Retention of the Human Rad9 Checkpoint Complex in Extraction-resistant Nuclear Complexes after DNA Damage

Matthew A Burtelow,Scott H Kaufmann,Larry M Karnitz

doi:10.1074/jbc.m001244200

Abstract

Studies in yeasts and mammals have identified many genes important for DNA damage-induced checkpoint activation, including Rad9, Hus1, and Rad1; however, the functions of these gene products are unknown. In this study we show by immunolocalization that human Rad9 (hRad9) is localized exclusively in the nucleus. However, hRad9 was readily released from the nucleus into the soluble extract upon biochemical fractionation of un-irradiated cells. In contrast, DNA damage promptly converted hRad9 to an extraction-resistant form that was retained at discrete sites within the nucleus. Conversion of hRad9 to the extraction-resistant nuclear form occurred in response to diverse DNA-damaging agents and the replication inhibitor hydroxyurea but not other cytotoxic stimuli. Additionally, extraction-resistant hRad9 interacted with its binding partners, hHus1 and an inducibly phosphorylated form of hRad1. Thus, these studies demonstrate that hRad9 is a nuclear protein that becomes more firmly anchored to nuclear components after DNA damage, consistent with a proximal function in DNA damage-activated checkpoint signaling pathways.

Highlights

In eukaryotes, DNA damage activates complex cellular responses that initiate DNA repair and slow or block progression through the cell cycle
Rad1 has homology with Ustilago maydis Rec1 [40], which is a checkpoint protein and a 3Ј-5Ј exonuclease, suggesting that Rad1 may participate in DNA metabolic events that are required for checkpoint activation
We show that in response to DNA damage the hRad91⁄7hHus11⁄7hRad1 checkpoint complex redistributes into a less extractable, chromatin-bound form, suggesting that these proteins have a proximal function in the DNA damage-signaling pathway

Summary

Introduction

DNA damage activates complex cellular responses that initiate DNA repair and slow or block progression through the cell cycle (reviewed in Refs. 1– 6). DNA damage promptly converted hRad9 to an extraction-resistant form that was retained at discrete sites within the nucleus. Conversion of hRad9 to the extraction-resistant nuclear form occurred in response to diverse DNA-damaging agents and the replication inhibitor hydroxyurea but not other cytotoxic stimuli.

Results

Conclusion