Human Homologs of Schizosaccharomyces pombe Rad1, Hus1, and Rad9 Form a DNA Damage-responsive Protein Complex

Abstract

DNA damage activates cell cycle checkpoints in yeast and human cells. In the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe checkpoint-deficient mutants have been characterized, and the corresponding genes have been cloned. Searches for human homologs of S. pombe rad1, rad9, and hus1 genes identified the potential human homologs hRad1, hRad9, and hHus1; however, little is known about the roles of these proteins in human cells. The present studies demonstrate that hRad1 and hHus1 associate in a complex that interacts with a highly modified form of hRad9, but hHus1 and hRad1 do not associate with hRad17. In addition to being a key participant in complex formation, hRad9 is phosphorylated in response to DNA damage. Together, these results suggest that hRad9, hRad1, and hHus1 are central components of a DNA damage-responsive protein complex in human cells.DNA damage triggers a variety of cellular responses in eukaryotic cells, including the induction of a regulatory signaling network that activates checkpoint controls (1-3). Checkpoint activation transiently blocks cell cycle progression by arresting cells in G 1 and G 2 /M and slowing progression through S phase. Genetic studies in the yeast Saccharomyces cerevisiae and Schizosaccharomyces pombe have identified many of the relevant players in DNA damage-induced checkpoint activation (4, 5). A common theme that emerges from these studies is that checkpoint-deficient yeast are dramatically more sensitive to genotoxins than their wild-type counterparts (1-3), suggesting that the checkpoint proteins play critical roles in cellular responses to DNA damage.Recent studies suggest that key checkpoint regulators may be conserved between yeast and humans. Cloning of the human gene mutated in AT (ATM) revealed that the ATM gene exhibited significant homology with the S. pombe rad3 (sprad3) and S. cerevisiae MEC1 (scMEC1) checkpoint genes (6). The corresponding proteins scMec1, spRad3, and Atm are protein kinases that share large stretches of homology, including a phosphatidylinositol 3-kinase-related kinase (PIKK) 1 domain. The PIKKs are also functionally conserved as mutation of scMEC1, sprad3, or ATM disrupts ionizing radiation (IR)-induced checkpoints and sensitizes the organisms to IR (7).The PIKKs are integral players in a tentative DNA damageinducible signaling pathway that interfaces with the cell cycle machinery (2, 3, 8). In this model, an unidentified sensor recognizes damaged DNA. Potential sensor candidates include the checkpoint proteins, spRad1, spRad9, spRad17, and spHus1. The sensor relays a signal to PIKK family members, which, at least in the case of ATM, are activated by IR (9, 10). In S. pombe, a PIKK-dependent pathway is required for DNA damage-induced activation of spChk1 (11), a protein kinase that phosphorylates Cdc25 and blocks its ability to activate Cdc2, thereby preventing cell cycle transition through the G 2 /M boundary.The similarities between yeast and humans extend beyond these proteins, as human homologs of sprad1 (12-14), spra...

The Rad9–Hus1–Rad1 (9–1–1) clamp activates checkpoint signaling via TopBP1

“…Following treatment with 10 mM HU for 1 h, transfected Rad9 −/− DT40 cells and parental (wild-type) DT40 cells were lysed, separated by SDS-PAGE, and sequentially immunoblotted for phospho-Ser 345 -Chk1, Chk1, and Rad9. The multiple bands present in the Rad9 immunoblots are due to various forms of phosphorylated Rad9 (Volkmer and Karnitz 1999). The dotted line indicates the juxtaposition of nonadjacent regions of the same gel.…”

“…Transfections were with empty vector (EV); vectors expressing S-tagged wild-type Rad9 (WT), Rad9-9A (9A), Rad9-9A fused to full-length TopBP1 (9A-TopBP1), Rad9-9A-TopBP1 in which the WDDP motif in the AD was deleted from TopBP1 (9A-TopBP1-⌬WDDP), Rad9-9A fused to the TopBP1 AD (9A-AD), a tailless Rad9 fused to the AD (⌬tail-AD), Rad17, or H2B fused to the TopBP1-AD (H2B-AD); or a vector expressing PCNA fused to the Rad9 tail (PCNA-Rad9 tail) or the TopBP1 AD (PCNA-AD). The multiple bands present in the Rad9 immunoblots are due to various forms of phosphorylated Rad9 (Volkmer and Karnitz 1999). Asterisk indicates nonspecific immunoreactive bands (A) or putative degradation products of the PCNA fusion proteins (D).…”

“…Bound proteins were sequentially immunoblotted for endogenous TopBP1 (top) and Rad9 (middle). The multiple bands present in the Rad9 immunoblots are due to various forms of phosphorylated Rad9 (Volkmer and Karnitz 1999). (Bottom) A portion of the lysate was also immunoblotted to demonstrate equal TopBP1 levels in all samples.…”

“…Wild-type, Rad9 −/− , and Rad17 −/− DT40 cells were grown as described previously (Kobayashi et al 2004). HEK293 cells (5 × 10 to 8 × 10 6 per transfection) were transfected by electroporation as described previously (Volkmer and Karnitz 1999) using a total of 40 µg of DNA per transfection and a 230-V, 20-msec pulse in a 0.4-cm cuvette with a BTX T 820 electroporator. Following electroporation, the cells were replated in DMEM supplemented with 10% FBS and cultured for 14-20 h before harvest.…”

The role of DNA exonucleases in protecting genome stability and their impact on ageing

“…Modified hRAD9, along with hRAD1 and hHus1, form the human 9-1-1 complex (Volkmer and Karnitz 1999), a heterotrimeric clamp complex structurally (Xu et al 2009) and functionally analogous to PCNA that is loaded onto damaged DNA tracts (Burtelow et al 2000;Carr 2002) by an RF-C like complex and is hyperphosphorylated in a damage-dependent manner, for example by ATM after ionising radiation (IR) (reviewed in Cox and Kearsey 2009). Such phosphorylation is required for IR-induced G1/S checkpoint activation (Chen et al 2001a) but also after hydroxyurea-induced replication stress or UV-induced DNA damage.…”