DNA repair pathways can enable tumour cells to survive DNA damage that is induced by chemotherapeutic treatments; therefore, inhibitors of specific DNA repair pathways might prove efficacious when used in combination with DNA-damaging chemotherapeutic drugs. In addition, alterations in DNA repair pathways that arise during tumour development can make some cancer cells reliant on a reduced set of DNA repair pathways for survival. There is evidence that drugs that inhibit one of these pathways in such tumours could prove useful as single-agent therapies, with the potential advantage that this approach could be selective for tumour cells and have fewer side effects.

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DNA repair pathways as targets for cancer therapy

The repair of DNA lesions that occur endogenously or in response to diverse genotoxic stresses is indispensable for genome integrity. DNA lesions activate checkpoint pathways that regulate specific DNA-repair mechanisms in the different phases of the cell cycle. Checkpoint-arrested cells resume cell-cycle progression once damage has been repaired, whereas cells with unrepairable DNA lesions undergo permanent cell-cycle arrest or apoptosis. Recent studies have provided insights into the mechanisms that contribute to DNA repair in specific cell-cycle phases and have highlighted the mechanisms that ensure cell-cycle progression or arrest in normal and cancerous cells.

Regulation of DNA repair throughout the cell cycle

https://www.cell.com/trends/cell-biology/pdf/S0962-8924(15)00142-7.pdf

Repair Pathway Choices and Consequences at the Double-Strand Break

DNA ends exposed after introduction of double-strand breaks (DSBs) undergo 5′–3′ nucleolytic degradation to generate single-stranded DNA, the substrate for binding by the Rad51 protein to initiate homologous recombination. This process is poorly understood in eukaryotes, but several factors have been implicated, including the Mre11 complex (Mre11–Rad50–Xrs2/NBS1), Sae2/CtIP/Ctp1 and Exo1. Here we demonstrate that yeast Exo1 nuclease and Sgs1 helicase function in alternative pathways for DSB processing. Novel, partially resected intermediates accumulate in a double mutant lacking Exo1 and Sgs1, which are poor substrates for homologous recombination. The early processing step that generates partly resected intermediates is dependent on Sae2. When Sae2 is absent, in addition to Exo1 and Sgs1, unprocessed DSBs accumulate and homology-dependent repair fails. These results suggest a two-step mechanism for DSB processing during homologous recombination. First, the Mre11 complex and Sae2 remove a small oligonucleotide(s) from the DNA ends to form an early intermediate. Second, Exo1 and/or Sgs1 rapidly process this intermediate to generate extensive tracts of single-stranded DNA that serve as substrate for Rad51. For years, researchers working on DNA repair have faced a conundrum: homologous recombination, a process that repairs double-strand DNA breaks, requires the production of single-stranded DNA tails with a 3′ end. Yet the obvious candidate for an exonuclease to function in this process is the Mre11 complex, which generates 5′ tailed molecules. Eleni Mimitou and Lorraine Symington now introduce some clarity into the situation. The cell's trick is to use a two-step process in which a short oligonucleotide is first removed from one strand by the Mre11 complex, and then the exonuclease ExoI and helicase Sgs1 load onto this substrate to generate the 3′-tailed molecule.

/pdf/sae2-exo1-and-sgs1-collaborate-in-dna-double-strand-break-510zhq46iq.pdf

Sae2, Exo1 and Sgs1 collaborate in DNA double-strand break processing

▪ Abstract This review focuses on eukaryotic translesion synthesis (TLS) DNA polymerases, and the emphasis is on Saccharomyces cerevisiae and human Y-family polymerases (Pols) η, ι, κ, and Rev1, as well as on Polζ, which is a member of the B-family polymerases. The fidelity, mismatch extension ability, and lesion bypass efficiencies of these different polymerases are examined and evaluated in the context of their structures.One major conclusion is that, despite the overall similarity of basic structural features among the Y-family polymerases, there is a high degree of specificity in their lesion bypass properties. Some are able to bypass a particular DNA lesion, whereas others are efficient at only the insertion step or the extension step of lesion bypass. This functional divergence is related to the differences in their structures. Polζ is a highly specialized polymerase specifically adapted for extending primer termini opposite from a diverse array of DNA lesions, and depending upon the DNA lesion, it ...

Eukaryotic translesion synthesis DNA polymerases: Specificity of structure and function

Homologous recombination is a ubiquitous process with key functions in meiotic and vegetative cells for the repair of DNA breaks. It is initiated by the formation of single-stranded DNA on which recombination proteins bind to form a nucleoprotein filament that is active in searching for homology, in the formation of joint molecules and in the exchange of DNA strands1. This process contributes to genome stability but it is also potentially dangerous to cells if intermediates are formed that cannot be processed normally and thus are toxic or generate genomic rearrangements. Cells must therefore have developed strategies to survey recombination and to prevent the occurrence of such deleterious events. In Saccharomyces cerevisiae, genetic data have shown that the Srs2 helicase negatively modulates recombination2,3, and later experiments suggested that it reverses intermediate recombination structures4,5,6,7. Here we show that DNA strand exchange mediated in vitro by Rad51 is inhibited by Srs2, and that Srs2 disrupts Rad51 filaments formed on single-stranded DNA. These data provide an explanation for the anti-recombinogenic role of Srs2 in vivo and highlight a previously unknown mechanism for recombination control.

The Srs2 helicase prevents recombination by disrupting Rad51 nucleoprotein filaments.

DNA helicases are involved in many aspects of DNA metabolism, including transcription, replication, recombination and repair. In the yeast Saccharomyces cerevisiae, the absence of the Sgs1 helicase results in genomic instability and accelerated ageing. In human cells, mutations in orthologues of SGS1 lead to Bloom (BS), Werner (WS) or Rothmund-Thomson (RTS) syndromes, which are rare, autosomal recessive diseases characterized by genetic instability associated with cancer predisposition. Although data concerning these human diseases are accumulating, there is still no clear idea of the function of the proteins involved. Here we show that sgs1Delta mutants are deficient in DNA repair and are defective for induced recombination events that involve homologous chromosomes. The role of homologous recombination is further evidenced in haploid cells in which both Sgs1p and Srs2p are absent. Yeast SRS2 encodes another DNA helicase involved in the maintenance of genome integrity. Our data suggest that some defects observed in BS, WS or RTS are the consequence of unrestrained recombination.

Homologous recombination is responsible for cell death in the absence of the Sgs1 and Srs2 helicases

Mutations in the Saccharomyces cerevisiae SRS2 gene were isolated in different genetic screens, namely suppression of the trimethoprim sensitivity of rad6 and rad18 mutants (29), suppression of the DNA damage sensitivity in rad18 cells (2), and hyperrecombination (3). The Srs2 protein shares homologies with the bacterial UvrD, Rep (2), and PcrA helicases. As predicted by the sequence, Srs2 was shown in vitro to have 3′-5′ DNA helicase activity (50).

Genetic studies showed that a number of deleterious phenotypes associated with srs2 null alleles are suppressed by deletions of the RAD51, RAD52, RAD55, and RAD57 genes, which are all involved in the formation of Rad51 nucleofilaments on single-stranded DNA (ssDNA) (1, 9, 26). These suppressed phenotypes include UV sensitivity and synthetic lethality with rad54 (17, 47, 52) and sgs1 (14) mutations, which both (most likely) affect later recombination steps (4, 13, 38). Links between Srs2 and homologous recombination were also revealed by the fact that srs2 null alleles suppress the γ-ray and methyl methanesulfonate (MMS) sensitivities conferred by several leaky alleles of RAD52 and RAD51 but have no suppressor effects on the corresponding deletions (9, 25, 42, 52). These phenotypes led to the proposal that Srs2 could destabilize recombination intermediates poisoned to some extent by the leaky recombination proteins (9). In the absence of Srs2, the leakiness of the proteins would allow efficient recombinational repair to occur. In support of this interpretation, Srs2 was recently shown to disrupt in vitro Rad51 nucleofilaments on ssDNA (27, 66). This activity might well account for the srs2 phenotypes cited above, which can be explained by the abnormal persistence of inappropriate recombination intermediates.

Several data indicate that Srs2 is active during replication. In the mitotic cycle, the expression of the gene is high during the S and G2 phases (17). Furthermore, Srs2 is phosphorylated during the S phase in response to checkpoint activation by DNA damages (36). Finally, Srs2 interacts with Pol32, a subunit of polymerase δ (23). This, together with the genetic interactions between srs2 and rad51 and the physical interaction between Srs2 and Rad51 proteins (27), might indicate that one role of Srs2 is to control recombination intermediates arising during replication and eventually to reverse them.

To find new clues on the role(s) of Srs2 in DNA metabolism, we performed a screen for mutations that are synthetically lethal with an srs2 deletion. Mutations in several genes were isolated. For most of them, the synthetic lethality with an srs2Δ mutation was suppressed by preventing homologous recombination by a RAD51 deletion. However, two mutants behaved differently in that synthetic lethality was not suppressed by a recombinational defect. One of these bore the ulp1-I615N mutation, a recessive temperature-sensitive allele of the essential ULP1 gene. Ulp1 is a protease specific for both the processing of the precursor of the ubiquitin-like Smt3 protein (an ortholog of the mammalian SUMO-1 protein) into its mature form and the cleavage of Smt3 from its conjugates. It is required for cell cycle progression, in which it plays a role in the G2/M cell cycle transition (32, 33). Several homologs of Ulp1 have now been found in different species, including Schizosaccharomyces pombe (63) and Drosophila melanogaster (31). Key roles of sumoylation are indicated by the fact that mammalian SUMO-1 is involved in a broad range of cellular processes, such as p53 and c-jun transcriptional activation, signal transduction, inflammatory and immune responses, and the control of cell growth (reviewed in reference 40). Sumoylation plays a role in proper protein localization, organizing and targeting promyelocytic leukemia nuclear bodies, and protein stabilization and has been proposed to be involved in apoptosis (reviewed in reference 53). SUMO conjugates of histone H4, which may mediate gene silencing, have also been reported (57). Numerous reports have shown close relations between SUMO-1 and genome integrity maintenance (reviewed in reference 46). Indeed, some human proteins involved in genome stability, e.g., the DNA topoisomerases I and II and the DNA helicase of the RecQ family involved in the Werner syndrome (Wrn), are sumoylated after DNA-damaging treatments (24). The Bloom syndrome helicase (Blm), which also belongs to the RecQ helicase family, interacts with the SUMO-1-conjugating enzyme Ubc9 and with Rad51 (see reference 19 for a review). A link between sumoylation and recombination has also been suggested by interactions of SUMO-1 or Ubc9 with Rad51 or Rad52 (54, 55). Finally, sumoylation, i.e., Smt3 conjugation, is believed to play a role during the S phase of the S. cerevisiae cell cycle, since PCNA gets either mono-ubiquitinated or sumoylated. The ubiquitin-Smt3 couple possibly acts like a regulatory switch that directs PCNA for alternative functions (21).

We describe here the phenotypes conferred by the ulp1-I615N mutation screened as synthetically lethal with an srs2 deletion. The mutant is thermosensitive, and at a permissive temperature, it presents severe growth defects, moderate UV sensitivity, a strong mitotic hyperrecombination phenotype, an impairment in meiosis, and an accumulation of Smt3 conjugates. The mutation was found to be synthetically lethal not only with srs2 but also with mutations in genes involved in the RAD51-dependent homologous recombination pathway (rad51, rad52, rad54, rad55, rad50, and mre11). These results, together with those of other experiments described below, led us to postulate that the ulp1 mutation affects replication rather than recombination, resulting in the accumulation of recombinogenic intermediates that require both homologous recombination and Srs2 to be correctly processed. We show that this mutant does indeed accumulate single-stranded interruptions in newly replicated DNA.

A new Saccharomyces cerevisiae strain with a mutant Smt3-deconjugating Ulp1 protein is affected in DNA replication and requires Srs2 and homologous recombination for its viability.

Christine Soustelle

Papers

The Srs2 helicase prevents recombination by disrupting Rad51 nucleoprotein filaments.

Homologous recombination is responsible for cell death in the absence of the Sgs1 and Srs2 helicases

A new Saccharomyces cerevisiae strain with a mutant Smt3-deconjugating Ulp1 protein is affected in DNA replication and requires Srs2 and homologous recombination for its viability.