engleski

In vivo evidence for Cas3 involvement in R-loops formation

CRISPR (clustered regularly interspaced short palindromic repeats) is a recently discovered defence mechanism against phages or other genetic elements in many bacteria and archea. It consists of arrays of repeats separated by spacers. Spacers are usually derived from foreign DNA such as phage or plasmids and these spacers provide resistance toward phage that carry the incorporated sequence. In the vicinity of the CRISPR region are located CRISPR-associated (cas) genes that are needed for the various stages in the CRISPR immunity mechanism. In Escherichia coli CRISPR DNA is transcribed into long precursor RNA (pre-crRNA) that is processed by Cascade proteins into smaller mature crRNA. Mature crRNA consists of one spacer and part of one repeat at its 5’ end and it is used as a template by Cas proteins for degradation (interference) of invading DNA. The mechanism for this RNA interference is not known, and in addition to Cascade proteins, Cas3 is probably required for this interference pathway. In this work we wanted to test the possible role of cas3 gene on R-loops formation in vivo. We investigated this possibility by examining how Cas3 affects plasmid yield of ColE1 based plasmid replicons, and on the efficiency of transformation with plasmids that carry wt and cas3 mutants into various bacterial strains. Our genetic analysis showed that cas3 gene is involved in plasmid yield in a manner different to recG that is known to disrupt R-loops. Our results also indicate genetic interactions of cas3 with rnhA and topA mutants. We propose that cas3 gene is involved in mediating interaction between DNA and RNA molecules, probably via R-loops formation.

cas3 ; R-loop ; CRISPR

nije evidentirano