Subtle alterations in PCNA-partner interactions severely impair DNA replication and repair.

The robustness of complex biological processes in the face of environmental and genetic perturbations is a key biological trait. However, while robustness has been extensively studied, little is known regarding the fragility of biological processes. Here, we have examined the susceptibility of DNA replication and repair processes mediated by the proliferating cell nuclear antigen (PCNA). Using protein directed evolution, biochemical, and genetic approaches, we have generated and characterized PCNA mutants with increased affinity for several key partners of the PCNA-partner network. We found that increases in PCNA-partner interaction affinities led to severe in vivo phenotypic defects. Surprisingly, such defects are much more severe than those induced by complete abolishment of the respective interactions. Thus, the subtle and tunable nature of these affinity perturbations produced different phenotypic effects than realized with traditional "on-off" analysis using gene knockouts. Our findings indicate that biological systems can be robust to one set of perturbations yet fragile to others.

Elimination of oxalate by fat sand rats (Psammomys obesus): wild and laboratory-bred animals compared.

Wild fat sand rats (Psammomys obesus) can feed exclusively on plants containing much oxalate, but little calcium; oxalate intake may exceed 300 mg/d, while calcium intake is approximately 30 mg/day. By contrast, for generations, laboratory bred P. obesus have been fed a low-oxalate (<100 mg/day), high-calcium (approximately 150 mg/day) rodent chow. We compared oxalate intake and excretion between wild and laboratory-bred animals, both fed the natural high-oxalate diet, to determine whether these different dietary histories are reflected in the animal's ability to eliminate dietary oxalate. Since both wild and laboratory-bred P. obesus harbor intestinal oxalate-degrading bacteria, we predicted that their oxalate intake and excretion would be similar. Indeed, we found no significant differences in oxalate intake or excretion between the groups fed either saltbush or alfalfa (p>0.05). However, due to the differences in dietary calcium intake between the two diets, in both groups only part (23-25%) of the ingested oxalate was excreted when the animals were fed the oxalate-rich saltbush, yet most (87-90%) was excreted when feeding on calcium-rich alfalfa. Thus, even after generations of feeding on a commercial low-oxalate diet, fat sand rats maintain intestinal oxalate-degrading bacteria that appear to increase in number and activity when presented with their natural diet.

Oxalate balance in fat sand rats feeding on high and low calcium diets.

Oxalate reduces calcium availability of food because it chelates calcium, forming the sparingly soluble salt calcium-oxalate. Nevertheless, fat sand rats (Psammomys obesus; Gerbillinae) feed exclusively on plants containing much oxalate. We measured the effects of calcium intake on oxalate balance by comparing oxalate intake and excretion in wild fat sand rats feeding on their natural, oxalate-rich, calcium-poor diet with commercially-bred fat sand rats feeding on an artificial, calcium-rich, oxalate-poor diet of rodent pellets. We also tested for the presence of the oxalate degrading bacterium Oxalobacter sp. in the faeces of both groups. Fat sand rats feeding on saltbush ingested significantly more oxalate than fat sand rats feeding on pellets (P < 0.001) and excreted significantly more oxalate in urine and faeces (P < 0.01 for both). However the fraction of oxalate recovered in excreta [(oxalate excreted in urine + oxalate excreted in faeces)/oxalate ingested] was significantly higher in pellet-fed fat sand rats (61%) than saltbush-fed fat sand rats (27%). We found O. sp. in the faeces of both groups indicating that fat sand rats harbor oxalate degrading bacteria, and these are able, to some extent, to degrade oxalate in its insoluble form.

Oxalate, calcium and ash intake and excretion balances in fat sand rats (Psammomys obesus) feeding on two different diets.

Fat sand rats Psammomys obesus feed exclusively on plants of the family Chenopodiaceae, which contain high concentrations of chloride salts (NaCl, KCl) and oxalate salts. Ingestion of large quantities of oxalate is challenging for mammals because oxalate chelates Ca(2+) cations, reducing Ca(2+) availability. Oxalate is a metabolic end-point in mammalian metabolism, however it can be broken-down by intestinal bacteria. We predicted that in fat sand rats microbial breakdown of oxalate will be substantial due to the high dietary load. In addition, since a high concentration of soluble chloride salts increases the solubility of calcium oxalate in solution, we examined whether a change in the intake of chloride salts affects microbial oxalate breakdown and calcium excretion in fat sand rats. We measured oxalate, calcium and other inorganic matter (ash) intake and excretion in fat sand rats feeding on two different diets: saltbush (Atriplex halimus), their natural diet, and goose-foot (Chenopodium album), a non-native chenopod on which fat sand rats will readily feed and that has a similar oxalate content to saltbush but only 2/3 of the ash content. In animals feeding on both diets, 65-80% of the oxalate ingested did not appear in urine or faeces. In animals consuming the more saline saltbush, significantly more oxalate was apparently degraded (p<0.001), while significantly less oxalate was excreted in urine (p<0.01) and in faeces (p<0.05). We propose, therefore, that fat sand rats rely on symbiotic bacteria to remove a large portion of the oxalates ingested with their diet, and that the high dietary salt intake may play a beneficial role in their oxalate and calcium metabolism.