Significance Base excision repair (BER) is one of the major DNA repair pathways used to fix a myriad of cellular DNA lesions. The enzymes involved in BER, including DNA polymerase β (Polβ), have been identified and characterized, but how they act together to efficiently perform BER has not been fully understood. Through gel electrophoresis, mass spectrometry, and kinetic analysis, we discovered that the two enzymatic activities of Polβ can be interlocked, rather than functioning independently from each other, when processing DNA intermediates formed in BER. The finding prompted us to hypothesize a modified BER pathway. Through conventional and time-resolved X-ray crystallography, we solved 11 high-resolution crystal structures of cross-linked Polβ complexes and proposed a detailed chemical mechanism for Polβ’s 5′-deoxyribose-5-phosphate lyase activity.

The RNA-guided CRISPR-associated (Cas) proteins Cas9 and Cas12a provide adaptive immunity against bacteriophage and function as powerful tools for genome editing in wide-ranging cell types.Here we present a third and fundamentally distinct RNA-guided platform, CRISPR-CasX, which uses a unique structure and mechanism for programmable double-stranded DNA cleavage.Biochemical and in vivo data demonstrate that CasX is active for E. coli and human genome modification.Eight cryo-EM structures of CasX in different states of assembly with its guide RNA and double-stranded DNA substrates reveal an extensive RNA scaffold and an unanticipated domain required for DNA unwinding.These data demonstrate how CasX activity arose through convergent evolution to establish an enzyme family that is functionally separate from both Cas9 and Cas12a.Archaea and bacteria utilize CRISPR-Cas systems (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins) for adaptive immunity against invading nucleic acids 1,2 .CRISPR arrays, consisting of repeated sequences interleaved with sequences acquired from foreign DNA, are templates for CRISPR RNAs (crRNAs) that guide a Cas nuclease to cleave complementary DNA sequences.In addition to their microbial functions, RNA-guided DNA binding and cutting have proven to be transformative tools for genome and epigenome editing across wide-ranging cell types and organisms [3][4][5] .Despite extensive effort, just two kinds of CRISPR-Cas nucleases, Cas9 and Cas12a (Cpf1), provide the foundation for this revolutionary technology 6,7 .Metagenomic analysis of microbial DNA from groundwater samples revealed a new protein, CasX (a placeholder name pending re-analysis of the class 2 CRISPR-Cas phylogeny) which is also referred as Cas12e 5 , that prevented bacterial transformation by plasmid DNA when expressed with cognate crRNAs targeting the plasmid 8 .Sequence analysis of CasX revealed no similarity to other CRISPR-Cas enzymes, except for the presence of a RuvC nuclease domain similar to that found in both Cas9 and Cas12a enzyme families as well as transposases and recombinases 8 .Phylogenetic analysis suggests that CasX arose from a TnpB-type transposase by an independent insertion event into ancestral CRISPR loci, distinct from Cas12a and the remaining type V effectors (Extended Data Fig. 1a).Consistent with this hypothesis, the CasX RuvC domain shares less than 16% identity to RuvC domains in either Cas9 or Cas12a (Extended Data Fig. 1b).This evolutionary ambiguity of CasX hinted that CasX may have a structure and molecular mechanism distinct from other CRISPR-Cas enzymes.However, without full reconstitution of the CasX enzyme, it was not possible to determine the basis of the previously reported plasmid interference activity.We demonstrate here that CasX is an RNA-guided DNA endonuclease that generates a staggered double-stranded break in DNA at sequences complementary to a 20-nucleotide segment of its guide RNA.We further find that CasX induces programmable, site-specific genome repression in E. coli and genome editing in human cells.Biochemical data shows that CasX is a hybrid enzyme containing elements of both Cas9 and Cas12a as well as novel RNA folds and protein domains, establishing this enzyme family as the third CRISPR-Cas system effective for genetic manipulation.The small size of CasX (<1000 amino acids), DNA cleavage characteristics and derivation from non-pathogenic microorganisms, offer important advantages over other CRISPR-Cas genome editing enzymes.Eight molecular

The MSMEG_4306 gene from Mycobacterium smegmatis encodes a protein of unknown function with 242 amino-acid residues that contains a conserved zinc-ribbon domain at its C-terminus. Here, the crystal structure of MSMEG_4306 determined by the single-wavelength anomalous dispersion method using just one zinc ion co-purified with the protein is reported. The crystal structure of MSMEG_4306 shows a coiled-coil helix domain in the N-terminal region and a zinc-ribbon domain in the C-terminal region. A structural similarity search against the Protein Data Bank using MSMEG_4306 as a query revealed two similar structures, namely CT398 from Chlamydia trachomatis and HP0958 from Helicobacter pylori, although they share only ∼15% sequence identity with MSMEG_4306. Based on comparative analysis, it is predicted that MSMEG_4306 may be involved in secretion systems, possibly by interacting with multiple proteins or nucleic acids.

The riboflavin biosynthesis pathway has been shown to be essential in many pathogens and is absent in humans. Therefore, enzymes involved in riboflavin synthesis are considered as potential antibacterial drug targets. The enzyme 3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBPS) catalyzes one of the two committed steps in the riboflavin pathway and converts d-ribulose 5-phosphate (Ru5P) to l-3,4-dihydroxy-2-butanone 4-phosphate and formate. Moreover, DHBPS is shown to be indispensable for Mycobacterium, Salmonella, and Helicobacter species. Despite the essentiality of this enzyme in bacteria, no inhibitor has been identified hitherto. Here, we describe kinetic and crystal structure characterization of DHBPS from Vibrio cholerae (vDHBPS) with a competitive inhibitor 4-phospho-d-erythronohydroxamic acid (4PEH) at 1.86-Å resolution. In addition, we also report the structural characterization of vDHBPS in its apo form and in complex with its substrate and substrate plus metal ions at 1.96-, 1.59-, and 2.04-Å resolution, respectively. Comparison of these crystal structures suggests that 4PEH inhibits the catalytic activity of DHBPS as it is unable to form a proposed intermediate that is crucial for DHBPS activity. Furthermore, vDHBPS structures complexed with substrate and metal ions reveal that, unlike Candida albicans, binding of substrate to vDHBPS induces a conformational change from an open to closed conformation. Interestingly, the position of second metal ion, which is different from that of Methanococcus jannaschii, strongly supports an active role in the catalytic mechanism. Thus, the kinetic and structural characterization of vDHBPS reveals the molecular mechanism of inhibition shown by 4PEH and that it can be explored further for designing novel antibiotics.