Abstract
Cas4 nucleases constitute a core family of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) associated proteins, but little is known about their structure and activity. Here we report the crystal structure of the Cas4 protein Pcal_0546 from Pyrobaculum calidifontis, which revealed a monomeric protein with a RecB-like fold and one [2Fe-2S] cluster coordinated by four conserved Cys residues. Pcal_0546 exhibits metal-dependent 5′ to 3′ exonuclease activity against ssDNA substrates, whereas the Cas4 protein SSO1391 from Sulfolobus solfataricus can cleave ssDNA in both the 5′ to 3′ and 3′ to 5′ directions. The active site of Pcal_0546 contains a bound metal ion coordinated by the side chains of Asp123, Glu136, His146, and the main chain carbonyl of Ile137. Site-directed mutagenesis of Pcal_0546 and SSO1391 revealed that the residues of RecB motifs II, III and QhXXY are critical for nuclease activity, whereas mutations of the conserved Cys residues resulted in a loss of the iron-sulfur cluster, but had no effect on DNA cleavage. Our results revealed the biochemical diversity of Cas4 nucleases, which can have different oligomeric states, contain [4Fe-4S] or [2Fe-2S] clusters, and cleave single stranded DNA in different directions producing single-stranded DNA overhangs, which are potential intermediates for the synthesis of new CRISPR spacers.
Highlights
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and associated proteins (Cas) represent the only adaptive microbial immunity system which protects microbial cells from invading viruses and plasmids [1,2]. This immunity system is based on the incorporation of short DNA sequences (30–50 nucleotides) from viral genomes or plasmids into the host chromosome, which are transcribed into guide RNAs (CRISPR RNAs or crRNAs) and direct Cas proteins to degrade DNAs or RNAs containing the complementary sequences [3,4,5,6]
SSO0001 and the Pyrobaculum Cas4 proteins belong to the DUF83 family, based on phylogenetic analysis they form two distinct protein groups (Supplementary Figure S2)
Iron-sulfur clusters are known to be important cofactors of many nucleic acid enzymes and binding proteins that function in DNA repair, replication, recombination, transcription, regulation, genome protection and include different nucleases, helicases, glycosylases, RNA polymerases, RNA methyltransferases and transcription factors [42,43,44]
Summary
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and associated proteins (Cas) represent the only adaptive microbial immunity system which protects microbial cells from invading viruses and plasmids [1,2] This immunity system is based on the incorporation of short DNA sequences (30–50 nucleotides) from viral genomes or plasmids into the host chromosome, which are transcribed into guide RNAs (CRISPR RNAs or crRNAs) and direct Cas proteins to degrade DNAs or RNAs containing the complementary sequences [3,4,5,6]. Cas proteins recognize foreign DNA and incorporate short DNA fragments (30–50 nucleotides) as new spacers separated by identical repeats of similar size into the CRISPR locus on the host chromosome These viralspecific spacers are transcribed during the expression step as long primary crRNAs and processed by other Cas proteins into a library of short crRNAs containing one spacer flanked by portions of the adjacent repeat sequences. In the CRISPR interference step, these short crRNAs guide different Cas proteins or complexes to recognize and degrade foreign DNAs or RNAs containing complementary sequences
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