Fission Yeast Rad17 Associates with Chromatin in Response to Aberrant Genomic Structures

Abstract

Fission yeast checkpoint protein Rad17 is required for the DNA integrity checkpoint responses. A fraction of Rad17 is chromatin bound independent of the other checkpoint proteins throughout the cell cycle. Here we show that in response to DNA damage induced by either methyl methanesulfonate treatment or ionizing radiation, increased levels of Rad17 bind to chromatin. Following S-phase stall induced by hydroxyurea or a cdc22 mutation, the chromatin-bound Rad17 progressively dissociates from the chromatin. After S-phase arrest by hydroxyurea in cds1⌬ or rad3⌬ cells or by replication mutants, Rad17 remains chromatin bound. Rad17 is able to complex in vivo with an Rfc small subunit, Rfc2, but not with Rfc1. Furthermore, cells with rfc1⌬ are checkpoint proficient, suggesting that Rfc1 does not have a role in checkpoint function. A checkpointdefective mutant protein, Rad17(K118E), which has similar nuclear localization to that of the wild type, is unable to bind ATP and has reduced ability in chromatin binding. Mutant Rad17(K118E) protein also has reduced ability to complex with Rfc2, suggesting that Lys 118 of Rad17 plays a role in Rad17-Rfc small-subunit complex formation and chromatin association. However, in the rad17.K118E mutant cells, Cds1 can be activated by hydroxyurea. Together, these results suggest that Rad17 binds to chromatin in response to an aberrant genomic structure generated from DNA damage, replication mutant arrest, or hydroxyurea arrest in the absence of Cds1. Rad17 is not required to bind chromatin when genomic structures are protected by hydroxyurea-activated Cds1. The possible checkpoint events induced by chromatin-bound Rad17 are discussed.

Histone H2A Phosphorylation Controls Crb2 Recruitment at DNA Breaks, Maintains Checkpoint Arrest, and Influences DNA Repair in Fission Yeast

“…The checkpoint responses in S. pombe require a group of six "checkpoint Rad proteins" (Rad1, Rad3, Rad9, Rad17, Rad26, and Hus1) that are thought to function as sensors of DNA replication arrest and DNA damage (11). Rad1, Rad9, and Hus1 have weak sequence similarities to proliferating cell nuclear antigen and form a ring-shaped complex (12,31,38). Rad17 has sequence similarity to replication factor C (RFC) proteins (32) and associates with other RFC subunits (38).…”

“…Rad1, Rad9, and Hus1 have weak sequence similarities to proliferating cell nuclear antigen and form a ring-shaped complex (12,31,38). Rad17 has sequence similarity to replication factor C (RFC) proteins (32) and associates with other RFC subunits (38). The RFC complex recruits proliferating cell nuclear antigen onto DNA; therefore, it has been proposed that the Rad17 complex loads a Rad1-Rad9-Hus1 complex onto sites of DNA damage.…”

Cell cycle checkpoint signaling through the ATM and ATR kinases

“…A second potential DNA-targeting subunit is Rad17 (S. pombe nomenclature). In fission yeast, rad17 + associates with chromatin throughout the yeast cell cycle, and this basal level of DNA-bound rad17 + is either increased or decreased in response to genotoxic stress, with the direction of change determined by the type of DNA damage incurred by these cells (Griffiths et al 2000;Kai et al 2001). In addition to its predicted role as a checkpoint clamp loader for the Rad1-Rad9-Hus1 complex (Thelen et al 1999;Venclovas and Thelen 2000), our group has shown that treatment of human cells with genotoxic agents triggers rapid associations of ATM and ATR with hRad17 (Bao et al 2001).…”

Identification of NPM-ALK interacting proteins by tandem mass spectrometry

“…Interestingly, proteins that bind to components of nuclear matrix such as lamin B1 (Luderus et al, 1992), centromere proteins (Payen et al, 1998), and RAD17 homolog (Kai et al, 2001) were also identified. A subset of these proteins may represent proteins that interact with the nucleophosmin component of the NPM-ALK fusion protein.…”