Interactions of the DNA Ligase IV-XRCC4 Complex with DNA Ends and the DNA-dependent Protein Kinase

Abstract As its name implies, the DNA dependent protein kinase (DNA-PK) requires DNA double-stranded ends for enzymatic activation. Here, I demonstrate that hairpinned DNA ends are ineffective for activating the kinase toward many of its well-studied substrates (p53, XRCC4, XLF, HSP90). However, hairpinned DNA ends robustly stimulate certain DNA-PK autophosphorylations. Specifically, autophosphorylation sites within the ABCDE cluster are robustly phosphorylated when DNA-PK is activated by hairpinned DNA ends. Of note, phosphorylation of the ABCDE sites is requisite for activation of the Artemis nuclease that associates with DNA-PK to mediate hairpin opening. This finding suggests a multi-step mechanism of kinase activation. Finally, I find that all non-homologous end joining (NHEJ) defective cells (whether deficient in components of the DNA-PK complex or components of the ligase complex) are similarly deficient in joining DNA double-stranded breaks (DSBs) with hairpinned termini.

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Xenopus Cds1 Is Regulated by DNA-Dependent Protein Kinase and ATR during the Cell Cycle Checkpoint Response to Double-Stranded DNA Ends

Molecular and Cellular Biology ◽

10.1128/mcb.24.22.9968-9985.2004 ◽

2004 ◽

Vol 24 (22) ◽

pp. 9968-9985 ◽

Cited By ~ 7

Author(s):

Troy D. McSherry ◽

Paul R. Mueller

Keyword(s):

Cell Cycle ◽

Protein Kinase ◽

Cell Cycle Checkpoint ◽

Dual Function ◽

Dependent Protein Kinase ◽

Double Stranded Dna ◽

Cycle Checkpoint ◽

Protein Dissociation ◽

Dependent Protein ◽

Dna Ends

ABSTRACT The checkpoint kinase Cds1 (Chk2) plays a key role in cell cycle checkpoint responses with functions in cell cycle arrest, DNA repair, and induction of apoptosis. Proper regulation of Cds1 is essential for appropriate cellular responses to checkpoint-inducing insults. While the kinase ATM has been shown to be important in the regulation of human Cds1 (hCds1), here we report that the kinases ATR and DNA-dependent protein kinase (DNA-PK) play more significant roles in the regulation of Xenopus Cds1 (XCds1). Under normal cell cycle conditions, nonactivated XCds1 constitutively associates with a Xenopus ATR complex. The association of XCds1 with this complex does not require a functional forkhead activation domain but does require a putative SH3 binding region that is found in XCds1. In response to double-stranded DNA ends, the amino terminus of XCds1 is rapidly phosphorylated in a sequential pattern. First DNA-PK phosphorylates serine 39, a site not previously recognized as important in Cds1 regulation. Xenopus ATM, ATR, and/or DNA-PK then phosphorylate three consensus serine/glutamine sites. Together, these phosphorylations have the dual function of inducing dissociation from the ATR complex and independently promoting the full activation of XCds1. Thus, the checkpoint-mediated activation of XCds1 requires phosphorylation by multiple phosphoinositide 3-kinase-related kinases, protein-protein dissociation, and autophosphorylation.

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