DNA damage kinase signaling: checkpoint and repair at 30 years

Abstract

From bacteria to mammalian cells, damaged DNA is sensed and targeted by DNA repair pathways. In eukaryotes, kinases play a central role in coordinating the DNA damage response. DNA damage signaling kinases were identified over two decades ago and linked to the cell cycle checkpoint concept proposed by Weinert and Hartwell in 1988. Connections between the DNA damage signaling kinases and DNA repair were scant at first, and the initial perception was that the importance of these kinases for genome integrity was largely an indirect effect of their roles in checkpoints, DNA replication, and transcription. As more substrates of DNA damage signaling kinases were identified, it became clear that they directly regulate a wide range of DNA repair factors. Here, we review our current understanding of DNA damage signaling kinases, delineating the key substrates in budding yeast and humans. We trace the progress of the field in the last 30 years and discuss our current understanding of the major substrate regulatory mechanisms involved in checkpoint responses and DNA repair.

Keywords: DNA damage response; DNA repair; checkpoint; kinase; mass spectrometry.

Figure 1. DNA damage signaling via PIKKs and checkpoint kinases in budding yeast and humans

DNA damage signaling is initiated at DNA structures that form during DNA damage or replication stress, including single‐strand DNA (ssDNA) and broken DNA ends. The apical PIKKs are recruited to these structures and become activated to initiate downstream signaling. Mec1/ATR is recruited to RPA‐coated ssDNA, while Tel1/ATM and DNA‐PKcs initially associate with DNA ends formed by double‐strand breaks. Adaptor proteins are often required to mediate the transfer of phosphorylation from apical to downstream checkpoint kinases. Apical and downstream checkpoint kinases function coordinately to mediate cellular responses to DNA damage, either directly or through the regulation of additional downstream kinases. PIKKs also target an extensive network of substrates independently of downstream checkpoint kinases.

Figure 2. Recruitment of DNA damage signaling kinases and adaptor proteins to DNA lesions: conserved features between budding yeast and humans

Phosphorylation and adaptor proteins play a key role in the recruitment of downstream checkpoint kinases. The colored ovals indicate phosphorylation events mediated by DNA damage signaling kinases (see kinase key). The orange lines indicate protein–protein interactions promoted by the indicated phosphorylation events (also methylation (me) or ubiquitylation (Ub)). Activation of the downstream checkpoint kinases by the apical PIKK kinases requires adaptor proteins (outlined in green). In most cases, these adaptor proteins act as scaffolds to directly bind to and recruit the downstream checkpoint kinase. The model, mostly based on extensive work in yeast, posits that the recruitment of the downstream checkpoint kinase to the proximity of the apical PIKK kinase enables the phosphorylation and activation of the downstream checkpoint kinase. In addition to activating the downstream checkpoint kinase, phosphorylation events mediated by the apical PIKK kinases are critical for scaffold assembly, often promoting protein–protein interactions. Accordingly, a conserved feature of several adaptor proteins in budding yeast and humans is the presence of protein domains responsible for binding phosphorylated proteins (FHA and BRCT domains). Notably, other kinases such as CDK and CK2 also catalyze phosphorylation events involved in adaptor recruitment, although these events are often not induced by DNA damage. For DNA‐PKcs, while this kinase has been implicated in the phosphorylation of H2AX and 53BP1, it does not seem to be involved in CHK2 phosphorylation.

Figure 3. Yeast‐to‐human parallels in core checkpoint responses mediated by DNA damage signaling

Substrate map highlighting the phosphorylation events involved in core DNA damage signaling responses in yeast and humans (see text for detailed discussion of each substrate). Conserved or functionally analogous phosphorylation events are positioned parallel to each another. The colored ovals indicate phosphorylation events mediated by DNA damage signaling kinases (see “kinase‐dependency” key). The arrows or lines that emanate from the colored ovals represent the role phosphorylation plays in regulating that protein (see “role of phosphorylation” key). Question marks indicate uncertainty, either in the functionality of the phosphorylation event or in the identity of the kinase or substrate. Arrows that impinge on CDK demonstrate how DNA damage signaling can indirectly inhibit CDK activity.

Figure 4. Yeast‐to‐human parallels in the regulation of DNA repair proteins by DNA damage signaling: substrates and mechanisms

Substrate map cataloging the DNA damage signaling events regulating DNA repair proteins (see text for detailed discussion of each substrate). Conserved or analogous substrates involved in related DNA repair pathways are positioned parallel to each other. The colored ovals indicate phosphorylation events mediated by DNA damage signaling kinases (see “kinase‐dependency” key). The arrows or lines that emanate from the colored nodes represent the role phosphorylation plays in regulating that protein (see “role of phosphorylation” key). Question marks indicate uncertainty, either in the functionality of the phosphorylation event or in the identity of the kinase or substrate.