Rad52 forms DNA repair and recombination centers during S phase

Abstract

Maintenance of genomic integrity and stable transmission of genetic information depend on a number of DNA repair processes. Failure to faithfully perform these processes can result in genetic alterations and subsequent development of cancer and other genetic diseases. In the eukaryote Saccharomyces cerevisiae, homologous recombination is the major pathway for repairing DNA double-strand breaks. The key role played by Rad52 in this pathway has been attributed to its ability to seek out and mediate annealing of homologous DNA strands. In this study, we find that S. cerevisiae Rad52 fused to green fluorescent protein (GFP) is fully functional in DNA repair and recombination. After induction of DNA double-strand breaks by ␥-irradiation, meiosis, or the HO endonuclease, Rad52-GFP relocalizes from a diffuse nuclear distribution to distinct foci. Interestingly, Rad52 foci are formed almost exclusively during the S phase of mitotic cells, consistent with coordination between recombinational repair and DNA replication. This notion is further strengthened by the dramatic increase in the frequency of Rad52 focus formation observed in a pol12-100 replication mutant and a mec1 DNA damage checkpoint mutant. Furthermore, our data indicate that each Rad52 focus represents a center of recombinational repair capable of processing multiple DNA lesions.

The F-Box Protein Dia2 Regulates DNA Replication

“…To examine the possibility that dia2 cells accumulate spontaneous DNA damage, we used a Rad52-YFP reporter strain. Rad52 is involved in recombinational repair and has been shown to localize to discrete nuclear foci when cells are treated with DNA-damaging agents (Lisby et al, 2001). We generated a dia2 Rad52-YFP strain and examined the cells using fluorescence microscopy.…”

“…We generated a dia2 Rad52-YFP strain and examined the cells using fluorescence microscopy. A deletion mutant of the checkpoint kinase Mec1 (suppressed by deletion of the ribonucleotide reductase inhibitor Sml1) was used as a positive control (Lisby et al, 2001). The dia2 mutant showed an increased number of Rad52-YFP foci over WT.…”

Correlation of Biochemical Properties with the Oligomeric State of Human Rad52 Protein

“…In yeast, distinct Rad52 foci that undoubtedly contain multiple protein rings form under conditions that result in DSBs, e.g. during meiosis and in S phase mitotic cells, and following exposure to ␥-irradiation (27). Also, co-localization of RAD52 group proteins, including Rad51, Rad52, and Rad54, is observed following DNA damage in mammalian cells (28 -31).…”

“…Also, co-localization of RAD52 group proteins, including Rad51, Rad52, and Rad54, is observed following DNA damage in mammalian cells (28 -31). Therefore, it appears that the ability of Rad52 to form higher order oligomeric complexes may be important to its function in promoting homologous recombination, and it has been suggested that such complexes may be required as an early step in the efficient catalysis of DNA repair via homologous recombination (27). Given the domain structure of the HsRad52 protein it may be that higher order oligomers of the protein are used in promoting complex formation among repair proteins and DNA through simultaneous interaction with multiple components.…”

Mapping genomic hotspots of DNA damage by a single-strand-DNA-compatible and strand-specific ChIP-seq method

“…Nuclear foci formed by fluorescent protein-tagged Rad52 have been widely used to assess the level of DNA damage in live cell imaging analysis (Lisby et al 2001;Du et al 2003). Unlike phospho-H2A(X), which is not found at the immediate vicinity of DSBs due to end resection (Shroff et al 2004), Rad52 is recruited to single-strand DNA (ssDNA) exposed by resection, in a replication protein A (RPA)-dependent manner (Lisby et al 2004).…”