The bacteriophage P and Escherichia coli DnaC proteins are known to recruit the bacterial DnaB replicative helicase to initiator complexes assembled at the phage and bacterial origins, respectively. These specialized nucleoprotein assemblies facilitate the transfer of one or more molecules of DnaB helicase onto the chromosome; the transferred DnaB, in turn, promotes establishment of a processive replication fork apparatus. To learn more about the mechanism of the DnaB transfer reaction, we investigated the interaction of replication initiation proteins with single-stranded DNA (ssDNA). These studies indicate that both P and DnaC contain a cryptic ssDNAbinding activity that is mobilized when each forms a complex with the DnaB helicase. Concomitantly, the capacity of DnaB to bind to ssDNA, as judged by UV-crosslinking analysis, is suppressed upon formation of a P⅐DnaB or a DnaB⅐DnaC complex. This novel switch in ssDNA-binding activity evoked by complex formation suggests that interactions of P or DnaC with ssDNA may precede the transfer of DnaB onto DNA during initiation of DNA replication. Further, we find that the O replication initiator enhances interaction of the P⅐DnaB complex with ssDNA. Partial disassembly of a ssDNA:O⅐P⅐DnaB complex by the DnaK͞DnaJ͞GrpE molecular chaperone system results in the transfer in cis of DnaB to the ssDNA template. On the basis of these findings, we present a general model for the transfer of DnaB onto ssDNA or onto chromosomal origins by replication initiation proteins.

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CRISPR-Cas systems provide bacteria with adaptive immunity against viruses. During spacer adaptation, the Cas1-Cas2 complex selects fragments of foreign DNA, called prespacers, and integrates them into CRISPR arrays in an orientation that provides functional immunity. Cas4 is involved in both the trimming of prespacers and the cleavage of protospacer adjacent motif (PAM) in several type I CRISPR-Cas systems, but how the prespacers are processed in systems lacking Cas4, such as the type I-E and I-F systems, is not understood. In Escherichia coli, which has a type I-E system, Cas1-Cas2 preferentially selects prespacers with 3′ overhangs via specific recognition of a PAM, but how these prespacers are integrated in a functional orientation in the absence of Cas4 is not known. Using a biochemical approach with purified proteins, as well as integration, prespacer protection, sequencing, and quantitative PCR assays, we show here that the bacterial 3′–5′ exonucleases DnaQ and ExoT can trim long 3′ overhangs of prespacers and promote integration in the correct orientation. We found that trimming by these exonucleases results in an asymmetric intermediate, because Cas1-Cas2 protects the PAM sequence, which helps to define spacer orientation. Our findings implicate the E. coli host 3′–5′ exonucleases DnaQ and ExoT in spacer adaptation and reveal a mechanism by which spacer orientation is defined in E. coli.

Edited by Charles E. Samuel CRISPR-Cas systems are RNA-based immune systems that protect many prokaryotes from invasion by viruses and plasmids. Type III CRISPR systems are unique, as their targeting mechanism requires target transcription. Upon transcript binding, DNA cleavage by type III effector complexes is activated. Type III systems must differentiate between invader and native transcripts to prevent autoimmunity. Transcript origin is dictated by the sequence that flanks the 3 end of the RNA target site (called the PFS). However, how the PFS is recognized may vary among different type III systems. Here, using purified proteins and in vitro assays, we define how the type III-B effector from the hyperthermophilic bacterium Thermotoga maritima discriminates between native and invader transcripts. We show that native transcripts are recognized by base pairing at positions ؊2 to ؊5 of the PFS and by a guanine at position ؊1, which is not recognized by base pairing. We also show that mismatches with the RNA target are highly tolerated in this system, except for those nucleotides adjacent to the PFS. These findings define the target requirement for the type III-B system from T. maritima and provide a framework for understanding the target requirements of type III systems as a whole. CRISPR arrays and CRISPR-associated (Cas) 2 genes provide prokaryotes with adaptive immunity to invading genetic elements such as bacteriophage (1). CRISPR arrays consist of short DNA sequences of foreign origin, called spacers, separated by host repeat sequences (2-4). Cas proteins assemble with CRISPR RNAs (crRNAs) to form effector complexes (5-7). These complexes identify and destroy invading nucleic acids that are complementary to their crRNAs (5, 8). CRISPR-Cas

The methyl-directed DNA repair efficiency of a series of M13mp9 frameshift heteroduplexes containing 1, 2, or 3 unpaired bases was determined by using an in vitro DNA mismatch repair assay. Repair of hemimethylated frameshift heteroduplexes in vitro was directed to the unmethylated strand; was dependent on MutH, MutL, and MutS; and was equally efficient on base insertions and deletions. However, fully methylated frameshift heteroduplexes were resistant to repair, while totally unmethylated substrates were repaired with no strand bias. Hemimethylated 1-, 2-, or 3-base insertion and deletion heteroduplexes were repaired by the methyl-directed mismatch repair pathway as efficiently as the G T mismatch. These results are consistent with earlier in vivo studies and demonstrate the involvement of methyl-directed DNA repair in the efficient prevention of frameshift mutations.In Escherichia coli, the fidelity of DNA replication is increased 100-fold by postreplication, methyl-directed DNA repair (10, 49,52,63). The misincorporated base on the newly replicated strand is distinguished from the correct base on the parent strand through the transient undermethylation of the daughter strand at 5'-GATC-3' sequences, which are subsequently methylated by the dam gene product (16,23,45). The repair efficiency of specific base pair mismatches varies considerably. Transition mismatches are repaired most efficiently, while some transversion mismatches are repaired relatively poorly or not at all by methyl-directed mismatch repair (12,30,61). In addition to Dam methylation, the products of the mutH, mutL, mutS, and uvrD genes are required for methyl-directed mismatch repair both in vivo (2,12,17,30,43,51) and in vitro (21, 36, 41, 43, 62, 64). By using an in vitro methyl-directed mismatch repair assay, the MutH, MutS, and MutL proteins of E. coli have been purified in a biologically active form (21,62,64). Both in vivo (37) and in vitro (64) studies suggest that strand discrimination occurs through the MutH-dependent nicking of the unmethylated strand at hemimethylated 5'-GATC-3' sites. MutS has been shown to bind base pair mismatches (28,61,62)

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Cyclohexene nucleic acids (CeNA) contain a cyclohexene ring instead of the normal-D-2'-deoxyribose. The cyclohexene oligonucleotide GTGTACAC was synthesized using phosphoramidite chemistry and standard protecting groups [1]. CeNA is stable against enzymatic degradation and induces RNaseH activity. CeNA also forms more stable duplexes with RNA than its natural analogues [2] [3]. Crystals of GTGTACAC were obtained at 289K by the hangingdrop vapour-diffusion technique. The crystals diffract to 1.7 Å resolution and belong to the trigonal space group R3 with unit-cell parameters a = 41.434 and c = 66.735 Å. The structure of a fully modified GTGTACAC sequence with left handed CeNA building blocks is presented. Particular interests concern the puckering of the sugar moiety, helical parameters and the hydration of the double helix.

The CRISPR‐Cas systems are a collection of RNA‐based adaptive immune systems that protect many prokaryotes from invasion by viruses and plasmids. CRISPR‐Cas systems are very diverse and can be organized into six types and more than 15 subtypes. Type III systems are unique as their targeting mechanism requires transcription of the target DNA. Type III effector complexes use small CRISPR RNAs (crRNAs) to identify complementary transcripts. Transcript binding simultaneously activates two nuclease domains, leading to the degradation of both the bound transcript and adjacent DNA. Like all other immune systems, CRISPR‐Cas systems must differentiate between self and non‐self in order to prevent autoimmunity, which could arise from targeting of the host CRISPR‐array. Type III systems use two mechanisms to identify self‐transcripts. Studies of the Type III‐A system from Staphylococcus epidermidis revealed that base pairing between the crRNA and the sequence flanking the 3′ end of RNA target site prevents autoimmunity. However, a study of the Type III‐B system from Pyrococcus furiosus revealed that a short sequence motif flanking the RNA target sequence is required for immunity and the absence of this motif in CRISPR arrays prevents autoimmunity. Neither of these mechanisms is well understood biochemically and it is not clear if these mechanisms are conserved within the subtypes. To address these points, we monitored RNA‐activated DNA cleavage by the Type III‐B effector complex from Thermotoga maritima with purified components. A series of RNA transcripts containing various target 3′‐flanking sequences were tested for their ability to activate DNA cleavage. We find that two factors are important in preventing autoimmunity in the T. maritima Type III‐B system: (1) base pairing between the crRNA and the target 3′‐flanking sequence of the transcript and (2) the presence of a G, which does not base pair with the crRNA, immediately adjacent to the 3′‐end of the RNA target sequence. These data suggest that the mechanisms used to avoid autoimmunity by Type III systems are not subtype‐specific and that identification of self transcripts is complex, involving base pairing and specific sequence recognition.Support or Funding InformationThis work was supported by National Institutes of Health grant GM097330 to S.B.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.