Fission yeast rad17: a homologue of budding yeast RAD24 that shares regions of sequence similarity with DNA polymerase accessory proteins.

Abstract

contributed equally to this work 4Corresponding author Following DNA damage or a block to DNA synthesis, checkpoint pathways act to arrest mitosis and prevent the attempted segregation of damaged or unreplicated DNA. The radl7 locus of Schizosaccharomyces pombe is one of seven known radiation-sensitive (rad) loci which are absolutely required to prevent mitosis following DNA damage in fission yeast. Six of these (radi, rad3, rad9, radl 7, rad26 and husi) are also required for the checkpoint which prevents mitosis from occurring before DNA replication is complete. We report here that the predicted radl7 gene product is a basic hydrophilic protein of 606 amino acids which contains five domains with sequence homology to replication factor C (RF-C)/activator 1 subunits. Western analysis and fusion with Green Fluorescent Protein indicate that the abundance and electrophoretic mobility of Radl7 is not significantly modified following a block to DNA synthesis or following DNA damage, and that Radl7 is localized in the nucleus. Radl7 function is not essential for growth, but is required for the function of the DNA structure-dependent checkpoints. Sitedirected mutagenesis has been used to demonstrate the biological significance of the RF-C/activator 1-related domains. These studies have also defined an element of the radiation sensitivity caused by loss of Radl7 function which is not associated with the radiationinduced G2 arrest defect seen in the radl7.d null mutant cells.

DNA damage checkpoint control in cells exposed to ionizing radiation

“…These protein complexes have functional homologs in DNA replication, and it is thought that they function similarly as part of the checkpoint response. The RAD17-RFC complex comprises RAD17 and the four small subunits of replication factor C (RFC; RFC2, RFC3, RFC4, RFC5) (Griffiths et al, 1995;Green et al, 2000;Lindsey-Boltz et al, 2001). RFC, which includes subunits RFC1-5, recognizes and binds single-strand/ double-strand DNA junctions and functions to load on the DNA a homotrimeric protein with a clamp-like structure needed for increased processivity for DNA polymerases d and e (Stillman, 1994;Brush and Kelly, 1996).…”

Genetic evidence for a role of Saccharomyces cerevisiae Mph1 in recombinational DNA repair under replicative stress

“…Upstream of Rad53, Rad9 and two more protein complexes signal the presence of DNA damage and fully activate Rad53 (De la Torre-Ruiz et al, 1998;Sanchez et al, 1999;Rouse and Jackson, 2002). One complex is a replication factor C (RFC)-like complex, where Rad24 interacts with four small RFC-subunits (Griffiths et al, 1995); the other complex, formed by Rad17, Ddc1 and Mec3 proteins, shows similarity to the sliding clamp PCNA (Venclovas and Thelen, 2000). In case of DNA damage the PCNA-like structure is loaded onto DNA by the RFC-like complex (Longhese et al, 1998, Melo et al, 2001.…”

Mitotic DNA damage and replication checkpoints in yeast

“…They may also be directly involved in the recognition of DNA damage, since several are similar to proteins involved in DNA metabolism (Table 1). Rad1 sp is similar to the Ustilago Rec1 exonuclease, the putative human Rad1 sp homolog (hRad1) displays exonuclease activity [15,16•], and Rad17 sp has limited similarity to replication factor C (RFC) [17]. Furthermore, S. cerevisiae homologs of Rad1 and Rad17 have been implicated in the processing of DNA damage [18].…”