Abstract
BackgroundThe Cas4 family endonuclease is a component of the adaptation module in many variants of CRISPR-Cas adaptive immunity systems. Unlike most of the other Cas proteins, Cas4 is often encoded outside CRISPR-cas loci (solo-Cas4) and is also found in mobile genetic elements (MGE-Cas4).ResultsAs part of our ongoing investigation of CRISPR-Cas evolution, we explored the phylogenomics of the Cas4 family. About 90% of the archaeal genomes encode Cas4 compared to only about 20% of the bacterial genomes. Many archaea encode both the CRISPR-associated form (CAS-Cas4) and solo-Cas4, whereas in bacteria, this combination is extremely rare. The solo-cas4 genes are over-represented in environmental bacteria and archaea with small genomes that typically lack CRISPR-Cas, suggesting that Cas4 could perform uncharacterized defense or repair functions in these microbes. Phylogenomic analysis indicates that both the CRISPR-associated cas4 genes are often transferred horizontally but almost exclusively, as part of the adaptation module. The evolutionary integrity of the adaptation module sharply contrasts the rampant shuffling of CRISPR-cas modules whereby a given variant of the adaptation module can combine with virtually any effector module. The solo-cas4 genes evolve primarily via vertical inheritance and are subject only to occasional horizontal transfer. The selection pressure on cas4 genes does not substantially differ between CAS-Cas4 and solo-cas4, and is close to the genomic median. Thus, cas4 genes, similarly to cas1 and cas2, evolve similarly to ‘regular’ microbial genes involved in various cellular functions, showing no evidence of direct involvement in virus-host arms races. A notable feature of the Cas4 family evolution is the frequent recruitment of cas4 genes by various mobile genetic elements (MGE), particularly, archaeal viruses. The functions of Cas4 in these elements are unknown and potentially might involve anti-defense roles.ConclusionsUnlike most of the other Cas proteins, Cas4 family members are as often encoded by stand-alone genes as they are incorporated in CRISPR-Cas systems. In addition, cas4 genes were repeatedly recruited by MGE, perhaps, for anti-defense functions. Experimental characterization of the solo and MGE-encoded Cas4 nucleases is expected to reveal currently uncharacterized defense and anti-defense systems and their interactions with CRISPR-Cas systems.
Highlights
The Cas4 family endonuclease is a component of the adaptation module in many variants of CRISPR-Cas adaptive immunity systems
Phylogenomic analysis of the Cas4 protein family As reported previously, the majority of the Cas4 proteins belong to two families, namely, COG1468 and COG4343; the latter is specific for subtype I-A CRISPR-Cas systems and is known as Csa1 [11]
We focused primarily on these three major families of Cas4 homologs and disregarded a few other, less common PD-DExK families that have been reported in the vicinity of several CRISPR-cas loci [11]
Summary
The Cas family endonuclease is a component of the adaptation module in many variants of CRISPR-Cas adaptive immunity systems. Cas is one of the core CRISPR-associated (Cas) proteins, which is implicated in the adaptation phase in several subtypes of CRISPR-Cas systems [1,2,3,4] During this phase of the CRISPR response, a segment of invading DNA (protospacer), usually from a virus or plasmid, is selected, typically, based on the presence of a protospacer adjacent motif (PAM) [5, 6]. The protospacer is incorporated by the Cas1-Cas adaptation complex into a CRISPR array, most often, next to the first, 5′-terminal repeat. Similar to the AddB nuclease, Cas is thought to form recombinogenic 3′ ssDNA overhangs in the protospacers, facilitating their subsequent incorporation into the CRISPR array as dsDNA spacers [16]. In an independent study, only the 5′-3′ exonuclease activity has been detected for SSO1391 as well [16]
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