Elda Cannavo

Methylation‐induced G2/M arrest requires a full complement of the mismatch repair protein hMLH1

The mismatch repair (MMR) gene hMLH1 is mutated in ∼50% of hereditary non‐polyposis colon cancers and transcriptionally silenced in ∼25% of sporadic tumours of the right colon. Cells lacking hMLH1 display microsatellite instability and resistance to killing by methylating agents. In an attempt to study the phenotypic effects of hMLH1 downregulation in greater detail, we designed an isogenic system, in which hMLH1 expression is regulated by doxycycline. We now report that human embryonic kidney 293T cells expressing high amounts of hMLH1 were MMR‐proficient and arrested at the G2/M cell cycle checkpoint following treatment with the DNA methylating agent N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG), while cells not expressing hMLH1 displayed a MMR defect and failed to arrest upon MNNG treatment. Interestingly, MMR proficiency was restored even at low hMLH1 concentrations, while checkpoint activation required a full complement of hMLH1. In the MMR‐proficient cells, activation of the MNNG‐induced G2/M checkpoint was accompanied by phosphorylation of p53, but the cell death pathway was p53 independent, as the latter polypeptide is functionally inactivated in these cells by SV40 large T antigen.

Sae2 promotes dsDNA endonuclease activity within Mre11-Rad50-Xrs2 to resect DNA breaks

To repair double-strand DNA breaks by homologous recombination, the 5′-terminated DNA strand must first be resected, which generates 3′ single-stranded DNA overhangs. Genetic evidence suggests that this process is initiated by the Mre11–Rad50–Xrs2 (MRX) complex. However, its involvement was puzzling, as the complex possesses exonuclease activity with the opposite (3′ to 5′) polarity from that required for homologous recombination. Consequently, a bidirectional model has been proposed whereby dsDNA is first incised endonucleolytically and MRX then proceeds back to the dsDNA end using its 3′ to 5′ exonuclease. The endonuclease creates entry sites for Sgs1–Dna2 and/or Exo1, which then carry out long-range resection in the 5′ to 3′ direction. However, the identity of the endonuclease remained unclear. Using purified Saccharomyces cerevisiae proteins, we show that Sae2 promotes dsDNA-specific endonuclease activity by the Mre11 subunit within the MRX complex. The endonuclease preferentially cleaves the 5′-terminated dsDNA strand, which explains the polarity paradox. The dsDNA end clipping is strongly stimulated by protein blocks at the DNA end, and requires the ATPase activity of Rad50 and physical interactions between MRX and Sae2. Our results suggest that MRX initiates dsDNA break processing by dsDNA endonuclease rather than exonuclease activity, and that Sae2 is the key regulator of this process. These findings demonstrate a probable mechanism for the initiation of dsDNA break processing in both vegetative and meiotic cells.

Characterization of the Interactome of the Human MutL Homologues MLH1, PMS1, and PMS2

Postreplicative mismatch repair (MMR) involves the concerted action of at least 20 polypeptides. Although the minimal human MMR system has recently been reconstituted in vitro, genetic evidence from different eukaryotic organisms suggests that some steps of the MMR process may be carried out by more than one protein. Moreover, MMR proteins are involved also in other pathways of DNA metabolism, but their exact role in these processes is unknown. In an attempt to gain novel insights into the function of MMR proteins in human cells, we searched for interacting partners of the MutL homologues MLH1 and PMS2 by tandem affinity purification and of PMS1 by large scale immunoprecipitation. In addition to proteins known to interact with the MutL homologues during MMR, mass spectrometric analyses identified a number of other polypeptides, some of which bound to the above proteins with very high affinity. Whereas some of these interactors may represent novel members of the mismatch repairosome, others appear to implicate the MutL homologues in biological processes ranging from intracellular transport through cell signaling to cell morphology, recombination, and ubiquitylation.

Expression of the MutL Homologue hMLH3 in Human Cells and its Role in DNA Mismatch Repair

The human mismatch repair (MMR) proteins hMLH1 and hPMS2 function in MMR as a heterodimer. Cells lacking either protein have a strong mutator phenotype and display microsatellite instability, yet mutations in the hMLH1 gene account for approximately 50% of hereditary nonpolyposis colon cancer families, whereas hPMS2 mutations are substantially less frequent and less penetrant. Similarly, in the mouse model, Mlh1-/- animals are highly cancer prone and present with gastrointestinal tumors at an early age, whereas Pms2-/- mice succumb to cancer much later in life and do not present with gastrointestinal tumors. This evidence suggested that MLH1 might functionally interact with another MutL homologue, which compensates, at least in part, for a deficiency in PMS2. Sterility of Mlh1-/-, Pms2-/-, and Mlh3-/- mice implicated the Mlh1/Pms2 and Mlh1/Mlh3 heterodimers in meiotic recombination. We now show that the hMLH1/hMLH3 heterodimer, hMutLgamma, can also assist in the repair of base-base mismatches and single extrahelical nucleotides in vitro. Analysis of hMLH3 expression in colon cancer cell lines indicated that the protein levels vary substantially and independently of hMLH1. If hMLH3 participates in MMR in vivo, its partial redundancy with hPMS2, coupled with the fluctuating expression levels of hMLH3, may help explain the low penetrance of hPMS2 mutations in hereditary nonpolyposis colon cancer families.

Biallelic somatic inactivation of the mismatch repair gene MLH1 in a primary skin melanoma

Inactivation of mismatch repair (MMR) genes has been linked to the hereditary nonpolyposis colon cancer syndrome and to a subset of sporadic cancers. A phenotypic characteristic of tumors with defective MMR is microsatellite instability (MSI). Although MSI has been reported in a proportion of cutaneous melanomas, inactivation of MMR genes in this tumor type has not been detected thus far. We recently described a human melanoma cell line, PR‐Mel, and a cutaneous metastasis from the same patient, which displayed a MMR defect, and showed high MSI. Here we report that in the PR‐Mel cell line both MLH1 alleles are somatically inactivated. One allele is lost through a chromosomal deletion of the region 3p21–24, whereas the remaining allele harbors a G → A transition at position −1 of the acceptor splice site of intron 15, leading to the in‐frame skipping of exon 16. The primary melanoma of the PR patient shows loss of heterozygosity at the BAT21 microsatellite marker, located in the MLH1 gene, and does not express the MLH1 and PMS2 proteins. Moreover, it harbors the same mutation detected in the PR‐Mel cells. These results demonstrate that biallelic inactivation of MLH1 had occurred in the primary melanoma of the PR patient and suggest that disruption of MMR might have had a role in the development of the melanoma. This is the first report in which genetic defects leading to disruption of MMR function in a human melanoma have been identified.

High Expression of the Mismatch Repair Protein MSH6 Is Associated With Poor Patient Survival in Melanoma

OBJECTIVES The outcome of patients with primary melanoma (PM) cannot be completely explained based on currently adopted clinical-histopathologic criteria. In this study, we evaluated the potential prognostic value of mismatch repair protein expression in PMs. METHODS We examined the immunohistochemical staining of mismatch repair proteins in 18 benign nevi and 101 stage I to III PMs and investigated their association with tumor clinicopathologic variables and melanoma mortality. RESULTS Expression of MSH2, MLH1, and PMS2 was high in benign nevi and reduced in a subset of PMs. Conversely, MSH6 expression was absent or extremely low in benign nevi and increased in a subset of PMs. In the multivariate analysis, including sex, age, Breslow thickness, and ulceration, high MSH6 expression in PMs (ie, immunostaining in >20% of tumor cells) was significantly associated with an increased risk of melanoma mortality (relative risk, 3.76; 95% confidence interval, 1.12-12.70). CONCLUSIONS MSH6 protein expression can be a valuable marker to improve prognosis assessment in PMs.

Regulatory control of DNA end resection by Sae2 phosphorylation

DNA end resection plays a critical function in DNA double-strand break repair pathway choice. Resected DNA ends are refractory to end-joining mechanisms and are instead channeled to homology-directed repair. Using biochemical, genetic, and imaging methods, we show that phosphorylation of Saccharomyces cerevisiae Sae2 controls its capacity to promote the Mre11-Rad50-Xrs2 (MRX) nuclease to initiate resection of blocked DNA ends by at least two distinct mechanisms. First, DNA damage and cell cycle-dependent phosphorylation leads to Sae2 tetramerization. Second, and independently, phosphorylation of the conserved C-terminal domain of Sae2 is a prerequisite for its physical interaction with Rad50, which is also crucial to promote the MRX endonuclease. The lack of this interaction explains the phenotype of rad50S mutants defective in the processing of Spo11-bound DNA ends during meiotic recombination. Our results define how phosphorylation controls the initiation of DNA end resection and therefore the choice between the key DNA double-strand break repair mechanisms.It has previously been established that DNA end resection in yeast and in humans is under CDK control. Here the authors explain how phosphorylation regulates the capacity of Sae2 — the yeast orthologue of human CtIP — to promote DNA end resection.