Many individuals with multiple or large colorectal adenomas, or early-onset colorectal cancer (CRC), have no detectable germline mutations in the known cancer predisposition genes. Using whole-genome sequencing, supplemented by linkage and association analysis, we identified specific heterozygous POLE or POLD1 germline variants in several multiple adenoma and/or CRC cases, but in no controls. The susceptibility variants appear to have high penetrance. POLD1 is also associated with endometrial cancer predisposition. The mutations map to equivalent sites in the proof-reading (exonuclease) domain of DNA polymerases ε and δ, and are predicted to impair correction of mispaired bases inserted during DNA replication. In agreement with this prediction, mutation carriers’ tumours were microsatellite-stable, but tended to acquire base substitution mutations, as confirmed by yeast functional assays. Further analysis of published data showed that the recently-described group of hypermutant, microsatellite-stable CRCs is likely to be caused by somatic POLE exonuclease domain mutations.

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Stalled replication forks produced by three different ways of depleting deoxynucleoside triphosphate showed different capacities to undergo "replication fork reversal." This reaction occurred at the stalled forks generated by hydroxyurea treatment, was impaired under thermal inactivation of ribonucleoside reductase, and did not take place under thymine starvation.Stalled replication forks create the need for replication reactivation, and different ways of restarting replication have been proposed (19). In several replication mutants, the stalled forks generated upon the inactivation of the mutant enzyme are reversed and result in the formation of a Holliday junction (HJ) adjacent to a DNA double-strand end, a reaction called "replication fork reversal" (RFR) (Fig. 1A) (19,29,30). In a rec-proficient background, this intermediary could be processed without generating DNA double-strand breaks (DSBs) by using the recombination proteins RecBCD and RecA and by the HJ-specific resolvase RuvABC (Fig. 1B) (16). In contrast, in the absence of RecBCD activity (Fig. 1C), resolution of the RFR-produced HJ is done by RuvABC resolvase and leads to fork breakage. These particular DSBs are dependent on RuvABC activity in a recB-deficient background.To verify the RFR process, a recB-deficient background should be used (i) to inhibit the degradation or the recombinational repair of the DNA tail created by the regression of the fork (20) (Fig. 1B), allowing RuvABC resolvase to transform this tail in a DSB, and (ii) to inhibit the repair of the DSBs generated by RuvABC resolvase (Fig. 1C). According to the RFR model, the occurrence of this process at the stalled forks can be verified by testing whether there is an increase of DSBs in a recB-deficient background and determining whether these DSBs are dependent on RuvABC resolvase activity by measuring the levels of DSBs in recB-and recB ruvABC-deficient backgrounds (Fig. 1C) (29). The occurrence of RFR at the stalled forks has been verified by this system in several replication mutants, such as in the helicase mutants rep and dnaBts (18,29), in the holD G10 mutant (6), in the dnaEts mutant at 42°C, and in the dnaNts mutant at 37°C (11).If RFR does not take place at the stalled fork, at least two situations may arise. On the one hand, there would be an increased level of DSBs independent of RuvABC activity and generated by another, unknown endonuclease (Fig. 1E), as in the case of dnaBts recB ruvABC at 42°C in the absence of RecA protein (30). On the other hand, there would be no increase in the level of DSBs, probably because the stalled forks are not susceptible to the endonuclease action, and the restarting of the forks would take place without the generation of fork breakage. This situation has been described in gyrB mutants (10) and when ter replication termination sequences were placed at ectopic positions on the bacterial chromosome (3).Using the system described above, in the present work we studied the fate of the stalled replication forks caused by deoxynucleoside triphosph...

Background: CRL4Cdt2 requires that a substrate bind to proliferating cell nuclear antigen (PCNA) on DNA prior to ligase recruitment, but the precise role of PCNA is unclear. Results: A specific PCNA residue is required for destruction of CRL4 Cdt2 substrates. Conclusion: CRL4Cdt2 recognizes a composite surface composed of PCNA and substrate residues. Significance: This is the first ubiquitin ligase whose substrate recognition requires creation of a bipartite substrate surface.

Cdt1 plays a critical role in DNA replication regulation by controlling licensing. In Metazoa, Cdt1 is regulated by CRL4Cdt2-mediated ubiquitylation, which is triggered by DNA binding of proliferating cell nuclear antigen (PCNA). We show here that fission yeast Cdt1 interacts with PCNA in vivo and that DNA loading of PCNA is needed for Cdt1 proteolysis after DNA damage and in S phase. Activation of this pathway by ultraviolet (UV)-induced DNA damage requires upstream involvement of nucleotide excision repair or UVDE repair enzymes. Unexpectedly, two non-canonical PCNA-interacting peptide (PIP) motifs, which both have basic residues downstream, function redundantly in Cdt1 proteolysis. Finally, we show that poly-ubiquitylation of PCNA, which occurs after DNA damage, reduces Cdt1 proteolysis. This provides a mechanism for fine-tuning the activity of the CRL4Cdt2 pathway towards Cdt1, allowing Cdt1 proteolysis to be more efficient in S phase than after DNA damage.

The observed lengthening of the C period in the presence of a defective ribonucleoside diphosphate reductase has been assumed to be due solely to the low deoxyribonucleotide supply in the nrdA101 mutant strain. We show here that the nrdA101 mutation induces DNA double-strand breaks at the permissive temperature in a recB-deficient background, suggesting an increase in the number of stalled replication forks that could account for the slowing of replication fork progression observed in the nrdA101 strain in a Rec ؉ context. These DNA double-strand breaks require the presence of the Holliday junction resolvase RuvABC, indicating that they have been generated from stalled replication forks that were processed by the specific reaction named "replication fork reversal." Viability results supported the occurrence of this process, as specific lethality was observed in the nrdA101 recB double mutant and was suppressed by the additional inactivation of ruvABC. None of these effects seem to be due to the limitation of the deoxyribonucleotide supply in the nrdA101 strain even at the permissive temperature, as we found the same level of DNA double-strand breaks in the nrdA ؉ strain growing under limited (2-g/ml) or under optimal (5-g/ml) thymidine concentrations. We propose that the presence of an altered NDP reductase, as a component of the replication machinery, impairs the progression of the replication fork, contributing to the lengthening of the C period in the nrdA101 mutant at the permissive temperature.Ribonucleoside diphosphate reductase (NDP reductase) is the only specific enzyme required for the enzymatic formation of deoxyribonucleotides (dNTPs), the precursors of DNA synthesis in Escherichia coli. NDP reductase is a 1:1 complex of two nonidentical subunits called proteins R1 and R2, encoded by genes nrdA and nrdB, respectively (for a review, see reference 3). The best-known defective NDP reductase mutant of E. coli contains a thermolabile R1 subunit encoded by the nrdA101 allele. The activity of the enzyme measured in crude extracts of nrdA101 strains is limited to 6% of the wild-type activity at 25°C (6), and the dNTP pool is lower than wild type even at permissive temperatures (16). Our laboratory has shown that the presence of the nrdA101 allele lowers the replication rate of the mutant strain at the permissive temperature, as a nrdA101 mutant replicates the chromosome in 154 min at 30°C, while a nrdA ϩ strain does so in 98 min (10). Regarding the detrimental effect of the nrdA101 allele on the activity of the enzyme, this DNA replication effect is assumed to be due to the decrease in the NDP reductase activity as a dNTP provider. However, NDP reductase has been proposed as a component of the replication hyperstructure (10), and consequently the structure of the NDP reductase encoded by the nrdA101 allele might provoke a structural alteration of the replication hyperstructure that could also contribute to the lengthening of the C period in the mutant. A possible consequence of an altered replication hyperstr...

Summary Synthesis of dNTPs is required for both DNA replication and DNA repair and is catalyzed by ribonucleotide reductases (RNR), which convert ribonucleotides to their deoxy forms [1, 2]. Maintaining the correct levels of dNTPs for DNA synthesis is important for minimising the mutation rate [3-7], and this is achieved by tight regulation of ribonucleotide reductase [2, 8, 9]. In fission yeast, ribonucleotide reductase is regulated in part by a small protein inhibitor, Spd1, which is degraded in S phase and after DNA damage to allow up-regulation of dNTP supply [10-12]. Spd1 degradation is mediated by the activity of the CRL4Cdt2 ubiquitin ligase complex [5, 13, 14]. This has been reported to be dependent on modulation of Cdt2 levels which are cell cycle regulated, peaking in S phase, and which also increase after DNA damage in a checkpoint-dependent manner [7, 13]. We show here that Cdt2 levels fluctuations are not sufficient to regulate Spd1 proteolysis and that the key step in this event is the interaction of Spd1 with the polymerase processivity factor PCNA, complexed onto DNA. This mechanism thus provides a direct link between DNA synthesis and ribonucleotide reductase regulation.

NDP reductase activity can be inhibited either by treatment with hydroxyurea or by incubation of an nrdA ts mutant strain at the non-permissive temperature. Both methods inhibit replication, but experiments on these two types of inhibition yielded very different results. The chemical treatment immediately inhibited DNA synthesis but did not affect the cell and nucleoid appearance, while the incubation of an nrdA101 mutant strain at the nonpermissive temperature inhibited DNA synthesis after more than 50 min, and resulted in aberrant chromosome segregation, long filaments, and a high frequency of anucleate cells. These phenotypes are not induced by SOS. In view of these results, we suggest there is an indirect relationship between NDP reductase and the chromosome segregation machinery through the maintenance of the proposed replication hyperstructure.