free and bound states. In addition, the S. cerevisiae DNA recombinase Rad51 and E. coli single-stranded binding protein (SSB) also promote removal of RPA from ssDNA. These results reveal an unanticipated exchange between bound and free RPA suggesting a binding mechanism that can confer exceptionally slow off rates, yet also enables rapid displacement through a direct exchange mechanism that is reliant upon the presence of free ssDNA-binding proteins in solution. Our results indicate that RPA undergoes constant microscopic dissociation under all conditions, but this is only manifested as macroscopic dissociation (i.e. exchange) when free proteins are present in solution, and this effect is due to mass action. We propose that the dissociation of RPA from ssDNA involves a partially dissociated intermediate, which exposes a small section of ssDNA allowing other proteins to access to the DNA.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Homologous recombination allows for the regulated exchange of genetic information between two different DNA molecules of identical or nearly identical sequence composition, and is a major pathway for ...the repair of double-stranded DNA breaks. A key facet of homologous recombination is the ability of recombination proteins to perfectly align the damaged DNA with homologous sequence located elsewhere in the genome. This reaction is referred to as the homology search and is akin to the target searches conducted by many different DNA-binding proteins. Here I briefly highlight early investigations into the homology search mechanism, and then describe more recent research. Based on these studies, I summarize a model that includes a combination of intersegmental transfer, short-distance one-dimensional sliding, and length-specific microhomology recognition to efficiently align DNA sequences during the homology search. I also suggest some future directions to help further our understanding of the homology search. Where appropriate, I direct the reader to other recent reviews describing various issues related to homologous recombination.
Homologous recombination (HR) is a crucial pathway for double-stranded DNA break (DSB) repair. During the early stages of HR, the newly generated DSB ends are processed to yield long single-stranded ...DNA (ssDNA) overhangs, which are quickly bound by replication protein A (RPA). RPA is then replaced by the DNA recombinase Rad51, which forms extended helical filaments on the ssDNA. The resulting nucleoprotein filament, known as the presynaptic complex, is responsible for pairing the ssDNA with homologous double-stranded DNA (dsDNA), which serves as the template to guide DSB repair. Here, we use single-molecule imaging to visualize the interplay between human RPA (hRPA) and human RAD51 during presynaptic complex assembly and disassembly. We demonstrate that ssDNA-bound hRPA can undergo facilitated exchange, enabling hRPA to undergo rapid exchange between free and ssDNA-bound states only when free hRPA is present in solution. Our results also indicate that the presence of free hRPA inhibits RAD51 filament nucleation, but has a lesser impact upon filament elongation. This finding suggests that hRPA exerts important regulatory influence over RAD51 and may in turn affect the properties of the assembled RAD51 filament. These experiments provide an important basis for further investigations into the regulation of human presynaptic complex assembly.
CRISPR-Cas adaptive immune systems protect bacteria and archaea against foreign genetic elements. In Escherichia coli, Cascade (CRISPR-associated complex for antiviral defense) is an RNA-guided ...surveillance complex that binds foreign DNA and recruits Cas3, a trans-acting nuclease helicase for target degradation. Here, we use single-molecule imaging to visualize Cascade and Cas3 binding to foreign DNA targets. Our analysis reveals two distinct pathways dictated by the presence or absence of a protospacer-adjacent motif (PAM). Binding to a protospacer flanked by a PAM recruits a nuclease-active Cas3 for degradation of short single-stranded regions of target DNA, whereas PAM mutations elicit an alternative pathway that recruits a nuclease-inactive Cas3 through a mechanism that is dependent on the Cas1 and Cas2 proteins. These findings explain how target recognition by Cascade can elicit distinct outcomes and support a model for acquisition of new spacer sequences through a mechanism involving processive, ATP-dependent Cas3 translocation along foreign DNA.
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•Cascade can locate targets through PAM-dependent and PAM-independent pathways•PAM-dependent recognition enables direct recruitment of the Cas3 translocase/nuclease•PAM-independent recognition requires Cas1-Cas2 for Cas3 recruitment•Cas1-Cas2 serve as trans-acting factors that regulate Cas3 activities
Single-molecule analysis of the bacterial Cascade complex reveals two distinct pathways leading to differential regulation of Cas3 DNA translocase and nuclease activities.
Genetic recombination occurs in all organisms and is vital for genome stability. Indeed, in humans, aberrant recombination can lead to diseases such as cancer. Our understanding of homologous ...recombination is built upon more than a century of scientific inquiry, but achieving a more complete picture using ensemble biochemical and genetic approaches is hampered by population heterogeneity and transient recombination intermediates. Recent advances in single-molecule and super-resolution microscopy methods help to overcome these limitations and have led to new and refined insights into recombination mechanisms, including a detailed understanding of DNA helicase function and synaptonemal complex structure. The ability to view cellular processes at single-molecule resolution promises to transform our understanding of recombination and related processes.
Homologous recombination (HR) mediates the exchange of genetic information between sister or homologous chromatids. During HR, members of the RecA/Rad51 family of recombinases must somehow search ...through vast quantities of DNA sequence to align and pair single-strand DNA (ssDNA) with a homologous double-strand DNA (dsDNA) template. Here, we use single-molecule imaging to visualize Rad51 as it aligns and pairs homologous DNA sequences in real time. We show that Rad51 uses a length-based recognition mechanism while interrogating dsDNA, enabling robust kinetic selection of 8-nucleotide (nt) tracts of microhomology, which kinetically confines the search to sites with a high probability of being a homologous target. Successful pairing with a ninth nucleotide coincides with an additional reduction in binding free energy, and subsequent strand exchange occurs in precise 3-nt steps, reflecting the base triplet organization of the presynaptic complex. These findings provide crucial new insights into the physical and evolutionary underpinnings of DNA recombination.
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•Single-molecule imaging reveals how Rad51 presynaptic complexes interrogate dsDNA•Rad51 quickly samples and rejects any DNA lacking 8-nt tracts of microhomology•Rad51 stably captures DNA harboring 8 nt of microhomology•Subsequent strand invasion occurs in precise 3-nt steps
Single-molecule imaging with ssDNA curtains reveals how the Rad51/RecA family of DNA recombinases make use of short tracts of microhomology to interrogate and align homologous DNA sequences.
Condensin plays crucial roles in chromosome organization and compaction, but the mechanistic basis for its functions remains obscure. We used single-molecule imaging to demonstrate that Saccharomyces ...cerevisiae condensin is a molecular motor capable of adenosine triphosphate hydrolysis–dependent translocation along double-stranded DNA. Condensin’s translocation activity is rapid and highly processive, with individual complexes traveling an average distance of ≥10 kilobases at a velocity of ~60 base pairs per second. Our results suggest that condensin may take steps comparable in length to its ~50-nanometer coiled-coil subunits, indicative of a translocation mechanism that is distinct from any reported for a DNA motor protein. The finding that condensin is a mechanochemical motor has important implications for understanding the mechanisms of chromosome organization and condensation.
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