Isolation of the Schizosaccharomyces pombe RAD54 homologue, rhp54 +, a gene involved in the repair of radiation damage and replication fidelity

Abstract

The RAD54 gene of Saccharomyces cerevisiae encodes a putative helicase, which is involved in the recombinational repair of DNA damage. The RAD54 homologue of the fission yeast Schizosaccharomyces pombe, rhp54+, was isolated by using the RAD54 gene as a heterologous probe. The gene is predicted to encode a protein of 852 amino acids. The overall homology between the mutual proteins of the two species is 67% with 51% identical amino acids and 16% similar amino acids. A rhp54 deletion mutant is very sensitive to both ionizing radiation and UV. Fluorescence microscopy of the rhp54 mutant cells revealed that a large portion of the cells are elongated and occasionally contain aberrant nuclei. In addition, FACS analysis showed an increased DNA content in comparison with wild-type cells. Through a minichromosome-loss assay it was shown that the rhp54 deletion mutant has a very high level of chromosome loss. Furthermore, the rhp54 mutation in either a rad17 or a cdc2.3w mutant background (where the S-phase/mitosis checkpoint is absent) shows a significant reduction in viability. It is hypothesized that the rhp54+ gene is involved in the recombinational repair of UV and X-ray damage and plays a role in the processing of replication-specific lesions.

Novel functional requirements for non-homologous DNA end joining in Schizosaccharomyces pombe

“…In contrast, S.cerevisiae wild‐type cells are inefficient at repairing blunt‐ended DSBs by NHEJ and no further reduction is observed in YKU deletion strains (Boulton and Jackson, 1996b). In agreement with the independence of our NHEJ assay from homologous recombination activities, we found that rhp54 Δ , a strain defective in homologous recombination (Muris et al ., 1996), is proficient in NHEJ (Figure 1B). We generated pku70 and lig4 deletion strains and found that the frequency of rejoining of blunt DSBs is decreased ∼1000‐fold in these cells compared with wild‐type cells (isogenic pku70 + and lig4 + ) (Figure 1B).…”

A novel SMC protein complex in Schizosaccharomyces pombe contains the Rad18 DNA repair protein

“…Occasional double‐strand breaks occur during DNA replication and these are thought to be repaired by recombination. Cells deficient in the recombination‐repair genes rhp51 or rhp54 grow poorly (Muris et al ., 1993, 1996), presumably because they have difficulty in dealing with damage arising during replication. Double mutants of rhp51 or rhp54 with the DNA damage checkpoint genes are inviable (Muris et al ., 1996), showing that checkpoint‐mediated mitotic delay is necessary for the cell to deal with this kind of replicative stress.…”

Inflammation-induced DNA damage, mutations and cancer

“…Additionally, if a polymerase encounters a SSB during replication, the replication machinery cannot synthesize past the gap, and the replication fork collapses, leaving a one-ended DSB (Figure 7B) (113). Similarly, replication-blocking lesions (such as εA and εC) can cause DSBs (114)(115)(116), possibly because the stress of a replication fork encountering a lesion that cannot be bypassed causes breakage of the backbone, though the exact mechanism(s) for this breakage remains unclear (117,118). Both DSBs and SSBs have been observed in cells co-cultured with activated macrophages, confirming that phagocyte-associated RONS can cause these lesions (119).…”

“…As mentioned earlier, an important source of DSBs during inflammation is broken replication forks, which generate only one DSB end (see Figure 7), and thus cannot be accurately repaired by NHEJ. Therefore, lesions that cause replication fork breakdown (including replication blocking lesions and SSBs) necessitate HR to restore the replication fork (114,115,180). Notably, many of the intermediates of the BER pathway are SSBs, so if replication forks encounter these breaks before BER is completed, HR may be initiated as well (Figure 8) (111,181).…”