(NAS Colloquium) Links Between Recombination and Replication: Vital Roles of Recombination

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Colloquium on Links Between Recombination and Replication: Vital Roles of Recombination

Fig. 1. Simplified steps of Okazaki fragment maturation during DNA lagging strand synthesis (for further details see refs. 3–5). In rad27∆ cells, unprocessed replication intermediates could be resolved either by using an alternative process involving Dna2, Exo1, RNase H(35), and Polδ activities or by the Rad52 recombinational repair pathway.

Class I bypass process(es) includes several mechanisms for the removal of the initiator RNA; these have been explored by a combination of genetical and biochemical approaches in S. cerevisiae. The current view is that the combined action of RNase H(35) and Rad27 activities in large part accounts for the processing of Okazaki fragments. This process is underscored by the observation that simultaneous deletion of the RNH35 and RAD27 genes has a strong synergistic effect, resulting in extremely slow growth of double mutant cells (25). In the absence of Rad27p alone, it is envisaged that RNase H(35), which normally eliminates the RNA primer up to the last ribonucleotide, could remove the remaining monoribonucleotide at low efficiency, which therefore would facilitate subsequent DNA ligation (25). In addition, a recent study showed that in vitro the human Exo1 protein, a functional homolog of the Schizosaccharomyces pombe and S. cerevisiae Exo1 5′–3′ exonuclease (26, 27), was as efficient in removal of ribonucleotides as deoxynucleotides (28). This result and the observation that the exo1∆ rad27∆ double mutant exhibits synthetic lethality or sublethality at 30°C (29) indicates that Exo1p (also involved in mismatch repair) is a good candidate to allow the survival of the rad27∆ single mutant and rad27∆ rnh35∆ double mutant (28). Other structure-specific nucleases that may be functionally redundant also have been investigated. The Fen1/Rad27 protein belongs to a family of nucleases and is related to the S. cerevisiae exonucleases Rad2 (30) and Exo1 (27, 31), which are implicated in repair and recombination, as well as to two other proteins, Yen1 of unknown function, and Din7 with a mitochondrial function (32). Single or multiple deletions of the RAD27, RAD2, YEN1, and DIN7 genes were not found to confer synthetic lethality or synergistic effects on cell viability, indicating that none of these genes substitutes for the rad27∆ deficiency (22, 33). Additional studies have shown that overexpression of Exo1 (29) or Dna2, which has helicase (34) and flap endonuclease activity (10, 35), can compensate for the rad27∆ growth defect at 37°C. This result suggests that these proteins, alone or with other partners, are able to overcome the replication defect, either by their direct action on replication intermediates (reviewed in ref. 4) or as essential players in the alternative pathway(s). In addition, the synthetic lethality of rad27∆ with the pol3–01 mutation affecting the 5′–3′ exonuclease proofreading domain of DNA polymerase δ also suggests an additional mean to process the intermediate DNA structures accumulating at the border between two Okazaki fragments (16, 36).

Class II processes include those that compensate for the defect of rad27∆ by channeling blocked replication intermediates into the homologous recombinational repair pathway. This proposal is consistent with the hyperrecombinogenic phenotype of rad27∆ cells and the significant result that rad27∆ exhibits synthetic lethality in combination with deletions of several genes of the Rad52 epistasis group (12, 37). The colethality of the rad27∆ mutation with deletions of several genes of DNA damage checkpoint pathways, including RAD9, RAD17, RAD24, and MEC3 (13, 38), is also consistent with the accumulation of single-stranded DNA in rad27∆ cells (21), and possibly of double-strand breaks (DSBs) (12), which must be processed by the DNA repair machinery to avoid the lethal segregation of damaged or broken chromosomes. In this paper, we review and present additional evidence, based on extensive synthetic lethality approaches, for the essential role of the homologous recombinational-repair pathway (Rad52 pathway) in the survival of rad27∆ mutants. This analysis provides an extended example of the links between replication, repair, and recombination processes.

Materials and Methods

Media and Sporulation Conditions. Growth and sporulation of yeast cells were performed by standard methods (39). Standard medium (yeast extract/peptone/dextrose) was used for vegetative growth. For sporulation, cells were grown in presporulation media at 23°C or 30°C, washed in water, resuspended, and incubated in sporulation medium (1% potassium acetate supplemented with required amino acids) at 23°C or 30°C.

Yeast Strains. The names of diploids used in this study are shown in Tables 1 and 2. The origin and complete genotype of each can be supplied on request. In most cases, strains are of the S288C background, including the collection of single-deletion strains derived from FY1679 [MATa/MATα, ura3–52/ura3–52, trp1∆63/TRP1, leu2∆1/LEU2, his3∆200/HIS3 (40)] and used in the EUROFAN deletion project, and the collection derived from BY4741 (MATa, his3∆1, leu2∆0, met15∆0, ura3∆0) and BY4742 (MATα, his3∆1, leu2∆0, lys2∆0, ura3∆0) strains (41) and used for the international systematic S. cerevisiae gene disruption project (42). In FY and BY strains (obtained from EUROSCARF, Frankfurt), deleted genes are replaced with the KanMX marker (providing resistance to G418). These strains were found to be highly compatible with our laboratory MGD strains, which also share the S288C background (43), based on the high viability of meiotic products from hybrid crosses. All mre11 strains (44, 45) are of the SK1 background. The sgs1∆ strain, provided by S.Gangloff (Commissariat à l’Energie Atomique, Paris), is a W303 derivative. The rad27∆ strains used for synthetic lethality assays are FW2612 [MATα rad27::HIS3 (12)], FW2617 [MATa rad27::HIS3 (12)], and LSY702–2A (MATa rad27::TRP1) and LSY702–6B (MATα rad27::TRP1), which are W303 derivatives (37). We constructed the SK1 strains ORD5902–1A (MATa rad27::URA3, ura3, trp1, arg4∆Hpa1) and ORD5902–1C (MATα rad27::URA3, ura3) by one-step gene replacement of the RAD27 locus with the rad27::URA3 EcoRISphI restriction fragment of plasmid pMR∆rad27::URA3 (2). All RAD27 disruptions in this study were verified by Southern blot analysis, and the expected relevant phenotypic traits, such as cell division morphology, temperature sensitivity, and colethality with a rad51 deletion, also were confirmed.

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