Structure and function of a novel endonuclease acting on branched DNA substrates

Abstract

We show that Pyrococcus abyssi PAB2263 (dubbed NucS (nuclease for ss DNA) is a novel archaeal endonuclease that interacts with the replication clamp PCNA. Structural determination of P. abyssi NucS revealed a two-domain dumbbell-like structure that in overall does not resemble any known protein structure. Biochemical and structural studies indicate that NucS orthologues use a non-catalytic ssDNA-binding domain to regulate the cleavage activity at another site, thus resulting into the specific cleavage at double-stranded DNA (dsDNA)/ssDNA junctions on branched DNA substrates. Both 3 0 and 5 0 extremities of the ssDNA can be cleaved at the nuclease channel that is too narrow to accommodate duplex DNA. Altogether, our data suggest that NucS proteins constitute a new family of structure-specific DNA endonucleases that are widely distributed in archaea and in bacteria, including Mycobacterium tuberculosis.

Modulation of the Pyrococcus abyssi NucS Endonuclease Activity by Replication Clamp at Functional and Structural Levels

“…The crystal structure of PabNucS revealed a self-assembled dimer with a dumbbell-like organization that does not resemble any known protein structures (8,9). PabNucS is composed of 2 domains.…”

The Gratuitous Repair on Undamaged DNA Misfold

“…Similarly, Deletions in PCNA, RPA, and the Bloom protein (BLM), a 3'-5' helicase can also increase CNG repeat expansion or deletion, which reportedly interacts with FEN-1 in cleaving flaps. Recently NucS from Pyrococcus abyssi was found to be the equivalent of FEN-1 that cleaves the flapped DNA in Okazaki frangment processing in the lagging strand DNA replication (Ren et al, 2009;Creze et al, 2011). SLX1 and SLX4 are other structure-specific endonucleases acting as heteromer that cleave branched DNA substrates, particularly simple-Y, 5'-flap, or replication fork structures.…”

Archaeal DNA Repair Nucleases

“…Crystallographic structure of Pab2263 has been solved (Ren et al, 2009;Ren et al, 2007) and is the first representative of the DUF91 family. Pab2263 is composed of two independent domains, separated by a long and flexible linker (~28Å).…”

“…In Pab2263, the potential binding site involves two patches of three consecutive basic residues, two conserved aromatic residues and a conserved arginine. High affinity ssDNA binding activity of the N-terminal domain was desmonstrated using site-directed mutagenesis and EMSA experiments (Ren et al, 2009). The N-terminal domain displays a large hydrophobic patch exposed to the C-terminal domain, and is involved in the dimerisation of the protein.…”

“…In high concentration of NucS, nuclease activity can invade to the double stranded DNA regions, suggesting that NucS proteins carry a weak helicase and/or unmelting activity of dsDNA. Important observation is that addition of PCNA directs the cleavage activity of P. abyssi NucS towards the ss/ds DNA junctions, thus increasing the cleavage specificity (Ren et al, 2009). Pab2263-NucS is a founding member of a new family of structure-specific DNA endonuclease.…”