In eukaryotes, the CRISPR-Cas9 system has now been widely used as a revolutionary genome engineering tool. However, in prokaryotes, the use of nuclease-mediated genome editing tools has been limited to negative selection for the already modified cells because of its lethality. Here, we report on deaminase-mediated targeted nucleotide editing (Target-AID) adopted in Escherichia coli. Cytidine deaminase PmCDA1 fused to the nuclease-deficient CRISPR-Cas9 system achieved specific point mutagenesis at the target sites in E. coli by introducing cytosine mutations without compromising cell growth. The cytosine-to-thymine substitutions were induced mainly within an approximately five-base window of target sequences on the protospacer adjacent motif-distal side, which can be shifted depending on the length of the single guide RNA sequence. Use of a uracil DNA glycosylase inhibitor in combination with a degradation tag (LVA tag) resulted in a robustly high mutation efficiency, which allowed simultaneous multiplex editing of six different genes. The major multi-copy transposase genes that consist of at least 41 loci were also simultaneously edited by using four target sequences. As this system does not rely on any additional or host-dependent factors, it may be readily applicable to a wide range of bacteria.

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A DNA polymerase is encoded by the deep-sea vent phage NrS-1. NrS-1 has a unique genome organization containing genes that are predicted to encode a helicase and a single-stranded DNA (ssDNA)-binding protein. The gene for an unknown protein shares weak homology with the bifunctional primase-polymerases (prim-pols) from archaeal plasmids but is missing the zinc-binding domain typically found in primases. We show that this gene product has efficient DNA polymerase activity and is processive in DNA synthesis in the presence of the NrS-1 helicase and ssDNA-binding protein. Remarkably, this NrS-1 DNA polymerase initiates DNA synthesis from a specific template DNA sequence in the absence of any primer. The de novo DNA polymerase activity resides in the N-terminal domain of the protein, whereas the C-terminal domain enhances DNA binding.

Heterochromatin protein 1s (HP1s) are nonhistone chromosomal proteins that play a direct role in the formation and maintenance of heterochromatin structure. Similarly to Caenorhabditis elegans, silkworms possess holocentric chromosomes, in which diffused kinetochores extend along the length of each chromosome. We have isolated two silkworm HP1 homologues, BmHP1a and BmHP1b. Cytological analysis showed a unique localization of BmHP1s during cell division, in which these proteins first appear to dissociate from the chromosomes, but then return to enclose the chromosomes during metaphase. BmHP1s formed homo- and hetero-dimers and interacted with BmSu(var)3-9, which is a methyltransferase for histone H3 lysine 9 (H3K9). We further showed, using a silkworm cell-based reporter system, that BmHP1b had higher transcriptional repression activity than BmHP1a, whereas BmHP1a interacted more strongly with BmSu(var)3-9 than did BmHP1b. These results suggest that silkworm HP1a and HP1b may play different roles in heterochromatin formation in holocentric silkworm chromosomes.

Background: Flap endonucleases remove 5Ј-single-stranded DNA termini. Results: T7 gene 6 exonuclease is a flap endonuclease that cleaves 5Ј-single-stranded termini one nucleotide into the duplex. Conclusion:The flap endonuclease activity catalyzes the removal 5Ј-single-stranded tails arising from duplex DNA. Significance: The specificity of gene 6 protein identifies it as a flap endonuclease that can remove unusual structures of recombination and replication.