Many bacterial clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated (Cas) systems employ the dual RNA–guided DNA endonuclease Cas9 to defend against invading phages and conjugative plasmids by introducing site-specific double-stranded breaks in target DNA. Target recognition strictly requires the presence of a short protospacer adjacent motif (PAM) flanking the target site, and subsequent R-loop formation and strand scission are driven by complementary base pairing between the guide RNA and target DNA, Cas9–DNA interactions, and associated conformational changes. The use of CRISPR–Cas9 as an RNA-programmable DNA targeting and editing platform is simplified by a synthetic single-guide RNA (sgRNA) mimicking the natural dual trans-activating CRISPR RNA (tracrRNA)–CRISPR RNA (crRNA) structure. This review aims to provide an in-depth mechanistic and structural understanding of Cas9-mediated RNA-guided DNA targeting and cleavage. Molecular insights from biochemical and structural...

CRISPR-Cas9 Structures and Mechanisms.

DNA mismatch repair (MMR) is a highly conserved biological pathway that plays a key role in maintaining genomic stability. The specificity of MMR is primarily for base-base mismatches and insertion/deletion mispairs generated during DNA replication and recombination. MMR also suppresses homeologous recombination and was recently shown to play a role in DNA damage signaling in eukaryotic cells. Escherichia coli MutS and MutL and their eukaryotic homologs, MutSalpha and MutLalpha, respectively, are key players in MMR-associated genome maintenance. Many other protein components that participate in various DNA metabolic pathways, such as PCNA and RPA, are also essential for MMR. Defects in MMR are associated with genome-wide instability, predisposition to certain types of cancer including hereditary non-polyposis colorectal cancer, resistance to certain chemotherapeutic agents, and abnormalities in meiosis and sterility in mammalian systems.

/pdf/mechanisms-and-functions-of-dna-mismatch-repair-11cevavj74.pdf

Mechanisms and functions of DNA mismatch repair

Type II CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems use an RNA-guided DNA endonuclease, Cas9, to generate double-strand breaks in invasive DNA during an adaptive bacterial immune response. Cas9 has been harnessed as a powerful tool for genome editing and gene regulation in many eukaryotic organisms. We report 2.6 and 2.2 angstrom resolution crystal structures of two major Cas9 enzyme subtypes, revealing the structural core shared by all Cas9 family members. The architectures of Cas9 enzymes define nucleic acid binding clefts, and single-particle electron microscopy reconstructions show that the two structural lobes harboring these clefts undergo guide RNA-induced reorientation to form a central channel where DNA substrates are bound. The observation that extensive structural rearrangements occur before target DNA duplex binding implicates guide RNA loading as a key step in Cas9 activation.

/pdf/structures-of-cas9-endonucleases-reveal-rna-mediated-e5eo54au8k.pdf

Structures of Cas9 Endonucleases Reveal RNA- Mediated Conformational Activation

CRISPR-Cas systems that provide defence against mobile genetic elements in bacteria and archaea have evolved a variety of mechanisms to target and cleave RNA or DNA(1). The well-studied types I, II ...

The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA

DNA mismatch repair: functions and mechanisms.

Most known nucleotidyl-transfer enzymes use two metal ions for catalysis, but some enzymes use only one divalent cation in their active sites. A comparative analysis of previously available structural data reveals that the one-metal-ion enzymes use a similar mechanism to coordinate their single metal ion, which corresponds, functionally and structurally, to metal ion B in the two-metal-ion enzymes. Nucleotidyl-transfer enzymes, which synthesize, degrade and rearrange DNA and RNA, often depend on metal ions for catalysis. All DNA and RNA polymerases, MutH-like or RNase H–like nucleases and recombinases, and group I introns seem to require two divalent cations to form a complete active site. The two-metal-ion mechanism has been proposed to orient the substrate, facilitate acid-base catalysis and allow catalytic specificity to exceed substrate binding specificity attributable to the stringent metal-ion (Mg2+ in particular) coordination. Not all nucleotidyl-transfer enzymes use two metal ions for catalysis, however. The ββα-Me and HUH nucleases depend on a single metal ion in the active site for the catalysis. All of these one- and two metal ion–dependent enzymes generate 5′-phosphate and 3′-OH products. Structural and mechanistic comparisons show that these seemingly unrelated nucleotidyl-transferases share a functionally equivalent metal ion.

An equivalent metal ion in one- and two-metal-ion catalysis

Structure of an activated DNA-PK and its implications for NHEJ

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/j.febslet.2004.11.047

Portraits of a Y-family DNA polymerase.

Protein–nucleic acid interactions: from A(rgonaute) to X(PF)

Site-specific recombination in plane view.

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