DNA constantly undergoes chemical modifications, or DNA damage, that can distort genetic information. All organisms possess efficient pathways for the repair of the damage of the DNA. Critical elements of these pathways are nucleases that recognize and specifically cleave DNA structures: four-way DNA junctions (Holliday junctions), DNA flaps and structures corresponding to replication forks. In this presentation the structure and mechanism of selected structure-selective nucleases will be discussed.
Nucleotide excision repair (NER) is a general DNA repair pathway that detects and corrects a wide spectrum of different DNA modifications. Its critical step is the excision of a DNA fragment that contains the lesion. This step involves two nucleases – one of them is XPG (or Rad2 in yeast). It specifically recognizes a junction between single-stranded and double-stranded DNA in so-called DNA bubbles – structures generated during earlier stages of NER. We have solved a series of crystal structures of Rad2 in complex with DNA substrates (1). They revealed that Rad2 does not specifically recognize the single-stranded part of the substrate but rather binds the last exposed base pair of the double-stranded portion of the DNA. Rad2/XPG is closely related to other structure-specific nucleases: FEN1, EXO1 and GEN1 but it is the only member of the group that is capable of the cleavage of DNA bubbles. The structures offer an explanation for this unique specificity. An element called helical arch adopts a different structure in Rad2 than in FEN1 and EXO1. It no longer blocks the exit from the active site which allows Rad2 to accommodate substrates without a free 5’ end such as DNA bubbles.
Homologous recombination is another fundamental DNA repair pathway. It uses homologous regions of the DNA to drive the repair of particularly dangerous DNA lesions: DNA breaks and interstrand cross-links. One of the intermediates of this pathway are four-arm DNA structures called Holliday junctions. They need to be removed and one way to achieve it is through the action of specialized nucleases called resolvases. In bacteria the canonical resolvase is RuvC. It functions as a dimer which symmetrically cleaves the DNA using two active sites. We solved the first crystal structure of RuvC in complex with its DNA substrate at 3.75 Å resolution and verified the structural model using thiol-based site-specific cross-linking (2). The structure showed that the HJ is in a novel tetrahedral conformation with two phosphates 1 nt from the HJ exchange point interacting with the active sites. Two helices forming the RuvC dimer also participate in the stabilization of the exchange point. The mode of DNA binding by RuvC is very different from phage resolvases for which complex crystal structures had been solved previously, indicating that multiple modes of HJ recognition evolved.
In Eukaryotes one of the HJ removal mechanisms involves SLX1 and MUS81-EME1 nucleases whose concerted action is coordinated by scaffolding protein SLX4. We have solved the first crystal structure of yeast Slx1 and its complex with Slx4-interacting domain termed CCD (3). The structures demonstrated that the GIY-YIG nuclease and RING finger domains present in Slx1 form a compact structure reinforced by a long alpha-helix. Slx1 alone is inactive and it forms a homodimer in crystal and in solution in which the nuclease active site is blocked, offering a likely mechanism to control its promiscuous nuclease activity. Once the Slx1 binds the Slx4 CCD, the active becomes exposed and the nuclease is activated. Therefore, Slx1 is active only when bound to Slx4 platform which controls its activity.
References:
(1) Mietus M, et al.(2014) Nucleic Acids Res., 42(16):10762-75.
(2) Górecka KM, et al.(2013) Nucleic Acids Res., 41(21):9945-55
(3) Gaur V, et al.(2015) Cell Reports, in press