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OwnerJulie A. MAUPIN, Huiyong JIA +1inventors
InventorsJulie A. MAUPIN, Huiyong JIA, Swathi DANTULURI
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Here a new type of nuclease complex composed of RecJ3/4 subcomplex and aRNase J from H. volcanii is disclosed. This complex is found to transiently associate with Cdc48a and Uhls in the presence of ATP. As the ratio of RecJ3/4 subcomplex to aRNase J is increased in the reconstitution of this complex in vitro, the activities of aRNascJ arc found to shift from endonuclease and 5′-3′ exonuclease to primarily 3′-5′ exonuclease with preference for RNA to ssDNA. Phenotypic analysis of mutant strains reveals aRNase J is essential and Cdc48a, RecJ3, and RecJ4 are important for recovery of cells from DNA damaging agents including those that introduce double-strand breaks (DSBs). The disclosure here suggests a novel nuclease system that can control degradation process and direction of RNA as well as ssDNA in vitro.
A nuclease complex system comprising a nuclease and a set of proteins for controlling the nuclease to control the degradation of a single stranded nucleic acid, wherein the nuclease is aRNase J, wherein the set of nuclease-controlling proteins comprises RecJ3 and RecJ4, and wherein the single stranded nucleic acid is RNA or ssDNA.
The nuclease complex system of any one of the preceding claims,
The nuclease complex system of claim, wherein RecJ3 optionally has a tag at its C-terminal or N-terminal, wherein the tag can be selected from hexa-histidine (6× His), glutathione S-transferase (GST), FLAG, streptavidin-binding peptide (SBP), Strep II, maltose-binding protein (MBP), calmodulin-binding peptide (CBP), chitin-binding domain (CBD), HA, c-Myc, and the like.
The nuclease complex system of claim, wherein RecJ3 has His tag at its N terminal.
The nuclease complex system of claim, wherein RecJ3 optionally has a tag at its C-terminal or N-terminal, wherein the tag can be selected from hexa-histidine (6× His), glutathione S-transferase (GST), FLAG, streptavidin-binding peptide (SBP), Strep II, maltose-binding protein (MBP), calmodulin-binding peptide (CBP), chitin-binding domain (CBD), HA, c-Myc, and the like.
The nuclease complex system of claim, wherein RecJ4 has StrepII tag at its C terminal.
An in vitro nuclease activity titration kit comprising RecJ3, RecJ4, and aRNaseJ proteins individually packed in a container as powder or concentrated solution form, which are at least partially purified, wherein the kit further comprises concentrated reaction buffer having pH of about 7.2-7.7, Mg2+, Mn2+, and high salt to make a final concentration of about 2M NaCl in the reaction solution, reaction stop solution comprising EDTA and/or TPEN, and nuclease activity titration substrates comprising RNAs or ssDNA labeled with 6-carboxyfluorescein (6-FAM) at 5′ end or 3′ end, and RNAs labeled internally with phosphorothioate and at 3′ end with 6-FAM.
A nucleic acid strand encoding aRNase J with a StrepII tag at its C-terminal (aRNase J-StrepII).
A nucleic acid strand encoding RecJ3 with a His6 tag at its N-terminal (His6-RecJ3).
A nucleic acid strand encoding recJ4 with a StrepII tag at its C-terminal (RecJ4-StrepII).
A method of controlling direction of nucleotide degradation in a sample comprising a single-stranded DNA or RNA, the method comprising providing to the sample an amount of (i) RecJ3/J4 RNase from Haloferax volcanii and/or (ii) aRNase J from Haloferax volcanii;
Dependencies are inferred from the claim text.