Yet another job for Dna2: Checkpoint activation

Abstract

Mec1 (ATR in humans) is the principal kinase responsible for checkpoint activation in response to replication stress and DNA damage in Saccharomyces cerevisiae. Checkpoint initiation requires stimulation of Mec1 kinase activity by specific activators. The complexity of checkpoint initiation in yeast increases with the complexity of chromosomal states during the different phases of the cell cycle. In G1 phase, the checkpoint clamp 9-1-1 is both necessary and sufficient for full activation of Mec1 kinase whereas in G2/M, robust checkpoint function requires both 9-1-1 and the replisome assembly protein Dpb11 (human TopBP1). A third activator, Dna2, is employed specifically during S phase to stimulate Mec1 kinase and to initiate the replication checkpoint. Dna2 is an essential nuclease-helicase that is required for proper Okazaki fragment maturation, for double-strand break repair, and for protecting stalled replication forks. Remarkably, all three Mec1 activators use an unstructured region of the protein, containing two critically important aromatic residues, in order to activate Mec1. A role for these checkpoint activators in channeling aberrant replication structures into checkpoint complexes is discussed.

Keywords: ATR; Dna2; Mec1; Nuclease; Replication checkpoint.

Figure 1. The structure and functions of Dna2

(A) A schematic representation of the domain structure of S. cerevisiae and human Dna2 nuclease/helicase. The nuclease and helicase domains are depicted in green and blue, respectively. The N-terminal unstructured domain of yeast Dna2 is in light brown. The N-terminal domain of human Dna2 is shorter than that of its yeast counterpart and does not contain an appreciable unstructured region. The cysteines that coordinate the iron-sulfur domain as well as the Trp and Tyr that mediate Mec1^ATR activation are indicated. (B) Some of the functions of Dna2 in maintenance of nuclear genome stability. Left panel, Dna2 mediates processing of long flaps during Okazaki fragment maturation. Middle panel, Dna2 works in conjunction with Sgs1/BLM helicase in a pathway of DSB end resection. Right panel, Dna2 prevents reversal of replication forks. Additional details are found in the text. (C) The role of Dna2 during Okazaki fragment maturation. On the lagging strand, displacement synthesis by Pol δ generates flaps of 1–2nt that are cleaved by FEN1 to create a ligatable nick (short flap pathway, top of figure). Some flaps escape FEN1 cleavage and grow long enough to bind RPA (long flap pathway, bottom of figure). These long flaps require initial cleavage by Dna2 to allow final processing by FEN1 (flap trimming). Conditions or mutations that promote generation of long flaps (Pol δ exonuclease deficiency or fen1-Δ) are dependent on a functional Dna2, whereas FEN1 overexpression, deletion of the POL32 subunit of Pol δ or deletion of PIF1 promote the short flap pathway and show less dependence on Dna2 [27]. Dna2 bound to long flaps may trigger the checkpoint by activating Mec1^ATR.

Figure 2. Mec1^ATR activation during different cell cycle phases in S. cerevisiae

Top panel, 9-1-1 (orange) is the sole activator of Mec1 in G₁ phase. Middle panel, In G2/M, Mec1 can be activated through two redundant pathways that are separable. The first involves direct activation by 9-1-1 and depends on two aromatic residues in the unstructured C-terminus of the Ddc1 subunit of 9-1-1. The second pathway relies on activation by Dpb11 (in blue), although 9-1-1 is still required for Dpb11 recruitment. Bottom panel, Dna2 (green), 9-1-1 (orange) and Dpb11 (blue) act in a redundant fashion to stimulate Mec1 upon replication stalling induced by hydroxyurea. Dna2 is depicted as bound to long flaps, while 9-1-1 and thereby Dpb11 bind 5′-ssDNA-dsDNA junctions.